信息编号11120201至11120250间共44条。
☉ 11120201:Lingering Post-Katrina MH Problems May Be `New Normal' for Children
Only a coordinated effort by psychiatrists and mental health professionals can meet the ongoing needs of Katrina's youngest survivors. Many Louisiana children affected by Hurricane katrina show signs of resilience today, but many others display continuing psychiatric symptoms 10 months after the storm. "Among key issues that affect children are their ability to trust adults to keep them safe," said psychologist Joy Osofsky, Ph.D., a professor of pediatrics and psychiatry at the Louisiana State University (LSU) Health Sciences Center. She spoke at a meeting convened by the Substance Abuse and Mental Health Services Administration in New Orleans in May. "Children's reaction mirrors their parents'. Whenever it rains, whenever there's any kind of storm, like a recent tornado, we see lots of separation anxiety." Some kids are clingy, worry about what is going to happen, talk repeatedly about the hurricane, or are upset, afraid, or sad when thinking about the event, she said. They feel that nothing is fun and have a hard time getting along with friends and family now. The hardest-hit group may be high school students, who not only miss their friends but also worry about their ability to get into college. Screening organized in St. Bernard Parish by Osofsky and her husband, Howard Osofsky, M.D., Ph.D., professor and chair of psychiatry at LSU, found that 54 percent of children met criteria for mental health referral, and 14 percent directly requested counseling. They also learned that 34 percent of the youngsters were separated from their primary caregiver, 14 percent had friends who had died because of the storm, 45 percent had parents who were unemployed, and 33 percent had symptoms of PTSD or depression. Even today, many students don't live with their parents because the adults are working elsewhere, said Joy Osofsky. "We're seeing a `slow burn' of PTSD and continuing symptoms with a slow recovery and a continued desolation," she said. "Some behavioral and emotional reactions may be `normal' or represent a `new normal' after such a widespread disaster with continuing anxiety." Young people are anxious about the ongoing uncertainty in their lives, an anxiety compounded by the onset of the new hurricane season that began on June 1. Many have had trouble concentrating in school and have become disruptive. Schools that take a zero-tolerance approach to behavioral problems expel children who act out. On the positive side, children want to resume normal life and their prehurricane activities, enjoy going back to school and being with other children, and show more resilience with support from their parents. In her work immediately after the storm, Osofsky found that her training in trauma work was a help, but more important was just being there for young people, she said. "Listening, gathering information is a key. When they heard their own concerns, they felt better." As part of the Louisiana Spirit program that he heads, Howard Osofsky said, children returning to schools that reopened in St. Bernard and Orleans parishes were screened, resiliency-building programs were set up, and treatment provided when needed. Even before the storm, many children in New Orleans lived in areas of preexisting trauma, with subsistence-level poverty, crime, and bad general health, said Jay koonce, M.S.W., L.C.S.W., clinical manager of a substance abuse treatment center in Austin, Texas. koonce helped coordinate mental health services for evacuees in that city. "They endured a traumatizing experience of being victimized by nature and abandoned by society. They experienced trauma, on top of trauma, on top of trauma." Koonce's watchword for helping evacuees was: "Meet them where they are." He found that mental health professionals could either wait in designated areas for guests (as he called the evacuees) to self-refer or be brought in by police or medical staff, or they could take to the shelter floor and roam about, finding people who needed help. The latter approach was much more productive, he said. Going to those who needed help remained the favored tactic in Austin. In the months after the storm, as people moved out of shelters, students were enrolled in the city's public schools. A Communities-in-the-Schools katrina Project was established, funded by private corporations for a year to coordinate school-based counselors and case managers. During the school year, the school was the place to connect with families, said koonce. Now that school is out, the home is the locus of contact. "Asking parents if they need psychological help may meet with resistance, but we have more success if we say to them, `Why don't you come in and let's talk about how to help your kids?' then open up the discussion to include the parents' mental health and how it affects their children," he said....查看详细 (4922字节)

☉ 11120202:Law Opens MCO Panels To `Any Willing Provider'
Mental health advocates hail the latest, and surprisingly unopposed, expansion of "any willing provider" laws into Vermont. Among the first "any willing provider" measures aimed specifically at mental health clinicians, a recently enacted vermont law mandates that insurers provide identical reimbursement, regardless of whether treatment is provided by a member of its provider network. The measure was hailed by vermont mental health advocates as the latest refinement of the state's 1997 mental health parity law, which required insurers to provide comparable coverage for mental and other medical conditions. In the years since approval of that legislation, the state's mental health clinicians found managed care networks effectively limited mental health care for many residents by not covering the services of established clinicians for patients new to the insurance network. "These days insurance coverage often changes when people change jobs or move, so people often lose access to their mental health or substance abuse health care provider because they are not in the new insurer's [provider] network," said ken Libertoff, director of the vermont Association for Mental Health. "By passing this legislation, vermont reaffirms the right of health care consumers to receive treatment from practitioners of their choice as long as they are licensed and certified by the state of vermont." Research indicates that in mental health care, the relationship between the patient and clinician is an important variable in effective treatment, Libertoff said. Patient Choice Preserved Supporters described the bill (H 404) as part of a series of initiatives designed to preserve patient choice and ensure access to necessary and appropriate treatment options. The measure passed after a collaborative legislative push by state professional associations—including the vermont Psychiatric Association and vermont Medical Society—and the major patient advocacy groups. The measure's brief language requires health insurance plans to include in their networks "any licensed mental health or substance abuse [care] provider located within the geographic coverage area of the health benefit plan if the provider is willing to meet the terms and conditions for participation established by the health insurer." "This bill was a response to concerns about access to comprehensive treatment for people with mental illness and substance abuse disorders," said David Fassler, M.D., legislative representative for the vermont Psychiatric Association and a member of APA's Board of Trustees. "It eliminates the use of restrictive or closed panels by insurance companies and/or their managed care intermediaries." Supporters said the need for the measure was found in estimates that 20 percent to 30 percent of the state's population had a diagnosable mental health condition, but only 7 percent received treatment, which indicated the presence of barriers to care. The law, effective July 1, allows psychiatrists or mental health professionals to join a network or provider panel if they are willing to accept the same terms and conditions applicable to other participants. The requirement that clinicians agree to insurers' terms related to care and fees has caused some consternation among physicians over similar laws in other states. Short-Term Impact Unclear The law may have some negative impact on in-network mental health care clinicians in the short term because it eliminates any clinician or insurer monopolies on who gets to treat patients, Libertoff said. However, over the long term the measure will benefit patients, who have been forced to pay out of pocket to continue receiving care from the same clinician after they switched insurers. The bill easily passed both chambers and faced no formal opposition from insurers, which vigorously opposed broader any-willing-provider measures in other states. The industry's opposition culminated in the 2003 Supreme Court decision in Kentucky Association of Health Plans v. Miller, which found any-willing-provider state laws were not barred under the federal Employee Retirement Income Security Act of 1974 (ERISA). Insurers had argued that such laws were barred by ERISA, which preempts state laws regarding employer-provided health plans. Although no vermont insurers responded to calls from Psychiatric News, supporters of the law credited their lack of opposition to the broad support for the measure, the state's progressive politics, and early support from vermont Gov. Jim Douglas (R). Douglas signed the measure on May 4. Opponents of more general any-willing-provider laws in other states have argued that the measures would increase costs and reduce coverage options for consumers by limiting health plans' ability to negotiate with clinicians for discounted rates. Disruptions to insurers' provider networks would also disrupt their quality controls for clinicians, insurers have insisted. Quality-control measures by insurers, Libertoff said, have been found to be largely perfunctory, with few detailed examinations of the quality of care given by individual clinicians. Supporters of the bill highlighted statistics showing that the state's largest managed behavioral health panel represented fewer than 50 percent of licensed clinicians in the state, and other panels represent even fewer. They also pointed out that there are no comparable panel restrictions for primary care physicians. A copy of the law is posted at ....查看详细 (5658字节)
☉ 11120203:Interpersonal Relationships Predict Course of BPD
The quality of their relationships with children may help predict how patients with borderline personality disorder will fare in treatment. This is in line with reports indicating that for some patients motherhood leads to positive self-esteem, while others can be immobilized by the difficulties of childrearing. The quality of a patient's current interpersonal relationships and history of childhood trauma are factors that can aid clinicians in rendering a prognosis for treatment of borderline personality disorder (BPD). That was a new finding from the study "Predictors of Two-year Outcome for Patients With Borderline Personality Disorder" published in the May American Journal of Psychiatry. Somewhat more predictably, the longitudinal follow-up of 161 patients with BPD found that the strongest predictor of two-year outcome was severity of baseline psychopathology as measured by the number of BPD diagnostic criteria and functional disability. But the finding that an assessment of interpersonal stability can aid in the prediction of outcome was something of a surprise. "Interpersonal relationships have not been examined very often as prognostic variables," lead author John Gunderson, M.D., told Psychiatric News. "In our study, we looked at interpersonal relations alongside the other two factors—impulsivity and affective instability. These other two factors have often been looked at in prior research. "The overall quality of the borderline patients' interpersonal relationships proved to be a stronger predictor of course than either their affective instability or impulsivity," said Gunderson, a professor of psychiatry at Harvard Medical School and director of the Center for Treatment and Research on Borderline Personality Disorder at McLean Hospital. "That interpersonal relations have predictive value is clinically useful and adds to prior research. This means that clinicians should recognize that their patients with the most chaotic, stormy, and frequent interpersonal disturbances are not likely to get well soon." Impulsivity Data Surprising Gunderson added that the finding that impulsivity was not predictive was surprising, since it had emerged as a solid predictor in other studies. "We don't really understand why it wasn't here," he said. "Our sample was pretty representative, and our assessments were reliable and used standard measures. Perhaps longer-term follow-up will tell us more." In the study, 160 patients were recruited from four clinical sites of the Collaborative Longitudinal Personality Disorders Study. Patients were assessed at baseline and at six, 12, and 24 months with the Structured Clinical Interview for DSM-IV Axis I disorders; the Diagnostic Interview for DSM-IV Personality Disorders, a modified version of that instrument; the Longitudinal Interval Follow-Up Evaluation; and the Childhood Experiences Questionnaire–Revised. Three Categories Assessed The patients were assessed for a range of variables, grouped into three categories—psychiatric history, developmental experiences, and presenting phemonenology. Psychiatric history variables found to be predictive were length of previous hospitalizations and early age of psychiatric contact. Developmental experiences predictive of poor outcome included childhood abuse, parental underinvolvement, being a victim of father-daughter incest, parental divorce, parental brutality, childhood separations or losses, and disturbed maternal relationships. Predictor variables within presenting phenomenology traits were of three subtypes: severity, comorbidity, and personality. Severity variables previously reported to predict poor outcome include having low Global Assessment Scale scores, meeting a greater number of bipolar disorder criteria, and presenting a greater number of Axis II diagnoses at follow-up. Comorbidity variables found to be predictive of poor outcome include substance abuse and depression or "dysphoria." Comorbid personality predictor variables of poor outcome include those related to schizotypal, antisocial, and paranoid personality disorders. Better outcome was related to comorbid obsessive-compulsive personality disorder. Personality trait variables that predicted outcome were impulsivity and affective instability. Two-year outcome was assessed according to measures of global functioning and number of borderline personality disorder criteria. The clearest and strongest finding from the study was that the greater the severity level of dysfunction and psychopathology at baseline, the worse the outcome. yet Gunderson drew attention to the fact that the study did not yield overwhelmingly conclusive results for any of the predictors. "A rather sobering message is that none of the predictors is very strong," he told Psychiatric News. "The heterogeneous and often quite positive course of borderline patients remains hard to predict with confidence." Nevertheless, the relevance of the quality and stability of current interpersonal relationships is a finding that will aid clinicians in determining prognosis and treatment. Related to this was a finding that among BPD patients who are parents, the quality of relationships with their children was also predictive. This is consistent with clinical reports that some patients with BPD find motherhood a source of positive self-esteem and security, whereas others can be immobilized by the conflicts related to nurturing or disciplining their children, according to the report. "Clinicians have often been exposed to borderline patients who found the stress of mothering overwhelming," Gunderson said. "Whenever they felt angry at their child, they felt they were bad and became self-destructive. This study sheds light on the other side of this. Many borderline patients have found the mothering experience a source of stability and self-esteem. They are less apt to reappear in treatment settings. knowing this has made me more circumspect about advising women with borderline personality disorder whether to have children or not." "Predictors of 2-Year Outcome for Patients With Borderline Personality Disorder" is posted at ....查看详细 (6304字节)

☉ 11120204:Psychiatrists Search for Links Between Religion, Illness
Could substance abuse be viewed as a spiritual quest? Could a spiritual vacuum underlie personality disorders? Psychiatrists propose these bold concepts at APA's 2006 annual meeting. Many people—psychiatrists and others—believe that psychiatry has not been religion friendly. After all, Freud was an avowed atheist, and only about one-third of American psychiatrists say they carry religious beliefs into their everyday lives, according to a study cited at APA's 2006 annual meeting in the symposium "A Research Agenda for DSM-V Concerning Religious and Spiritual Issues in the Diagnostic Process." Mary Lynn Dell, M.D.: We need to think about the value of religion and spirituality to youth coping with illness, death, or other stressors. William Narrow, M.D.: Religion and spirituality might be viewed as complementary or alternative medicine. Joan Arehart-Treichel Nonetheless, during the past decade or so, the possible impact of religion and spirituality on mental health has sparked interest among psychiatrists (Psychiatric News, March 19, 2004; June 18, 2004; November 4, 2005). And material presented at the symposium, which was sponsored by the APA Corresponding Committee on Religion, Spirituality, and Psychiatry, should kindle interest further. Little research has been conducted on the possible influence of religion and spirituality on mental health, symposium speakers agreed—at least compared with the extensive research that has been conducted on other mental health topics. For example, information about the influence of religion and spirituality on psychopathology among youth is limited to only a few studies, Mary Lynn Dell, M.D., an associate professor of psychiatry at Emory University, noted. In addition, studies that have been conducted have produced results that often conflict. For instance, at least 76 studies have been conducted on the relationship between religion and anxiety, Gerrit Glas, M.D., Ph.D., a professor of psychiatry and philosophy at Leiden University in the Netherlands, reported. Of these, 35 found less anxiety among religious people; 10 found more, and the rest found either no link or produced mixed results. As for studies exploring the possible influence of religion on obsessive-compulsive disorder, including religious obsessions, contradictory outcomes have also emerged, he said. Role in Depression Uncertain The role of religion and spirituality in depression is likewise in question. One study, Dell pointed out, suggested that religious involvement might shield adolescents against depression. In contrast, Dan Blazer II, M.D., Ph.D., a professor of psychiatry at Duke University, and his colleagues found, in a community-based study, that subjects who identified their religious affiliation as Pentecostal had a higher rate of major depression than did the overall population. However, the Pentecostal subjects came from a lower socioeconomic background. So other factors related to psychological stress may have contributed to their depression more than their religious beliefs did, Blazer explained. Also unclear is whether religious belief can help people cope with trauma, said Samuel Thielman, M.D., Ph.D., director of the Office of Mental Health Services at the U.S. Department of State. Studies have produced both positive and negative results in this domain. For example, one investigation found that trauma made people more religious; another found that it did not. Even with the paucity of research on religion, spirituality, and mental well-being, and in the face of conflicting research results, one symposium speaker—Marc Galanter, M.D., a professor of psychiatry at New york University—tapped research he has conducted on the subject to make a provocative suggestion. He and his group have found that substance-abuse patients tend to score high in spiritual needs—that is, in a thirst or reaching out for a God, the arts, humanism, nature, or something that is transcendent for them. He and his team have also learned that physicians recovering from alcoholism rated Alcoholics Anonymous, which is spiritually oriented, very highly. So substance abuse and its treatment might be viewed, at least in certain circumstances, as a spiritual quest, he maintained. Daring Proposal Offered Another speaker, C. Robert Cloninger, M.D., a professor of psychiatry and genetics at Washington University and director of the university's Sansone Center for Well-Being, made an even more audacious hypothesis—that a spiritual vacuum underlies personality disorders. Harm avoidance, novelty seeking, and the search for social approval are some of the emotional needs that people have, Cloninger explained, and they activate more primitive parts of the human brain such as the amygdala. Yet these emotional needs have to be regulated by certain character traits that activate the rational part of the brain, the prefrontal cortex. These traits include being self-directed (responsible), cooperative (flexible, helpful), and self-transcendent (compassionate). Furthermore, people with personality disorders tend to lack self-regulation of these character traits and thus can be irritating and unlikable. This lack of self-regulation, Cloninger asserted, can be characterized as "a deficit in their spiritual perspective, which leads to patterns of thought, feeling, and behavior that can be described as vices like pride, lust, and greed." Some 19th-century psychiatrists also held the same view, he pointed out. At the beginning of the 19th century, the antisocial personality was referred to as "moral insanity" or "loss of self-government." Benjamin Rush said that people with personality disorders are "insensitive to the suffering of others." Cloninger, in fact, proposes that if psychiatrists helped individuals with personality disorders develop a fuller spiritual perspective, it might "expand their awareness of the intangible connections among people...." Such an expanded awareness may not only lead to improvement in their character and behavior, but also "make their lives more meaningful and satisfying." "This is fascinating, a totally different approach," audience member Carl Bell, M.D., commented. Bell is president and C.E.O. of the Community Mental Health Council in Chicago. And in a sense, spirituality might be viewed as a complementary or alternative treatment, William Narrow, M.D., the symposium discussant, pointed out. Narrow is associate director of APA's Division of Research and director of research for DSM-V. Yet there is no doubt, Narrow added, that much more research needs to be conducted to determine the roles that religion an information might eventually bolster psychiatrists' efforts to diagnose, treat, and even prevent various mental illnesses....查看详细 (6862字节)
☉ 11120205:Positive Psychosis Symptoms Linked to Violence Risk
Command hallucinations and persecutory delusions are associated with episodes of violence among a large sample of people with schizophrenia, but serious violence by such individuals is infrequent. Though violent behavior is the exception rather than the rule among people with schizophrenia, one large study found that positive psychotic symptoms such as persecutory ideation and grandiosity were associated with an increased risk of serious violence. In contrast, negative symptoms such as passivity and social withdrawal were associated with a decreased risk of violent behavior. "I think these findings reinforce the view that violence risk reduction should be an important goal and component of community-based treatment for schizophrenia and that risk reduction needs to focus on clinical as well as nonclinical factors that may contribute to violence," one of the study's investigators, Marvin Swartz, M.D., told Psychiatric News. Swartz is a professor and head of the Division of Social and Community Psychiatry at Duke University Medical Center. Nonclinical factors related to violence in the sample included residing in restrictive housing (such as a halfway house, psychiatric hospital, or jail) or with family, not feeling "listened to" by family members, and a recent history of police contact. Data came from baseline interviews of 1,410 people with schizophrenia enrolled in the National Institute of Mental Health Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study. The CATIE study was a randomized trial conducted between January 2001 and December 2004 at 56 clinical sites in 24 states to investigate treatment effectiveness and outcomes among patients with schizophrenia. Each study site screened inpatients and outpatients for study eligibility. Those who were deemed to be appropriate for participation had adequate decision-making capacity and were receiving suboptimal treatment with antipsychotic medications due to problems with efficacy or tolerability. During a baseline assessment and before the patients were randomized to experimental treatments for the CATIE study, lead investigator Jeffrey Swanson, Ph.D., and colleagues assessed the patients for violent behavior in the prior six months. He is an associate professor in the Department of Psychiatry and Behavioral Sciences at Duke. To assess the generalizability of the sample, Swanson compared participants with a "quasi-random" sample of 1,413 patients enrolled in the Schizophrenia Care and Assessment Program, an observational study of schizophrenia treatment in the United States. Though the CATIE sample had a lower proportion of minority patients, the two samples were similar in other demographic characteristics, age, and a variety of clinical characteristics. The similarities provide "some confidence that the CATIE project's randomized, controlled-trial design did not result in a biased selection of more severely ill and impaired patients," the authors noted. They used the MacArthur Community violence Interview to measure minor and serious violence. Researchers defined minor violence as a simple assault without injury or weapon use (shoving or slapping another person, for example) and serious violence as assault using a lethal weapon or resulting in injury, threat with a lethal weapon, or sexual assault. Researchers gathered information on violent acts by the subjects through patient self-reports and family collateral information (when available). Researchers also assessed patients at baseline for factors such as available social support, severity of illness, and awareness of mental health problems, among others. They also used the Structured Clinical Interview for Axes I and II of DSM-IV Disorders–Patient Edition to confirm schizophrenia diagnoses and assess childhood conduct problems. In addition, they used the Positive and Negative Syndrome Scale (PANSS) to rate severity of psychotic symptoms. Among the 1,410 patients, the six-month prevalence of any violence was 19.1 percent: 219 patients (15.5 percent) reported minor violence, while 51 (3.6 percent) reported serious violence. Both Clinical, Nonclinical Factors Play Role Swanson found that serious violence was associated with several clinical and nonclinical factors. For instance, those who scored above the median on the PANSS for positive psychosis symptoms had 2.71 times the risk for serious violence as those with lower scores. Patients who scored above the median for negative psychotic symptoms had about a quarter of the risk as those who scored below this point. People with suspiciousness and persecutory delusions also had an increased risk of engaging in serious violence (odds ratio 1.46, p....查看详细 (9021字节)

☉ 11120206:Parents' Verbal Abuse Leaves Long-Term Legacy
Parental verbal abuse may wound children's psyches so deeply that the effects remain apparent in young adulthood. Such abuse may wreak psychological havoc greater than that caused by physical abuse. With an M.B.A. degree under her belt, 24-year-old "Jaime" (not her real name) should have glowing job prospects in Chicago. But she harbors memories that erode her self-confidence and make her bristle with anger—memories of her father shouting at her, during drunken rages, that she was ugly and of little value. Indeed, verbal abuse during childhood can scar people deeply, a new study suggests. It was headed by Martin Teicher, M.D., Ph.D., director of the Developmental Biopsychiatry Research Program at McLean Hospital, which is affiliated with Harvard Medical School. Results were published in the June American Journal of Psychiatry. Although the injurious effects of child physical and sexual abuse have been the subject of considerable inquiry, not much attention has been paid to the possibly noxious effects of verbal abuse on children. More than 500 young adults were recruited via advertisements to participate in the study. Each subject was evaluated for childhood exposure to verbal abuse, physical abuse, sexual abuse, and domestic violence. Each subject was also assessed for current anxiety, depression, anger-hostility, and symptoms of dissociation. The researchers then looked to see if there were any associations between verbal abuse during childhood and current anxiety, depression, anger-hostility, and symptoms of dissociation; between other types of abuse during childhood and these current psychological problems; and how any associations for verbal abuse might compare with associations for other types of childhood abuse. The strength of the association between maltreatment history and current psychological difficulties was determined by calculating the effect sizes and 95-percent confidence intervals for the differences between subjects who had no exposure to maltreatment and subjects exposed to the maltreatment categories. "Effect size is a more valuable measure for assessing the impact of an experience than the p value, which is strongly affected by group size," Teicher and his coworkers explained in their report. Childhood verbal abuse had a relatively weak association with current anxiety, the investigators found, but it had moderate to strong links with current depression, anger-hostility, and dissociative symptoms. Moreover, these links were stronger than those for being a victim of physical abuse during childhood. They were comparable to those for witnessing domestic violence during childhood and for being sexually abused by a nonfamily member during childhood. The only form of child abuse that had a stronger link with current depression and dissociative symptoms than childhood verbal abuse was being sexually abused by a family member. And even it had a weaker connection with current anger-hostility than did childhood verbal abuse. These results, of course, do not prove that childhood verbal assaults can cause psychological consequences in early adulthood since the investigation was of a retrospective rather than a prospective nature. Nonetheless, Teicher and his coworkers believe that it may well be the case. As they concluded in their report, childhood verbal abuse is "a potent form of maltreatment." But perhaps the most interesting findings to emerge from their study came when they examined the links between more than one type of child abuse and current psychological difficulties. They found, for example, an extraordinarily powerful link between the combination of verbal abuse and witnessing domestic violence and current dissociative symptoms. "This finding is consonant with studies that suggest that emotional abuse may be a more important precursor of dissociation than is sexual abuse," Teicher and his team said. Indeed, the connections that Teicher and his group found between various combinations of child abuse and current psychological difficulties were so potent that they often equaled or exceeded the link between familial sexual abuse during childhood and current mental health. "This is of great importance," the researchers noted, "as it suggests that combined exposure to less blatant forms of abuse may be just as deleterious as the most egregious acts we confront." The study was funded by the National Institute of Mental Health and the National Institute on Drug Abuse. "Sticks, Stones, and Hurtful Words: Relative Effects of Various Forms of Childhood Maltreatment" is posted at ....查看详细 (4724字节)
☉ 11120207:Old But New
I read with interest the article "Residency Program Combines Child, General Psychiatry" in the March 3 issue. I was surprised that this combined training seemed like a new and suddenly creative concept. Thomas Anders, M.D., wrote, "Now we have regulatory concurrence that integrated training is feasible. We are left with how to implement this." Certainly, one place to look for proof is Duke University Medical Center. Drs. John Fowler and Ewald Busse had a four-year combined program for child psychiatry training already in place when I entered in 1970. The general residents were part of the same program, starting their child rotations in year one. Thus, this "new" concept is at least 36-plus years old....查看详细 (718字节)
☉ 11120208:Spred-1 negatively regulates allergen-induced airw
T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic asthma. These cytokines transmit signals through the Janus kinase/signal transducer and activator of transcription (STAT) and the Ras–extracellular signal-regulated kinase (ERK) signaling pathways. Although the suppressor of cytokine signaling (SOCS) family proteins have been shown to regulate the STAT pathway, the mechanism regulating the ERK pathway has not been clarified. The Sprouty-related Ena/VASP homology 1–domain-containing protein (Spred)-1 has recently been identified as a negative regulator of growth factor–mediated, Ras-dependent ERK activation. Here, using Spred-1–deficient mice, we demonstrated that Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness, without affecting helper T cell differentiation. Biochemical assays indicate that Spred-1 suppresses IL-5–dependent cell proliferation and ERK activation. These data indicate that Spred-1 negatively controls eosinophil numbers and functions by modulating IL-5 signaling in allergic asthma. Abbreviations used: AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BMEo, BM–derived eosinophil; EGFP, enhanced GFP; ERK, extracellular signal-regulated kinase; PAS, periodic acid-Schiff; Spred, Sprouty-related EVH1-domain-containing protein. H. Inoue, R. Kato, and S. Fukuyama contributed equally to this work. Asthma is characterized by a variable degree of airflow obstruction, airway hyperresponsiveness (AHR; defined by enhanced airflow obstruction in response to nonspecific stimuli), mucus overproduction, and chronic airway inflammation. Numerous eosinophils and lymphocytes infiltrate peribronchial tissues in asthmatics. Th2 cells are the predominant lymphocyte population that infiltrates the airways of asthmatics, and the cytokine products of Th2 cells play essential roles in airway eosinophilia, AHR, and serum IgE in animal models (1). Eosinophils are produced in bone marrow, and recent observations in both mice and humans suggest that pulmonary allergen exposure results in both increased output of eosinophils from hemopoietic tissues and increased migration of these cells to the lung (2–4). It is the accumulation of activated eosinophils during the late-phase response to allergen exposure that ultimately results in progressive inflammatory tissue damage. In addition, pulmonary eosinophilia in response to allergen challenge is associated with elevated levels of eosinophil-derived proteins in both the lungs and peripheral blood (5, 6). However, the specific mechanisms that alter eosinophilopoiesis in asthma are poorly understood. Eosinophil production is predominantly regulated by the Th2 cytokine, IL-5 (7, 8). IL-5 receptor consists of IL-5–specific -chain and the common ?-chain that is shared by the IL-3 and GM-CSF receptor (9). In addition to the JAK–STAT pathway, the Ras–extracellular signal-regulated kinase (ERK) pathway has also been implicated in signaling of IL-5 and other cytokines (10–12), and this pathway is shown to be important for IL-5–dependent cell survival (12). Therefore, the Ras-ERK signals seem to be important for eosinophilia in asthma; however, the regulation of this pathway and its contribution to the disease have not been clarified. ERK activation is initiated by binding of Grb2 to the phosphorylated tyrosine residues of the receptor or phosphorylated adaptor molecules such as Shc, FRS-2, IRS-1/2, SHP-2, and Gab-1. The complex of Grb2 and SOS activates Ras by GTP loading. Ras-GTP recruits Raf-1 to the plasma membrane (13, 14). Raf-1 is phosphorylated and activated by not well-defined kinases with complex regulatory mechanisms (15). Activated Raf phosphorylates and activates the dual-specific kinase MEK, which phosphorylates and activates ERK. The regulation of this pathway has been suggested to be quite important for cell proliferation and differentiation. Recently, Sprouty family proteins were identified as negative regulators for several growth factor-induced ERK activation including FGF and EGF (9, 16). Four Sprouty homologues are found in mammals. We cloned an additional Sprouty-related family of novel membrane bound molecules, Sprouty-related Ena/VASP homology 1–domain-containing proteins (Spreds; reference 17). Three members of Spreds were identified in mammals (18), which have a Sprouty-related COOH-terminal cysteine-rich domain in addition to the NH2-terminal Ena/VASP homology 1 domain. Like Sproutys, Spred-1, Spred-2, and Spred-3 also down-regulate Ras/ERK signaling. As Spred inhibits active Ras-induced ERK activation, Spred might modulate the unidentified activation steps of Raf by a novel mechanism. Spred/Sprouty family proteins have emerged as a negative regulator of the ERK pathway; however, details of their physiological function and molecular mechanism remain to be investigated. In the present paper, we generated Spred-1 knockout mice and examined the function of Spred-1 on the development of asthma and associated eosinophilia. We show that Spred-1–/– mice exhibited exaggerated allergen-induced AHR, eosinophilia, and mucus production in a murine allergic asthma model. Spred-1–/– mice also showed increased responsiveness, especially ERK signals, to IL-5 and subsequent overexpression of IL-13 in eosinophils. Thus, it is conceivable that the down-regulation of Spred-1 in the airways has a significant role in prolonged airway eosinophilia and asthma phenotypes. We propose the possibility that Spred-1 may present a novel therapeutic target for the treatment of asthma. Results Generation of Spred-1–/– mouse and assessment of T cell development We found that Spred-1 is highly expressed in hematopoietic cells (unpublished data). To examine the function of Spred-1 in Th1/Th2 cell differentiation and diseases, we generated mice lacking the Spred-1 gene by homologous recombination techniques (Fig. 1). To obtain the loss-of-function mutant, exons containing the kit-binding domain and Sprouty-related COOH-terminal cysteine-rich domains were deleted. Loss of Spred-1 protein expression was confirmed by Western blotting of the tissue extracts. Spred-1 protein was detected in the brain of the WT mice but not in that of the mutant mice. Offspring were born within the Mendelian expectation ratio from intercrosses of heterozygotes as well as incrosses of homozygotes. This indicates that Spred-1 is not necessary for fertility and development. Adult Spred-1–/– mice appeared to be healthy, except that a slight lower body weight, a shortened face, and a kinky tail, but they showed no apparent abnormalities in most organs (unpublished data). No abnormalities of peripheral hematopoietic cell population and number, CD4/CD8 profiles of spleen and thymus, or B cell development were observed in Spred-1–/– mice (unpublished data). Enhanced OVA-induced asthma phenotypes in Spred-1–/– mice Because the ERK pathway is shown to be involved in the Th1/Th2 balance (19–21), we investigated the role of Spred-1 in a Th2-type disease, bronchial asthma, using an OVA-induced asthma model. After systemic sensitization to OVA and aerosolized OVA challenges, airway responsiveness to acetylcholine aerosol was measured using an invasive technique (22). WT mice demonstrated AHR to acetylcholine, and Spred-1–/– mice exhibited significantly enhanced AHR compared with WT mice (Fig. 2, A and B). OVA sensitization and challenge resulted in an increased number of eosinophils in bronchoalveolar lavage (BAL) fluid in WT mice. Spred-1–/– mice after an OVA challenge displayed a further increase in eosinophils in BAL fluids compared with WT mice, although there was no significant difference in the number of lymphocytes and neutrophils (Fig. 2 C). There was no difference in OVA-induced asthma between C57BL/6 mice and WT (Spred-1+/+) littermates (unpublished data). Alcian blue/periodic acid-Schiff (PAS) staining was performed to examine the levels of mucus hyperproduction. After OVA challenge, mild staining in the airways of WT mice cells was noted, and staining levels were increased significantly in the intrapulmonary bronchi of Spred-1–/– mice (Fig. 2, D and E), indicating enhanced goblet cell metaplasia in Spred-1–/– lung. In the absence of sensitization and challenges, no substantial differences were apparent in ARH to acetylcholine, inflammatory cells in BAL fluids, and Alcian blue/PAS staining cells in the airways between Spred-1–/– and WT mice. Th1/Th2 responses in Spred-1–/– T cells Enhanced AHR in Spred-1–/– mice may be explained by a simple increased Th2 response, although IL-4 levels were not altered. Therefore, we measured serum IgE levels that are determined by Th1/Th2 balance. The concentrations of total IgE and OVA-specific IgE in Spred-1–/– mice were similar to those in WT controls (Fig. 4 A). There were no significant differences in serum IgA levels among the groups (Fig. 4 A). CD4+ T cells were isolated from draining lymph node cells from paratracheal and mediastinal lymph nodes after OVA challenges and stimulated in vitro with anti-TCR plus anti-CD28 mAbs. IL-4, IL-5, IL-13, and IFN- production from CD4+ T cells was similar between Spred-1–/– and WT mice (Fig. 4 B). These data suggest that Spred-1 does not affect Th1/Th2 differentiation and cytokine production from T cells. To confirm these conclusions, we assessed the development of Th1 and Th2 cells from naive CD4+ T cells in WT and Spred-1–/– mice. T cells were stimulated with anti-TCR plus anti-CD28 mAbs, and the population of IL-4– or IFN-–producing cells was analyzed by intracellular FACS staining. The ratio of IL-4– and IFN-–producing T cells was comparable between Spred-1–/– and WT mice. IL-4 and IFN- levels in the culture supernatant were also comparable (unpublished data). We also tested the generation of Th1 and Th2 cells under a Th1- or Th2-skewed condition. Naive CD4+ T cells were stimulated with anti-TCR mAb in the presence of IL-4 plus anti–IL-12 mAb or of IL-12 plus anti–IL-4 mAb. There were no significant differences in the generation of IL-4–dependent Th2 cells or IL-12–dependent Th1 cells between Spred-1–/– and WT T cells (Fig. 4 C). These data suggested that the development of asthma phenotypes is enhanced in Spred-1–/– mice through the up-regulation of a limited repertoire of Th2 cytokines, such as IL-13, and that this is not due to the enhancement of antigen-specific Th2 immune responses. In addition to T cells, eosinophils produce Th1 and Th2 cytokines, including IL-5 and IL-13 (23, 24). Next, we investigated IL-13 expression in accumulated eosinophils in the airways of OVA-challenged mice (Fig. 3 B). Eosinophils were isolated from BAL fluids and incubated in vitro for the evaluation of IL-13 production. Airway eosinophils isolated from Spred-1–/– mice secreted significantly higher levels of IL-13 than those from WT mice (Fig. 3 B). These findings indicate that the IL-13 production from eosinophils is augmented in Spred-1–/– mice. Increased eosinophilia in response to IL-5 but not to IL-13 in Spred-1–/– mice It has been shown that IL-5 is critical for the induction of eosinophilia (25–27), as is IL-13 for the development of eosinophilic inflammation and AHR (28, 29). Thus, to elucidate the mechanism of enhanced OVA-induced asthma phenotypes and preferential IL-13 production in Spred-1–/– mice, we analyzed the response of eosinophils to these cytokines. Intratracheal administration of recombinant mouse IL-13 increased eosinophil counts in BAL fluids in WT mice; however, the level of eosinophilia after IL-13 instillation in Spred-1–/– mice was comparable to that in WT mice (unpublished data). Intraperitoneal injection of recombinant mouse IL-5 into WT mice induced an increase in the eosinophil number in peripheral blood as reported previously (30). In Spred-1–/– mice, IL-5 injection induced a prominent and prolonged increase in peripheral eosinophil counts (Fig. 5 A). To confirm the hyperresponsiveness of Spred-1–/– eosinophil progenitors to IL-5 in vitro, IL-5–dependent colonies were counted from the bone marrow and spleen of Spred-1–/– and WT mice. Bone marrow and splenic cells were cultured with IL-5 as the only supportive cytokine to enumerate eosinophil precursors. In this medium, the number of colonies from Spred-1–deficient bone marrow and splenic cells was more than those from WT cells (Fig. 5 B). These data suggest that Spred-1–/– mice contain more eosinophil progenitors than WT mice or that the hematopoietic progenitors of Spred-1–/– mice are more sensitive to IL-5. Because the Ras–ERK pathway has been shown to regulate eosinophil chemotaxis (31), we performed the chemotaxis experiment with eosinophils derived from bone marrow of Spred-1–/– or WT mice. Chemotaxis of eosinophils from Spred-1–/– mice was accelerated in response to eotaxin compared with WT mice (Fig. 5 C). These findings indicate that Spred-1–/– mice exhibit the hyperresponsiveness of eosinophil to IL-5 and to eotaxin and enhanced/sustained eosinophilia. Spred-1 inhibits IL-5–mediated ERK activation and cell proliferation IL-5 activates the propagation of signals principally via the JAK–STAT pathways, especially STAT5, and the Ras-MAPK pathways (32). To elucidate the molecular mechanism by which the inactivation of Spred-1 enhances IL-5 responses, we analyzed the JAK-STAT and Ras–MAPK pathways using an IL-5–dependent cell line, Y16 (33). Although forced expression of WT or C-Spred-1 did not affect IL-5–induced JAK2 and STAT5 activation, WT Spred-1 reduced Raf-1 and ERK activation, and the dominant negative C mutant of Spred-1 augmented Raf-1 and ERK activation (Fig. 6 A). Other tyrosine kinases such as Lyn and JAK2 have been shown to enhance eosinophil differentiation (34). Therefore, we analyzed the interaction of Spred-1 with Raf-1, Lyn, and JAK2 kinases in Y16 cells transfected with WT Flag-Spred-1. The expression levels of Lyn in Y6 cells was very low, so we could not conclude the interaction between Lyn and Spred-1. Raf-1 was coimmunoprecipitated with Spred-1 as reported previously (17). However, JAK2 was not coimmunoprecipitated with Spred-1 (Fig. 6 B). These data further support our proposal that the effect of Spred-1 is specific to the downstream of JAK2 (i.e., the Ras–Raf–ERK pathway). Next, we examined the effect of WT and C-Spred-1 on IL-5–dependent proliferation of Y16 cells. WT and C-Spred-1 cDNAs were introduced into Y16 cells with enhanced GFP (EGFP) using a bicistronic retrovirus vector pMY-IRES-EGFP (35). Because the infected cells expressed both EGFP and Myc-tagged Spred-1, the percentage of infected cells was determined as the EGFP-positive rate by flow cytometry. When Y16 cells were cultured in a medium containing 5 U/ml IL-5 for 4 wk, the proportion of control IRES-EGFP–infected cells was unchanged; however, the population of WT Spred-1–infected cells decreased, whereas that of C-Spred-1–infected cells increased (Fig. 6 C). The MEK inhibitor, U0126, blocked IL-5–induced proliferation of C-Spred-1–transfected Y16 cells as efficiently as parental Y16 cells (Fig. 6 D). These results suggest that the inactivation of Spred-1 enhances IL-5 responses through augmenting Ras–MAPK activity. To further confirm the effect of Spred-1 on IL-5–dependent signals in more physiological conditions, BM–derived eosinophil (BMEo) cells were analyzed (Fig. 7). Activations of Raf-1 and ERK2 were augmented in Spred-1–/– BMEos compared with WT BMEos, but JAK2 activation was not affected (Fig. 7). These results further supported that Spred-1 inhibits the Ras–ERK pathway downstream of IL-5/JAK2 and that the hyperresponsiveness of Spred-1–/– eosinophils to IL-5 may be explained by enhanced ERK activation. Discussion We have recently identified Spreds and determined that Spred-1 interacts with Ras and inhibits growth factor–induced Raf kinase activation (17). The present paper demonstrated that Spred-1 inactivation by a dominant negative mutant enhances IL-5 signals via the Ras–MAPK pathway and IL-5–induced cell proliferation, whereas Spred-1 overexpression suppresses IL-5–induced Ras–MAPK activity. Spred-1–deficient mice exhibit augmented eosinophilia in response to IL-5. It has been suggested that the Ras–MAPK pathway might regulate Th cell development (19–21, 36). By using mouse models, we demonstrated that Spred-1 inactivation increases allergen-induced expression of IL-13 and magnifies asthma phenotypes without affecting Th2 differentiation including IL-4 or IgE levels. We further demonstrated that IL-13 production by airway eosinophils is up-regulated in Spred-1–deficient mice after allergen challenges. IL-5 (25–27) and IL-13 (28, 29) are critical for the development of asthma. Therefore, these data suggest that Spred-1 plays an important regulatory role in asthma by modulating the signaling of a limited repertoire of Th2 cytokines. IL-4 has been suggested to be important for the generation of allergen-specific Th2 cells during sensitization (37), and the roles of Ras–MAPK activation in IL-4 production and Th2 differentiation have been investigated. Although some reported no effect on IL-4 production in T cells when inhibiting ERK activity (37), others report a decrease (19, 38) or increase in IL-4 expression and subsequent Th2 differentiation (20, 21). In the present paper, we showed that IL-4 and IFN- production in vitro from Th cells in response to anti-TCR from Spred-1–deficient mice is normal. Furthermore, the levels of IL-4 in the airways and serum IgE after allergen challenge in vivo in Spred-1–deficient mice were comparable to those in WT mice. Thus, we consider that Spred-1 may not influence Th2 differentiation. The higher numbers of IL-5–dependent colonies from the bone marrow and spleen from Spred-1–/– mice than those from WT mice suggest that Spred-1–deficient mice contain an increased number of eosinophil progenitors. The mechanism for this is not clear at present. Our paper shows that Spred-1 inactivation by a dominant negative mutant enhances IL-5 signals via the Ras–MAPK pathway and IL-5–induced cell proliferation using the IL-5–dependent murine B cell line, Y16. Therefore, Spred-deficient eosinophil progenitor cells may respond more efficiently to IL-5. However, we could not rule out the possibility that Spred-1–deficient mice contain a higher number of stem cells, which respond better to the stem cell factor. Allergen-induced eosinophilia is probably due to a higher response of progenitors to IL-5. IL-5 activates several kinases, including Btk, JAK2, Lyn, and Raf-1, as well as the phosphatase SHP2 (32, 39, 40). In eosinophils, JAK2 and Lyn appear to be important for cell proliferation and survival, whereas Raf-1 seems to play a central role in regulating cell function, such as degranulation, adhesion, and survival (41). Because IL-5–induced eosinophilia is markedly enhanced and prolonged in Spred-1–deficient mice, Spred-1 might affect eosinophil proliferation and/or survival in response to IL-5. Our present data suggest that Spred-1 regulates IL-13 expression through the inactivation of Ras–MAPK signals. This hypothesis is supported by the previous findings that eosinophils have the potential to produce IL-13 (24, 42) and that the Ras–ERK pathway mediates induction of IL-13 expression in T cells (43) and mast cells (44). The downstream functional target molecules of Ras–MAPK in IL-13 induction in response to IL-5 have not been clarified. It has been reported that the IL-5 receptor expression on airway eosinophils is down-regulated after an inhaled allergen challenge and that this is associated with a loss of IL-5 responsiveness (45, 46). However, it is unlikely that Spred-1 modulates IL-5 receptor expression because a specific inhibitor of MAPK kinase (MEK) 1 had no effect on the down-regulation of IL-5 receptor -chain in eosinophils (47). We demonstrated that the IL-5–mediated proliferation of Y16 cells is inhibited by WT Spred-1 transfection, whereas it is increased by C-Spred-1 in the present paper. Furthermore, a MEK inhibitor, U0126, blocked IL-5–induced proliferation in Y16 cells, and the inhibitory effect of U0126 was similar between parental Y16 cells and C-Spred-1 transfectants. These data suggest that Spred-1 regulates IL-5–dependent proliferation by modulating the ERK pathway. It has already been demonstrated that pharmacological inhibitor of ERK phosphorylation attenuates allergen-induced airway reactions (48). Our findings and the previous paper suggest that the Ras–ERK pathway is critical in the development of eosinophilic inflammation and AHR. Given the variety of cellular distribution of Spred-1 expression including airway epithelial cells, endothelial cells (49), hematopoietic progenitor cells (unpublished data), and eosinophils in the present paper, it is unlikely that all of the asthma phenotypes in Spred-1–/– mice are due to Spred-1 deficiency on eosinophils. In addition, other Spred family members might also regulate eosinophilia and allergen-induced AHR. However, our data clearly demonstrate that Spred-1–/– mice showed prominent airway eosinophilia and intact Th1/Th2 differentiation, and Spred-1–/– eosinophils are more sensitive to IL-5–induced proliferation and Raf-1/ERK activation, as well as eotaxin-induced migration in vitro. Therefore, it is possible that hyperresponsive eosinophils to IL-5 and eotaxin in Spred-1–/– mice may predominantly contribute to the airway pathology in these mice. Our studies provide evidence that Spred-1 is critical for the IL-5 response and IL-13 production and for allergen-induced asthma without influencing Th2 development. Although the relationship between total IgE and asthma prevalence is well known (50), an atopic status is not an associated finding in severe asthma (51). There is considerable evidence to support a critical role for the involvement of Th2 cytokines and eosinophils both in atopic and in nonatopic asthma. In particular, IL-5 is critical for eosinophilia, and IL-13 has the potential to modulate airway inflammation and AHR independently of IgE in animal models. Therefore, we propose that Spred-1 may be a useful therapeutic target to compensate for atopic and nonatopic asthma. Materials and Methods Mice A genomic library from the 129/SV mouse strain (Stratagene) was screened with a cDNA probe of the mouse Spred-1, and several overlapping positive clones, including from second to fourth exons, were identified. The targeting vector was constructed by replacing the fourth exon with a pgk-neo cassette while preserving 5-Kb (left arm) and 3-Kb (right arm) flanks of homologous sequences (Fig. 1 A). The hsv-TK gene was inserted for negative selection. Homologous recombination in murine embryonic stem cells was performed as described previously (52) and was confirmed by Southern blot analysis. The chimeric mice were backcrossed to C57BL/6 three times. The resultant F3 mice were intercrossed to obtain the offspring for analysis. Genomic PCR was performed as described previously (53). The following primer sets were used: Spred-1 WT, 5'-GCAGACTACAGACATCCGGACATGTGG-3' and 5'-CGCATGGCCCCAATGATACCGGCAAG-3'; Spred-1–/–, 5'-CGAGATCAGCAGCCTCTGTTCCACATAC-3' and 5'-CCAAGAGAGCTGAGGATGAACTCACCG-3'. RT-PCR To detect Spred-1, total RNA was extracted from the bone marrow, spleen, and brain using TRIzol reagent (GIBCO BRL). RT-PCR was performed using a GeneAmp RNA PCR kit (PE Biosystems) according to the manufacturer's instructions. The specific primers for Spred-1 were 5'-GATGAGCGAGGAGACGGCGAC-3' and 5'-GTCTCTGAGTCTCTCTCCACGGA-3'. Sensitization and challenge 10–12-wk-old C57BL/6 mice, Spred-1–/– mice, or their WT littermates were sensitized with intraperitoneal injections of 20 μg OVA (Grade V; Sigma-Aldrich) plus 2.25 mg aluminum hydroxide (Pierce Chemical Co.) on days 1 and 14. On days 26–28, mice received aerosol challenge containing either saline or 1% OVA for 20 min/d. Measurement of airway responsiveness On day 30, 36 h after the last aerosol challenge, mice were ventilated to measure AHR to acetylcholine aerosol as described previously (22, 54). Airway opening pressure was measured with a differential pressure transducer and continuously recorded. Stepwise increases in the acetylcholine dose were given with an ultrasonic nebulizer. All animal experiments were approved by the Committee on Animal Research, Faculty of Medicine, Kyushu University. The data were expressed as the provocative concentration 200 (PC200), the concentration at which airway pressure was 200% of its baseline value, and PC200 was calculated by log-linear interpolation for individual mice. Lower log PC200 values represent greater AHR. The serum levels of total and OVA-specific IgE were analyzed by ELISA with rat anti–mouse IgE (Serotec Ltd.). BAL and cytokine measurements Mice were exsanguinated with a lethal dose of pentobarbital, and their lungs were gently lavaged with 1 ml of 0.9% saline via a tracheal cannula. Total and differential BAL cell counts were performed as described previously (22). Samples were centrifuged at 2,000 revolutions/min for 10 min, and the supernatants were stored at –80°C. Mouse IL-4, IL-5, IL-13, and IFN- were quantified using ELISA kits (Biosource International) according to the manufacturer's protocols. Measurement of cytokine production in BAL fluid was assessed by ELISA assays. Histological assessment Lungs were fixed with 10% formalin, and tissue sections were stained with Alcian blue/PAS to determine the presence of mycin glycoconjugates (22). The numerical scores for the abundance of PAS-positive mucus-containing cells in each airway were determined as follows: 0, 75% (36). Collection of lung cells and lymph node cells Enzymatic digestion of the lungs was performed with collagenase type 1A, hyaluronidase, and DNase, and the samples were filtered through a 52-μm nylon mesh. The erythrocytes were removed by lysis. Cells were stained with FITC-labeled anti-NK1.1 mAb, PE-labeled anti-CD19 mAb, PerCP-labeled anti-CD3 mAb, and allophycocyanin-labeled anti-CD4 mAb. After flow cytometric analysis using a FACSCalibur with CellQuest software (BD Biosciences), the absolute numbers of lung immune cells were calculated. Draining lymph node cells were collected from paratracheal and mediastinal lymph nodes, and CD4+ T cells isolated with a magnetic cell sorter (Miltenyi Biotec) were stimulated with a combination of 30 μg/ml anti-TCR mAb (H57-597) and 1 μg/ml anti-CD28 mAb (PV-1) for 48 h. ELISA assays for cytokine production were performed as described previously (54). Cell purification and induction of helper T cells Naive CD4+ T cells isolated from spleens were stimulated with a combination of anti-TCR mAb and anti-CD28 mAb, and 10 U/ml rIL-12 and anti–IL-4 mAb (11B11) were added for Th1 development, while 100 U/ml rIL-4 and anti–IL-12 mAb (C15.6 and C17.8) were added for Th2 development. ELISA assays for cytokine production and intracellular cytokine staining were performed as described previously (54). In vitro colony assay Single cell suspensions were isolated from the bone marrow or spleen of WT and Spred-1–/– mice. The erythrocytes were removed by lysis using NH4Cl and 3 x 103 cells were plated in methylcellulose (Methocult3434; StemCell Technologies Inc.) containing 10 ng/ml of IL-5. On day 14, the numbers of colonies were counted microscopically. Isolation of lung eosinophils BAL was performed in OVA-sensitized and -challenged mice by three repeated lavages with RPMI 1640 containing penicillin-streptomycin at 25 cm H2O. The lavage fluid was collected and incubated for 30 min to remove macrophages from the cell suspension by adherence to plastic. Eosinophils were isolated by a negative selection strategy, removing B cells and T cells with Ab-conjugated magnetic beads (MACS; Miltenyi Biotec) specific for CD45-R (B220) and CD90 (Thy1.2), respectively. The purity of the recovered eosinophils was confirmed to be >98% by staining cytospin preparations with Diff-Quik (neutrophils comprise the contaminating cell populations). Eosinophils were cultured at 106 cells/ml for the indicated times. IL-5 and IL-13 treatment in vivo Recombinant murine IL-5 (10,000 U/d) was injected intraperitoneally into WT- or Spred-1–deficient mice for 4 d (30). Differential counts were performed by examination of blood smears stained with a modified Wright-Giemsa stain at various time points. IL-13 administration was performed as described previously (22). A recombinant murine IL-13 solution (0.5 g) or a vehicle solution was instilled intratracheally on days 1, 3, and 5. BAL was performed on day 6, 24 h after the last instillation, and BAL cell differentials were determined. Cell lines and cultures IL-5–dependent murine cell line, Y16 was cultured in an RPMI 1640 medium containing 5% FBS, 50 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 5 U/ml IL-5 (55). The retrovirus-packaging cell line PLAT-E was maintained in DMEM containing 10% FBS. Bone marrow cells from the femoral bone of Spred-1–/– mice or their WT littermates were cultured in RPMI 1640 supplemented with 100 U/ml murine IL-5, 30% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-mercaptoethanol for 2 wk to generate populations of BMEos that were >95% pure. Chemotaxis assay The chemotaxis assay was performed in a 96-well disposal chemotaxis plate (5-μm pore size; Neuro Probe). In brief, eotaxin was diluted in RPMI 1640 with 0.1% BSA and placed in the bottom wells (27 μl), 25 μl of cell suspension at 4 x 106 cells/ml was placed on the top well of the chamber, which was separated from the bottom well by a polycarbonate filter. The plate was incubated for 60 min at 37°C in a humidified incubator with 5% CO2. The cells remaining on top of the filters were absorbed off, the filter tops were carefully washed, and the plates were centrifuged to pellet all cells on the undersides of the filters. The filters were removed, and cells in the bottom wells were counted by light microscopy. Data are reported as a migration index, calculated as follows: (number of cells migrating to chemoattractant)/(number of cells migrating to vehicle). Cell proliferation assay Cell proliferation was assayed using a Cell Counting Kit (Dojindo). In brief, 103 Y16 cells were plated in 96-well plates in RPMI 1640 medium with IL-5 and cultured for 48 h. After adding WST-8 and 1-methoxy PMS, optical density for 450 nm was measured. Plasmids, transfection, and infection WT Spred-1 and COOH-terminal–truncated Spred-1 (C-Spred-1) were subcloned into pMY-IRES-GFP vectors for retrovirus infection as described previously (56). In brief, pMY-IRES-GFP vectors were transfected into a PLAT-E packaging cell line using the transfection reagent FuGENE 6 (Boehringer) to obtain the viruses (35). Y16 cells (2 x 105 cells) were infected with viruses on a RetroNectin (TaKaRa)-coated plate for 24 h in the presence of 5 U/ml IL-5. Cells were washed three times with PBS, resuspended in RPMI 1640, 5% FBS containing 5 U/ml IL-5, and incubated for the indicated times. 104 cells were analyzed for GFP fluorescence on a flow cytometer. Immunochemical analysis Immunoprecipitation and immunoblotting were performed using anti-STAT5, anti-ERK2 (Santa Cruz Biotechnology, Inc.), anti-Flag (M2; Sigma-Aldrich), anti–phospho-TAT5, anti–phospho-ERK2 (Cell Signaling), and anti–phospho-Ser338-Raf-1 (Upstate Biotechnology) antibodies as described previously (56, 57). Anti–Spred-1 antibody was prepared by immunizing rabbits (17). Statistical analysis Values were expressed as the mean ± SEM. Differences among groups were analyzed using unpaired Student's t tests or an analysis of variance together with a post-hoc Bonferroni analysis. Nonparametric data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test. p-values <0.05 were considered to be significant. Acknowledgments We thank Ms. Y. Kawabata and Y. Yoshiura for technical assistance, Dr. Y. Kikuchi-Ooe (Institute of Medical Science, University of Tokyo) for technical advice, and Ms. Y. Nishi for text preparation. This work was supported by Special Grants-in-Aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan; the Japan Health Science Foundation; the Haraguchi Memorial Foundation; the Mochida Memorial Foundation; the Kato Memorial Foundation; and the Uehara Memorial Foundation. The authors have no conflicting financial interests. Submitted: 29 March 2004 Accepted: 22 November 2004 References Seki, Y., H. Inoue, N. Nagata, K. Hayashi, S. Fukuyama, K. Matsumoto, O. Komine, S. Hamano, K. Himeno, K. Inagaki-Ohara, et al. 2003. SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses. Nat. Med. 9:1047–1054.、 Takaki, S., A. Tominaga, Y. Hitoshi, S. Mita, E. Sonoda, N. Yamaguchi, and K. Takatsu. 1990. Molecular cloning and expression of the murine interleukin-5 receptor. EMBO J. 9:4367–4374. Sasaki, A., T. Taketomi, T. Wakioka, R. Kato, and A. Yoshimura. 2001. Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor but not epidermal growth factor-induced ERK activation. J. Biol. Chem. 276:36804–36808. Yasukawa, H., H. Misawa, H. Sakamoto, M. Masuhara, A. Sasaki, T. Wakioka, S. Ohtsuka, T. Imaizumi, T. Matsuda, J.N. Ihle, and A. Yoshimura. 1999. The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J. 18:1309–1320. Related Article...查看详细 (34179字节)

☉ 11120209:Role of ?2-integrins for homing and neovasculariza
The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo–expanded EPCs as well as murine hematopoietic Sca-1+/Lin– progenitor cells express ?2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin– hematopoietic progenitor cells from ?2-integrin–deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the ?2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of ?2-integrins in postnatal vasculogenesis. T . Chavakis' present address is Experimental Immunology Branch, National Cancer Institute/National Institutes of Health, Bethesda, MD 20892. Abbreviations used: ?2–/–, ?2-integrin–deficient; EPC, endothelial progenitor cell; HUVEC, human umbilical vein endothelial cell; MNC, mononuclear cell; vWF, von Willebrand factor. The term vasculogenesis was originally introduced to describe the de novo formation of new vessels from angioblasts during embryonic development (1). Accumulating evidence suggests that vasculogenesis, mediated by circulating bone marrow–derived endothelial progenitor or hematopoietic stem cells, plays an important role in postnatal neovascularization of adult ischemic tissues (2–7). Human endothelial progenitor cells (EPCs) were initially characterized by the expression of the VEGF receptor 2 (VEGF R2; Flk-1) and a hematopoietic marker such as CD133 (6). EPCs are mobilized from the bone marrow during ischemia (8, 9) or exogenously by stimulation with cytokines such as VEGF and contribute to neovascularization of ischemic tissues (4, 8, 10) or tumors (11). Infusion of EPCs or isolated hematopoietic progenitor cells (e.g., murine Sca-1+/Lin– cells) augmented neovascularization of ischemic myocardium and limbs and improved left ventricular function after myocardial ischemia (12–15). EPCs are preferentially recruited to sites of ischemia and incorporated into vascular structures (2, 4, 8, 12, 16). The mechanisms of EPC homing to sites of ischemia are still unclear. Because integrins are mediating the homing of transplanted hematopoietic stem cells to the bone marrow (17) as well as the recruitment of inflammatory cells to sites of inflammation, we investigated the contribution of integrins and especially of ?2-integrins for homing and neovascularization capacity of EPCs and hematopoietic stem cells to areas of ischemia. Recruitment of inflammatory cells requires a coordinated sequence of multistep adhesive and signaling events, including selectin-mediated rolling, leukocyte activation by chemokines, integrin-mediated firm adhesion and diapedesis (18–22). During firm adhesion of leukocytes to the endothelium, members of the ?2-integrin family, LFA-1 (L?2, CD11a/CD18), Mac-1 (M?2, CD11b/CD18), and p150,95 (X?2, CD11c/CD18), as well as ?1-integrins on leukocytes interact with endothelial counterligands such as ICAM-1, VCAM-1, and surface-associated fibrinogen. Mac-1 also regulates leukocyte adhesion to provisional matrix substrates including fibrinogen, which is deposited at sites of inflammation and injury upon increased vascular permeability and damage (19, 20, 23). Because ?2-integrins are strongly expressed on EPCs, we studied the role of the ?2-integrins for homing and neovascularization capacity of peripheral blood–derived cultivated human EPCs, bone marrow–derived murine hematopoietic Sca-1+/Lin– as well as VEGF R2+/Lin– progenitor cells. Our results show that ?2-integrins mediate the adhesive interactions of EPCs to mature endothelial cells and to extracellular matrix proteins and are critical for chemokine-induced transendothelial migration of EPCs in vitro. In a mouse model of hind limb ischemia, using murine Sca-1+/Lin– hematopoietic progenitor cells from ?2-integrin–deficient (?2–/–) mice, we demonstrate that ?2-integrins are involved in the homing of hematopoietic progenitor cells to sites of ischemia and are critical for their neovascularization capacity. Alternately, preactivation of the ?2-integrins on EPCs by activating antibodies significantly augments the in vivo neovascularization capacity of EPCs, indicating a new therapeutic approach to promote homing of EPCs. Results EPCs express active ?2-integrins To characterize the expression of adhesion receptors on EPCs, we used a microarray assay comparing EPCs and human umbilical vein endothelial cells (HUVECs). The endothelial phenotype of the ex vivo–cultivated EPCs was confirmed by immunostaining, FACS analysis, and functional response to shear stress as described previously (12, 24, 25). Strikingly, EPCs expressed mRNA for the ?2-integrin subunit and for the corresponding CD11a, CD11b, and CD11c subunits, whereas mature endothelial cells showed only a very low mRNA expression of the ?2-integrins (Fig. 1 A). FACS analysis confirmed the surface expression of the ?2-integrin (CD18) and the CD11a, CD11b, and CD11c subunits (Fig. 1 B). Coexpression of the endothelial markers von Willebrand factor (vWF) and CD31 on ?2-integrin positive EPCs was demonstrated by FACS analysis (Fig. 1 B and not depicted). mRNA expression of eNOS and VE-cadherin confirmed the endothelial phenotype of the EPCs (Fig. 1 C and not depicted). In contrast with EPCs, mature endothelial cells (HUVECs) do not express the ?2-integrin subunit (CD18) on the cell surface (unpublished data). Because ?2-integrins undergo a conformational activation in the presence of divalent cations, such as Mn2+ (26), we investigated whether Mn2+ is capable of activating ?2-integrins on the surface of EPCs. For this purpose, we detected activated ?2-integrins by using specific antibodies, which recognize activation-dependent epitopes of the CD11a (mAb24) and CD11b (CBRM1/5) subunits (27, 28). Although the stimulation of EPCs with Mn2+ had no effect on the total protein expression of CD11a or CD11b subunits, it enhanced the expression of the activation-dependent epitopes on the CD11a and CD11b subunits (Fig. 2 A). These results demonstrate that EPCs express functionally active ?2-integrins on their surface. ?2-integrins mediate adhesion of EPCs on endothelial cell monolayers and extracellular matrix proteins To assess the functional capacity of ?2-integrins expressed by EPCs, we investigated their role in the adhesion of EPCs on mature endothelial cell monolayers (HUVECs) and on recombinant human ICAM-1 and fibrinogen in a static adhesion assay. Stimulation of EPCs with Mn2+ significantly increased the adhesion of EPCs to TNF-–prestimulated HUVECs. Addition of an inhibitory ?2-integrin antibody significantly blocked EPC adhesion to HUVECs (Fig. 2 B). VLA–4 as well as V?3-integrin can also mediate intercellular adhesive interactions by binding to VCAM-1 and PECAM-1, respectively (23, 29). Therefore, a synthetic VLA-4 inhibitor and a cyclic RGD peptide (established inhibitor of the V?3- and V?5-integrin) were engaged in these studies. However, these inhibitors did not significantly inhibit EPC adhesion to HUVECs (Fig. 2 B). An inhibitory VLA-4 antibody (clone HP2/1), as well as an inhibitory ?1-integrin antibody (clone 6S6), also had no effect on the adhesion of EPCs to HUVECs (unpublished data). These results demonstrate that EPC adhesion to endothelial cells is predominantly mediated by ?2-integrins expressed on EPCs. Endothelial ICAM-1 and extracellular matrix-associated fibrinogen are established ligands for the ?2-integrins (30–33). Therefore, we investigated whether EPCs are capable of binding to immobilized recombinant human ICAM-1 and fibrinogen via ?2-integrins. Indeed, stimulation with either Mn2+ or an activating ?2-integrin antibody (KIM185) induced adhesion of EPCs to immobilized human ICAM-1 and fibrinogen (Fig. 2, C and D). Adhesion induced by both stimuli was completely abolished in the presence of an inhibitory ?2-integrin antibody (Fig. 2, C and D). In contrast, an inhibitory ?1-integrin antibody had no effect on the adhesion of EPCs to human ICAM-1 (unpublished data). These results demonstrate that EPCs bind to fibrinogen and endothelial ICAM-1 in a ?2-integrin–dependent manner. Role of ?2-integrins for transmigration of EPCs We investigated the involvement of ?2-integrins in the transendothelial migration of EPCs in a transwell transmigration assay. Chemoattraction of EPCs by MCP-1, SDF-1, and VEGF significantly increased the transmigration rate of EPCs through HUVEC monolayers (Fig. 3). Addition of an inhibitory ?2-integrin antibody (anti-CD18) significantly reduced EPC transmigration, whereas an inhibitory ?1-integrin antibody (anti-CD29) and RGD peptides had no effect (Fig. 3). Moreover, an inhibitory VLA-4 antibody did not affect chemokine-induced transendothelial migration of EPCs (unpublished data). Thus, ?2-integrins, but not ?1-integrins, mediate chemokine and VEGF-induced transendothelial migration of EPCs (Fig. 3). The functional activity of the stem/progenitor cells to improve neovascularization was assessed by intravenous infusion of Sca-1+/Lin– bone marrow cells derived from either wild-type or ?2–/– mice into athymic mice 24 h after induction of limb ischemia. After 14 d, transplantation of Sca-1+/Lin– bone marrow cells from wild-type mice significantly enhanced the recovery of blood flow of the ischemic hind limbs of athymic mice as compared with ischemic hind limbs from untreated athymic mice (Fig. 5 B). In contrast, ?2–/– Sca-1+/Lin– bone marrow cells were significantly less effective for improving recovery of limb perfusion as compared with wild-type Sca-1+/Lin– bone marrow cells (Fig. 5 B). Moreover, histological evaluation of ischemic hind limbs of athymic mice 14 d after cell infusion revealed a significantly lower capillary density in mice receiving ?2–/– Sca-1+/Lin– bone marrow cells compared with mice receiving wild-type cells (Fig. 5 C). Furthermore, the number of incorporated male Sca-1+/Lin– cells in female recipients was determined by fluorescence in situ hybridization for the murine Y-chromosome of the infused male cells. The number of incorporated Y-chromosome positive cells was significantly lower for the infusion of ?2–/– cells as compared with the infusion of wild-type cells (Fig. 5, D and E). Similar results were obtained when using isolated VEGF R2+/Lin– bone marrow–derived cells. The majority of VEGF R2+/Lin– cells expressed CD18 (89 ± 9.6%). Moreover, VEGF R2+/Lin– cells derived from CD18–/– mice cells showed a significantly reduced capacity to augment blood flow after ischemia as compared with WT cells (WT 180 ± 15% of untreated control mice; CD18–/– cells: 125 ± 7% of untreated control mice). These results indicate that the ?2-integrins are involved in the homing of progenitor cells to ischemic tissues and their neovascularization capacity. Activation of the ?2-integrins improves in vivo homing and neovascularization capacity of EPCs Because ?2-integrins are involved in the homing of EPCs, we investigated whether preactivation of ?2-integrins may improve homing and neovascularization capacity of human EPCs in the mouse model of hind limb ischemia. Ex vivo–expanded human EPCs isolated from peripheral blood were pretreated with the ?2-integrin–activating antibody (KIM 185), which was shown before to enhance ?2-integrin–dependent adhesion of EPCs to endothelial ICAM-1 or fibrinogen (Fig. 2), and were subsequently infused into athymic mice. To be able to detect an increase in progenitor cell–mediated neovascularization, we used a reduced number of EPCs (105 cells), which is lower than previously published numbers (5 x 105 EPCs; reference 25), to yield a 50% improvement of neovascularization as compared with untreated mice. Preincubation of the EPCs with the activating ?2-integrin antibody resulted in a significantly enhanced neovascularization capacity of infused EPCs in comparison with control antibody-treated EPCs as assessed by laser Doppler imaging (Fig. 6 A). Incorporation of human EPCs was detected by confocal microscopy using antibodies directed against human HLA and the endothelial marker protein vWF (Fig. 6, B–D). Incorporation of ?2-activating antibody-treated EPCs into the ischemic muscle was increased in comparison with control antibody-treated EPCs (Fig. 6, B and C). Moreover, the numbers of capillaries and small arterioles (20–50 μm) were significantly augmented in mice treated with preactivated EPCs (Fig. 6, E and F; capillary density: EPC + control mAb: 0.77 ± 0.10 capillaries per myocyte; EPC + activating mAb: 1.32 ± 0.09; P = 0.003). Thus, an external activation of the ?2-integrins by an activating antibody before infusion is capable of improving the neovascularization capacity of EPCs. Discussion The data of the present paper underscore the importance of ?2-integrins for the proangiogenic activity of EPCs and bone marrow–derived progenitor cells. Specifically, our investigations revealed the following: (a) EPCs as well as hematopoietic stem/progenitor cells express ?2-integrins; (b) ?2-integrins expressed on EPCs can be activated by Mn2+ and can mediate the adhesion of EPCs to mature endothelial cells, to recombinant human ICAM-1, and to fibrinogen and the chemokine-induced transendothelial migration of EPCs; (c) ?2–/– animals display a neovascularization defect in the model of hind limb ischemia; (d) ?2-integrins are involved in the in vivo homing of progenitor cells to sites of ischemia and their vascular integration and significantly contributed to the neovascularization capacity of infused bone marrow Sca-1+/Lin– or VEGF R2+/Lin– progenitor cells in the mouse model of hind limb ischemia as demonstrated using the ?2–/– progenitor cell populations; (e) stimulation of ex vivo–expanded human EPCs by preincubation with an activating ?2-integrin antibody significantly enhanced the homing of EPCs to sites of ischemia and EPC-induced neovascularization. Therefore, the present paper unravels a novel function of the ?2-integrin subunit CD18 for neovascularization exceeding its well-known function in innate and adaptive immune responses. Increasing evidence suggests that ?2-integrins are not only expressed on differentiated leukocytes but also on hematopoietic stem/progenitor cells (34, 35). Our in vitro data suggest that ?2-integrins expressed on EPCs mediate homing functions such as endothelial adhesion and transmigration. Moreover, ?2-integrins contribute to the in vivo homing of bone marrow–derived progenitor cells to ischemic tissue. In line with these data, it has been reported previously that ?2-integrins mediate adhesion and transmigration of hematopoietic stem/progenitor cells (36–38). In a recent paper assessing in vivo homing of embryonic EPCs derived from cord blood, the circulating cells arrested within tumor microvessels extravasated into the interstitium and incorporated into neovessels, suggesting that adhesion and transmigration are involved in the recruitment of EPCs to sites of tumor angiogenesis (39). Thus, it is conceivable to speculate that ex vivo–expanded adult EPCs and hematopoietic stem/progenitor cells may engage similar pathways for recruitment to sites of ischemia and incorporation into newly forming vessels. Our in vivo data provide the first evidence for a direct participation of ?2-integrins in neovascularization processes and particularly in stem/progenitor cell–mediated, ischemia-induced vasculogenesis. Adamis and colleagues previously highlighted the role of ?2-integrins for corneal and choroidal angiogenesis induced by injury (40, 41). In both studies, ?2–/– mice displayed a reduced inflammation-associated angiogenic response after injury and these effects were associated with reduced inflammatory cell infiltrates (40, 41). Yet, no incorporation of leukocytes into new vessels was reported in these studies. Moreover, the same group demonstrated that, in the case of retinal ischemia, leukocyte–endothelial cell interactions contribute to the development of ischemia by inducing vascular obliteration via Fas ligand–mediated endothelial cell apoptosis (42). In contrast with these findings, our data provide evidence that, during hind limb ischemia, intravenous infusion of bone marrow hematopoietic progenitor cells leads to incorporation of the transplanted cells in newly formed vessels and to improvement of neovascularization in an at least partially ?2-integrin–dependent manner. As opposed to EPCs, infusion of inflammatory cells, such as monocytes/macrophages, had only a slight if any effect on the neovascularization of ischemic limbs in the model of hind limb ischemia in athymic mice (25). Thus, our results support a novel direct function of ?2-integrins in progenitor cell–induced vasculogenesis during ischemia, which is distinct from the indirect role of ?2-integrins in the inflammation-associated angiogenesis described by Adamis and colleagues (40, 41). Because the ?2–/– mice display no defect in the mobilization of progenitor cells (43), the neovascularization defect in the ?2–/– mice in the model of hind limb ischemia is most conceivably mediated by a homing defect of progenitor cells into ischemic tissue. Interestingly, our present data indicate that the recruitment of hematopoietic progenitor cells to sites of ischemia is mediated at least in part by different mechanisms compared with the homing of infused cells into the bone marrow of lethally irradiated recipient mice, which is predominantly mediated via 4?1 (17, 43). In this context, ?2-integrins only act in a synergistic manner together with the 4?1-integrin. Our finding that ?2-integrin deficiency does not completely block homing and neovascularization improvement after infusion of Sca-1+/Lin– bone marrow cells suggests that other mechanisms may additionally be involved in these processes. We cannot exclude that 4?1-integrin partially compensates for the lack of ?2-integrin during in vivo homing of Sca-1+/Lin– bone marrow cells. Interestingly, the homing of inflammatory cells during pneumonia or myocardial ischemia in ?2–/– mice is mediated by the 4?1-integrin (44, 45). Moreover, the initial cell arrest of embryonic progenitor cell homing during tumor angiogenesis was suggested to be mediated by E- and P-selectin and P-selectin glycoprotein ligand-1 (39). Yet, it is important to underscore that this work was performed with embryonic EPCs, whereas we used adult EPCs and bone marrow stem/progenitor cells. It is likely that different cell types may use distinct mechanisms for homing to sites of ischemia. In addition, it is well established that interactions of selectins with selectin–ligands mediate the rolling of cells on the surface of endothelial cells as the initial step of homing (21). Further studies are needed to elucidate a potentially synergistic role of other adhesion molecules and their counterligands for the multistep recruitment process of adult endothelial progenitor and stem cells to ischemic tissue. Regardless of potentially additive mechanisms involved in the recruitment of stem/progenitor cells to areas of ischemia, our data clearly demonstrate that preincubation of EPCs with a ?2-integrin–activating antibody markedly enhanced the incorporation of transplanted EPCs in vessels and the neovascularization of ischemic limbs. The peripheral blood–derived EPCs used in the present paper are already used in clinical trials to improve neovascularization in patients with ischemic heart diseases (15). Thus, our results could have important clinical implications as they disclose a mechanism to enhance homing of EPCs and, thereby, improve neovascularization capacity of infused EPCs. In summary, the present paper demonstrates for the first time a critical role of ?2-integrins in vitro and in vivo for homing and neovascularization capacity of endothelial progenitor and hematopoietic progenitor cells. Moreover, our results show that activation of ?2-integrins appears to be a feasible and promising tool to improve the efficacy of EPC-induced neovascularization. A better understanding of the homing mechanisms of EPCs may lead to the development of new therapeutic strategies for improvement of vasculogenesis in patients with ischemic diseases. Materials and Methods Cell culture Mononuclear cells (MNCs) were isolated by density–gradient centrifugation with Ficoll from peripheral blood of healthy human volunteers as described previously (46). Immediately after isolation, total MNCs (8 x 106 cells/ml medium; cell density 2.5 x 106 cells/cm2) were plated on culture dishes coated with 10 μg/ml human fibronectin (Sigma-Aldrich) and maintained in endothelial basal medium (Cambrex) supplemented with 1 μg/ml hydrocortisone, 12 μg/ml bovine brain extract, 50 μg/ml gentamycin, 50 ng/ml amphotericin B, 10 ng/ml epidermal growth factor, and 20% FCS. After 3 d, nonadherent cells were removed, and adherent cells were incubated in medium for another 24 h before initiation of the experiments. EPCs were characterized by dual staining for 1,1'–dioctadecyl–3,3',3'–tetramethylindo-carbocyanine–labeled acetyl low-density lipoprotein, lectin, and expression of endothelial markers KDR, VE-cadherin, and vWF (25). Sca-1+/Lin– cells were purified from BM MNCs from wild-type and CD18–/– mice by negative selection using a cocktail of biotinylated antibodies to lineage markers (Lineage cell depletion kit, mouse; Miltenyi Biotec) for 10 min at 4°C followed by antibiotin microbeads for 15 min (Miltenyi Biotec). The Lin– BM cells were incubated with anti–Sca-1 microbeads (Miltenyi Biotec) for 15 min and Sca-1+/Lin– BM cells were collected (7). To obtain VEGFR2+ Lin– cells, Lin– cells were incubated with biotinylated Flk-1 antibodies (DSB-X biotin protein labeling kit; Molecular Probes; antibody was obtained from BD Biosciences) for 30 min at 4°C followed by antibiotin microbeads for 15 min. HUVECs were purchased from Cambrex and cultured in endothelial basal medium supplemented with 1 μg/ml hydrocortisone, 12 μg/ml bovine brain extract, 50 μg/ml gentamycin, 50 ng/ml amphotericin-B, 10 ng/ml epidermal growth factor, and 10% FCS until the third passage. After detachment with trypsin, cells (4 x 105 cells) were grown in 6-cm cell culture dishes or 96-well plates for at least 18 h as described previously (47). Oligonucleotide microarrays, FACS 10 μg of total RNA was hybridized to the HG-U95Av2 microarray (9670 human genes; Affymetrix, Inc.). The standard protocol used for sample preparation and microarray processing is available from Affymetrix, Inc. Expression data were analyzed using Microarray Suite version 5.0 (Affymetrix, Inc.) and GeneSpring version 4.2 (Silicon Genetics). 3 x 105 human EPCs, peripheral blood, or isolated Sca-1+/Lin– cells were incubated for 30 min at 4°C with FITC- or PE-labeled antibodies (anti-CD11a, -CD11b, -CD11c, -CD18, –Sca-1, and –c-kit; BD Biosciences; anti-vWF was obtained from Acris) or CBRM1/5-antibody (BD Biosciences) for 30 min at 37°C. The mAb24 antibody (provided by N. Hogg, Cancer Research UK London Research Institute, London, England, UK) was incubated for 30 min at 4°C and detected with a secondary FITC-labeled goat anti–mouse antibody (DakoCytomation). Surface expression was quantified using a FACS Calibur (BD Biosciences). Adhesion, transmigration experiments Cell–cell adhesion Cell–cell adhesion was performed as described previously (48, 49). Confluent HUVEC monolayers were stimulated with TNF- (Sigma-Aldrich) for 24 h. Ex vivo–expanded EPCs were stained with Cell Tracker green-CMFDA (Molecular Probes) and were resolved in adhesion buffer (150 mM NaCl, 20 mM Hepes, 2 mM MgCl2, 0.05% BSA, pH 7.4). A total of 105 EPCs/well (in 100 μl adhesion buffer) was added to the HUVEC monolayers in the absence or presence of blocking monoclonal ?2-integrin antibodies (clone IB4; Qbiogene; or mAb 60.3; J. Harlan, University of Washington, Seattle, WA), murine isotype control antibodies (Qbiogene), inhibitory agents cyclic RGD peptide or VLA-4 inhibitor (4-[{2-methyl-phenyl}aminocarbonyl]aminophenyl)acetyl-fibronectin-CS-1 fragment (1980–1983). After 20 min of incubation (37°C), the plates were washed twice with adhesion buffer at room temperature to remove nonadherent cells. Adherent cell tracker green-labeled EPCs were quantified in triplicates on a fluorescence plate reader (Fluostat; BMG Lab Technologies). Cell–matrix adhesion Cell–matrix adhesion was performed as described previously (48, 49). 96-well plates were coated overnight (4°C) with 10 μg/ml human fibrinogen (Enzyme Research Laboratories) or soluble recombinant human ICAM-1 (Bender MedSystems) and blocked with 1% (wt/vol) BSA for 1 h at room temperature. Ex vivo–expanded human EPCs in adhesion buffer were seeded at 1.2 x 105 cells/well in 100 μl in the absence or presence of 2 mM MnCl2 or activating human ?2-integrin antibody (clone KIM185; M. Robinson, Celltech Ltd., Slough, England, UK) and were incubated with blocking ?2-integrin mAb (clone IB4 or mAb 60.3) or murine isotype control antibodies (Qbiogene) for 20 min at 37°C. After removal of nonadherent cells by two washing steps, adhesion was quantified in triplicates by counting adherent cells in five randomly selected fields per well (magnification, 20; Axiovert 100; Carl Zeiss MicroImaging, Inc.). Transmigration Transendothelial migration was performed as described previously (50) using 6.5-mm transwell filters with 8-μm pore size (Costar). After inserts were coated with 0.2% gelatin (Sigma-Aldrich), HUVECs were seeded on transwell filters and cultivated for 48 h before the experiments were performed in a humidified atmosphere (37°C, 5% CO2). At the beginning of the experiment, 600 μl of migration assay medium (serum-free RPMI 1640 in the absence or presence of MCP-1, SDF1, or VEGF; R&D Systems) was added to the lower compartment of the transwell system. EPCs (5 x 105 in 100 μL) were added to the top compartment in the presence or absence of 30 μg/ml of blocking anti–?2-integrin antibody (mAb 60.3), anti–?1-integrin antibody (clone 6S6; Chemicon), anti–4-integrin antibody (clone HP2.1; Immunotech), murine isotype control antibodies (Qbiogene), or RGD peptides. After 18 h at 37°C, the number of cells transmigrated to the bottom compartment was quantified in duplicates with a cell counter (CASY-Counter; Sch?rfe-System). All inserts were fixed and stained to confirm the confluence of the endothelial monolayer. Animal experiments Mice. 8-wk-old ?2–/– mice and their age-matched wild-type littermates (either 129/Sv or C57BL/6J) were generated as described previously (51). All mice were genotyped by Southern blot analysis (51) and maintained under pathogen-free conditions. Athymic NMRI nude mice (6–8 wk) were obtained from The Jackson Laboratory. The animal experiments were approved from the Regional Board of Land Hessen, Germany. Model of hind limb ischemia The proximal femoral artery, including the superficial and the deep branch as well as the distal saphenous artery, were ligated. In transplantation experiments, progenitor cells were intravenously injected in nude mice 24 h after induction of limb ischemia. Human EPCs were pretreated with 20 μg/ml activating ?2-integrin antibody (clone KIM 185) or isotype control antibody for 30 min at 37°C and washed twice to remove unbound antibodies before injection (105 EPCs/mouse). In some experiments, sex-mismatched murine Sca-1+/Lin– or Flk-1+/Lin– bone marrow cells from male ?2–/– or wild-type mice were used. After 2 wk, we calculated the ischemic (right) versus normal (left) limb blood flow ratio using a Laser Doppler blood flow imager (Moor Instruments). Histology. The capillary density and the number and size of conductant vessels in the semimembraneous and adductor muscles were determined using 8-μm cryosections. Endothelial cells were identified with the panendothelial marker MECA-32 followed by donkey anti–rat IgG Alexa488 or CD31-FITC (BD Biosciences). Injected human EPCs were identified by costaining for HLA-ABC (allophycocyanin labeled; BD Biosciences) and vWF (Acris). Male murine BM-derived cells were identified by fluorescence in situ hybridization for the murine Y-chromosome (Cy3-labeled probe: Cambio; reference 7). Nuclei were stained with Sytox (Molecular Probes). Images were obtained by confocal microscopy (LSM 510; Carl Zeiss MicroImaging, Inc.). Statistical analysis Continuous variables are expressed as mean ± SD or SEM. Comparisons between groups were analyzed by Student's t test (two-sided) or analysis of variance with Bonferroni adjustment for experiments with more than two subgroups (SPSS 11.5 software). p-values <0.05 were considered as statistically significant. Acknowledgments We thank M. Muhly-Reinholz, T. R?xe, and M. N?her for their excellent technical assistance. Moreover, we thank Drs. N. Hogg, J. Harlan, and M. Robinson for providing the antibodies mAb24, mAb60.3, and KIM185, respectively. This work was supported by the Forschergruppe 501 (He 3044/2-2 to C. Heeschen) and the Alfried Krupp-Stiftung (to S. Dimmeler). K. Sasaki was in part supported by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. The work of K. Scharffetter-Kochanek was funded in party by the Collaborative Research Center SFB497-C7, Ulm. The authors have no conflicting financial interests. Submitted: 12 July 2004 Accepted: 19 November 2004 References Chavakis, T., M. Hussain, S.M. Kanse, G. Peters, R.G. Bretzel, J.I. Flock, M. Herrmann, and K.T. Preissner. 2002. Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes. Nat. Med. 8:687–693 Rohnelt, R.K., G. Hoch, Y. Reiss, and B. Engelhardt. 1997. Immunosurveillance modelled in vitro: naive and memory T cells spontaneously migrate across unstimulated microvascular endothelium. Int. Immunol. 9:435–450 Scharffetter-Kochanek, K., H. Lu, K. Norman, N. van Nood, F. Munoz, S. Grabbe, M. McArthur, I. Lorenzo, S. Kaplan, K. Ley, et al. 1998. Spontaneous skin ulceration and defective T cell function in CD18 null mice. J. Exp. Med. 188:119–131....查看详细 (30715字节)
☉ 11120210:Reciprocal and dynamic control of CD8 T cell homin
T cell activation by intestinal dendritic cells (DC) induces gut-tropism. We show that, reciprocally, DC from peripheral lymph nodes (PLN-DC) induce homing receptors promoting CD8 T cell accumulation in inflamed skin, particularly ligands for P- and E-selectin. Differential imprinting of tissue-tropism was independent of Th1/Th2 cytokines and not restricted to particular DC subsets. Fixed PLN-DC retained the capacity to induce selectin ligands on T cells, which was suppressed by addition of live intestinal DC. By contrast, fixed intestinal DC failed to promote gut-tropism and instead induced skin-homing receptors. Moreover, the induction of selectin ligands driven by antigen-pulsed PLN-DC could be suppressed "in trans" by adding live intestinal DC, but PLN-DC did not suppress gut-homing receptors induced by intestinal DC. Reactivation of tissue-committed memory cells modified their tissue-tropism according to the last activating DC's origin. Thus, CD8 T cells activated by DC acquire selectin ligands by default unless they encounter fixation-sensitive signal(s) for gut-tropism from intestinal DC. Memory T cells remain responsive to these signals, allowing for dynamic migratory reprogramming by skin- and gut-associated DC. Abbreviations used: Ag, antigen; CFSE, carboxyfluorescein diacetate succinimidyl ester; FucT, fucosyltransferase; HI, homing index; IVM, intravital microscopy; MFI, mean fluorescence intensity; PLN, peripheral lymph nodes; PP, Peyer's patches; PSGL-1, P-selectin glycoprotein ligand-1. The online version of this article contains supplemental material. To function in immune protection, T cells must be able to leave the blood and reach virtually every tissue in the body. A critical step in this process is the adhesion and subsequent transmigration of lymphocytes through the endothelial barrier. This strictly regulated multistep process has been partially characterized, at least for some tissues and T cell subsets (1, 2). Whereas na?ve T cells express homing receptors that allow them to migrate to lymphoid organs, like LN, Peyer's patches (PP), and the spleen, they are normally excluded from nonlymphoid peripheral tissues (2, 3). However, once T cells have become activated by their cognate antigen (Ag), they change their pattern of adhesion receptors and acquire the capacity to migrate to extralymphoid sites (3, 4). T cells localizing to the small intestine lamina propria selectively express the integrin 4?7 and the chemokine receptor CCR9 (5, 6), homing molecules that are essential for efficient T cell migration into the small bowel (7–9). On the other hand, cutaneous effector–memory T cells express E- and P-selectin ligands and the chemokine receptors CCR4 and/or CCR10 (10), receptors critical for T cell homing into the skin (10–14). Therefore, the expression of gut- and skin-homing receptors establishes a dichotomy in the distribution of effector–memory T cells, compartmentalizing the immune responses into two major surface areas exposed to largely distinct sets of Ag. Several reports have shown that the route of Ag entry determines the differential acquisition of tissue-specific homing molecules on lymphocytes (15, 16). We and others have shown recently that DC from PP or mesenteric LN specifically induced the expression of the gut-homing molecules 4?7 and CCR9 on CD8 T cells as well as the capacity to migrate to the small bowel (9, 17–19). However, the factors that induce T cell acquisition of skin-migratory potential remain unknown. It has also not been determined if effector–memory T cells with gut- or skin-homing potential represent a stable, irreversible differentiation stage or if, alternatively, tissue-tropic memory cells can adapt their migratory commitment when they are reactivated in a different anatomic context. Here we show that na?ve CD8 T cells activated by DC from skin-draining LN express significantly higher levels of E- and P-selectin ligands, adhere better to skin venules in vivo, and home significantly more efficiently to inflamed skin as compared with CD8 T cells activated with DC from gut-derived lymphoid tissues. Furthermore, the induction of selectin ligands driven by antigen-pulsed peripheral lymph nodes (PLN)-DC could be suppressed "in trans" by adding live intestinal DC. Moreover, we show that effector–memory CD8 T cells with an already committed skin- or gut-homing phenotype can be reeducated to adopt new migratory preferences in accordance with the anatomic source of the most recently encountered DC. Results control of CD8 T cell homing by DC To study the effect of DC from skin- and gut-associated lymphoid tissues, na?ve CD8 T cells from P14xT-GFP TCR-transgenic mice were cocultured for different time intervals with Ag-pulsed DCs from skin-draining LN (PLN-DC) or PP (PP-DC). A kinetic analysis showed that at day 2 of coculture, the expression level of skin- and gut-homing molecules was in general low, and there were no significant differences between CD8 T cells activated with PLN-DC and those activated with PP-DC (Fig. 1 A). However, as early as day 3, CD8 T cells activated with PLN-DC expressed significantly higher levels of E- and P-selectin ligands, as compared with those activated with PP-DC. The levels of E- and P-selectin ligands decreased after day 4, although the differences between CD8 T cells activated with PLN-DC and those activated with PP-DC remained statistically significant until day 6 of coculture. CD8 T cells activated with PP-DC expressed significantly higher levels of 4?7 and CCR9 than those activated with PLN-DC starting from day 3, but CD8 T cells activated with PP-DC expressing high levels of 4?7 were observed only after 4–5 d of coculture (17). Although na?ve CD8 T cells expressed 4?7 detectably (Fig. 1 A), their mean fluorescence intensity (MFI) for this marker was uniformly low (unpublished data). Since 3 d of coculture was the minimal period required to observe significant differences in both skin- and gut-homing molecules between CD8 T cells activated with PLN-DC and those activated with PP-DC, we chose this time point to perform CFSE (carboxyfluorescein diacetate succinimidyl ester) dilution assays in order to correlate the expression of homing molecules to the number of cell divisions. As shown in Fig. 1 (B–D), differential expression of selectin ligands as well as 4?7 and CCR9 was observed on CD8 T cells that had experienced a similar number of cell divisions. Comparable results for all traffic molecules examined were obtained when we used CD8 T cells from P14xTCR–/– mice, indicating that the observed differences were not due to expansion of an initially rare nonna?ve T cell population (unpublished data). mRNA expression and chemotactic responsiveness of CD8 T cells activated with PLN-DC or PP-DC Selectin ligands are formed by post-translational modification of glycoproteins like P-selectin glycoprotein ligand-1 (PSGL-1) (20). PSGL-1 is constitutively present on T cells, and we have shown previously that it remains equivalently expressed on CD8 T cells activated with PLN-DC or PP-DC (17). To acquire the capacity to bind selectins, PSGL-1 must undergo post-translational modifications, including (1,3)-fucosylation (20). Since fucosyltransferase-VII (FucT-VII) is a key enzyme in this process (21), we compared FucT-VII mRNA expression on CD8 T cells activated with PLN-DC and those activated with PP-DC using real-time PCR (Fig. 2 A). CD8 T cells activated with PLN-DC expressed significantly higher levels of FucT-VII mRNA as early as 2 d after coculture and maintained these elevated levels throughout the observation period. There was no significant difference in mRNA levels for FucT-IV (a second enzyme that can generate fucosylated selectin ligands) or core-2 ?1,6-N-acetylglucosaminyltransferase (C2GlcNAcT-I), an enzyme that generates O-linked glycans that are required substrates for (1,3)-fucosylation of selectin ligands (20) (see Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20041645/DC1). The murine chemokine receptors CCR4 and CCR10 have been implicated in T cell homing to the skin (14). CD8 T cells activated with PLN-DC expressed significantly higher levels of CCR4 mRNA at day 4 as compared with those activated with PP-DC (Fig. 2 B), whereas CCR10 mRNA was virtually undetectable in all samples analyzed. Accordingly, neither CD8 T cells activated with PLN-DC nor those activated with PP-DC migrated above the background to the CCR10 agonists CCL27 or CCL28 (Fig. S1; unpublished data). Similarly, although CCR8 was recently shown to be expressed by the majority of T cells in normal human skin (22), neither CD8 T cells activated with PLN-DC nor those activated with PP-DC migrated toward the CCR8 ligand CCL1. However, both sets of cells migrated equally efficiently toward gradients of the CCR4 ligands CCL22 or CCL17 (Fig. 2 E; unpublished data). The integrin 4?7 is composed of two chains. We have shown previously that 4, E, ?1, and ?7 integrin chains are up-regulated on CD8 T cells activated with either PP-DC or PLN-DC, but the 4 chain is significantly higher on CD8 T cells activated with PP-DC (Fig. 2 F; Fig. S1; [17]). Concurrent with these observations, the mRNA levels for 4 were significantly higher in CD8 T cells activated with PP-DC than in those activated with PLN-DC (Fig. 2 C), but there was no difference in E, ?1, or ?7 mRNA expression (Fig. S1). In agreement with Fig. 1, CCR9 mRNA levels were also significantly higher on CD8 T cells activated with PP-DC, starting at day 4 of coculture (Fig. 2 D). CD8 T cells activated with PLN-DC adhere to cutaneous venules and migrate better to inflamed skin than those activated with PP-DC Since CD8 T cells activated with PLN-DC expressed both CCR4 and higher levels of E- and P-selectin ligands than those activated with PP-DC, we postulated that they should be better equipped to migrate to the skin than CD8 T cells activated with PP-DC. However, pilot experiments failed to detect homing of either CD8 T cells activated with PLN-DC or PP-DC to normal skin (unpublished data). This was not unexpected because T cell migration to noninflamed murine skin is known to be very low, even upon adoptive transfer of endogenous cutaneous LN-derived T cells that express P- and E-selectin ligands (13). Therefore, homing of effector T cells into cutaneous sites has mostly been examined in inflamed skin (13, 14, 19). Thus, we compared the migration of CD8 T cells activated with PLN-DC or PP-DC into the ear skin after induction of a delayed-type hypersensitivity response (13). After adoptive transfer of a 1:1 mixture of differentially labeled CD8 T cells activated with PLN-DC or PP-DC, both subsets were equivalently represented in the blood and spleen (homing index 1.0; Fig. 3 A). Consistent with earlier experiments (17), CD8 T cells activated with PP-DC localized significantly better to mesenteric LN, whereas CD8 T cells activated with PLN-DC were more frequently found in PLN. Most importantly, CD8 T cells activated with PLN-DC homed significantly more efficiently into inflamed skin than CD8 T cells activated with PP-DC, thus confirming that the surface phenotype of CD8 T cells activated with PLN-DC or PP-DC is meaningful to predict in vivo migratory behavior. To determine if the preferential accumulation of CD8 T cells activated with PLN-DC in skin correlated with their ability to adhere within cutaneous microvessels, we performed intravital microscopy (IVM) to analyze interactions between fluorescently tagged CD8 T cells activated with PLN-DC or PP-DC and the venular endothelium in mouse ears. Since ear skin venules express E- and P-selectin constitutively and support abundant leukocyte rolling under steady-state conditions (23), it was not necessary to induce inflammation for these experiments. Both CD8 T cells activated with PLN-DC or PP-DC interacted with cutaneous venules, but those activated with PLN-DC rolled at a significantly higher frequency than CD8 T cells activated with PP-DC (Fig. 3 B). Interestingly, although we did not detect homing of CD8 T cells activated with PLN-DC into normal skin, we consistently observed a low number of rolling CD8 T cells activated with PLN-DC that adhered firmly to noninflamed venules, whereas those activated with PP-DC did not arrest under these conditions (Fig. 3 C). It has been shown that P- and E-selectin fulfill different functional roles in leukocyte rolling, with P-selectin being mainly responsible for the initial tethering, while E-selectin reduces the rolling velocity (Vroll) (23, 24). Consistent with the higher expression of P- and E-selectin ligands, we found that the median Vroll of CD8 T cells activated with PLN-DC was about half that of those activated with PP-DC (Fig. 3 D). Moreover, the dwell time during which CD8 T cells activated with PLN-DC continued to roll in cutaneous microvessels was significantly longer than for those activated with PP-DC and it was inversely correlated to the Vroll (Fig. 3 E). This difference in dwell time might selectively allow CD8 T cells activated with PLN-DC to activate integrins necessary for firm arrest in response to presumably low abundance chemoattractants. Role of DC subsets in the expression of homing molecules Several DC subpopulations have been described in lymphoid tissues (25), and the composition and magnitude of each subset varies between different lymphoid organs (26). For example, we have reported previously that PLN-DC contain a higher proportion of CD8+ DC than PP-DC (17). Moreover, in agreement with a previous report (27), PP-DC have a higher proportion of cells that are negative/low for the classic DC subset markers B220, CD11b, CD8 (Fig. 4 A; unpublished data). Since different DC subsets may induce distinct effector activities in T cells (25), it was important to explore whether the differences between PP-DC and PLN-DC in the induction of skin- and gut-homing molecules on CD8 T cells were related to their subset composition. However, when na?ve T cells were separately cocultured with each FACS-sorted constituent subset, all PLN-DC subsets induced more selectin ligands than their respective PP-derived counterparts, although CD11b+ (myeloid) PLN-DC tended to induce somewhat fewer selectin ligands than other PLN-DC (Fig. 4, B and C). all PP-DC fractions induced higher levels of 4?7 on CD8 T cells than the corresponding PLN-DC subset (Fig. 4 D). Interestingly, CD11b+ DC from both PP and PLN induced higher levels of 4?7 on CD8 T cells than other DC (Fig. 4 D; P < 0.05 for CD11b+ PLN-DC versus all other PLN-DC subsets). We also analyzed the expression of LFA-1/CD11a, which is required for T cell adhesion in a multitude of tissues, and found that it was equivalently up-regulated on CD8 T cells, regardless of the activating DC subset (Fig. 4 E). In two additional experiments, we included sorted subsets of splenic DC in our analysis. Spleen-DC, like PLN-DC, induced higher levels of selectin ligands and lower expression of 4?7 on CD8 T cells as compared with PP-DC (see Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20041645/DC1). Of note, splenic CD11bhigh DC, unlike those from PLN, were not different from other splenic subsets in their imprinting capacity. Effect of Th1/Th2 cytokines on skin- and gut-homing receptor expression Having established that all DC subsets from intestinal and cutaneous lymphoid tissues possess the reciprocal capacity to imprint in T cells a gut- or skin-homing phenotype, respectively, we set out to examine possible communication pathways that might contribute to these effects. T cells that are polarized under Th1, but not Th2, conditions acquire the capacity to bind E- and P-selectin and migrate efficiently into inflamed skin (28). In addition, IL-12 (Th1 cytokine produced by DC) up-regulates, whereas IL-4 (Th2 cytokine) down-regulates the expression of FucT-VII in lymphocytes (29). Therefore, we asked whether the differential induction of E- and P-selectin ligands on CD8 T cells activated with PLN-DC versus PP-DC was dependent on these cytokines. To this end, CD8 T cells were cocultured with either PLN-DC or PP-DC in the presence or absence of blocking antibodies against IL-12 or IL-4. As shown in Fig. 5 (A and B), addition of blocking anti-IL-12 (or anti-IL-12 plus anti-IFN-) did not affect the generation of E- and P-selectin ligands on CD8 T cells activated with PLN-DC. However, the presence of exogenous IL-12 in culture media significantly increased the expression of selectin ligands on CD8 T cells activated with PLN-DC or PP-DC, and this additional effect was blocked by the concomitant addition of anti–IL-12. Although the inclusion of IL-4 in culture media decreased the expression of E-selectin ligands in all cocultures, a blocking antibody against this cytokine did not increase the expression of these ligands on CD8 T cells activated with PLN-DC or PP-DC. Although 4?7 expression on CD4 T cells has been associated with a Th1 phenotype (30), the differential induction of 4?7 by PLN-DC and PP-DC was not significantly affected by inhibition of IL-12 or IL-4 (Fig. 5 C). Nonetheless, addition of exogenous IL-12 tended to increase and IL-4 decreased 4?7 on both sets of activated T cells. On the other hand, CCR9 was not appreciably affected by anti–IL-12 or anti–IL-4, whereas exogenous IL-12 slightly increased its expression (Fig. 5 D). Interestingly, addition of IL-4 increased significantly the percentage and MFI of CCR9+ in all cocultures, although this effect of IL-4 was not observed on anti-CD3 plus anti-CD28 activated T cells (Fig. 5 D; unpublished data). Taken together, these results confirm and expand previous observations that Th1 and Th2 cytokines can influence the expression of T cell traffic molecules (28), but neither IL-4 nor IL-12 appear to be responsible for the DC-induced imprinting of skin- or gut-tropism. Effect of DC fixation on T cell imprinting Next, we asked whether DC require an intact metabolism to imprint tissue-tropism in T cells. Ag-pulsed PP-DC and PLN-DC were lightly fixed with glutaraldehyde before coculture with na?ve T cells. Consistent with earlier reports (31), fixed DC retained their normal capacity to activate T cells, evidenced by the degree of proliferation and viability of the resulting effector T cells. However, fixed PP-DC induced more ligands for E- (Fig. 6 A) and P-selectin (Fig. 6 B) on CD8 T cells than unfixed PP-DC, and fixed PLN-DC induced selectin ligands comparably to their unfixed counterparts. On the other hand, the ability to induce 4?7 (Fig. 6 C) and CCR9 (Fig. 6 D) expression on T cells was severely impaired when DC were fixed before coculture. Interestingly, the presence of Ag-pulsed unfixed but not fixed PP-DC antagonized the induction of both selectin ligands by fixed PLN-DC. Indeed, addition of unfixed PP-DC to cocultures with fixed PLN-DC induced higher levels of 4?7 and CCR9 on CD8 T cells. In summary, these results reveal fundamental differences in the cellular mechanisms of DC-induced imprinting of skin- versus gut-tropism; DC do not appear to require an active metabolism (and/or surface molecules whose function is destroyed by fixation) to induce selectin ligands on T cells. By contrast, the ability of PP-DC to induce a gut-homing phenotype and to suppress the expression of skin-homing receptors is exquisitely sensitive to fixation. The presence of PP-DC overrides PLN-DC induction of selectin ligands on T cells Intestinal DC can imprint gut homing even when T cells are stimulated with antibodies and the DC are not presenting an activating Ag (17, 18). Having observed that Ag-pulsed PP-DC can inhibit the induction of selectin ligands by fixed-PLN-DC (Fig. 6, A and B), we asked if the mere presence of PP-DC without a specific Ag could modulate "in trans" the acquisition of skin traffic molecules on CD8 T cell activated with Ag-pulsed (and unfixed) PLN-DC, and vice versa. As shown in Fig. 6 E, PP-DC markedly suppressed the induction of E- and P-selectin ligands by peptide-pulsed PLN-DC. Interestingly, high concentrations of unpulsed PLN-DC had a potentiating effect on the induction of selectin ligands during Ag stimulation by a standard number of PLN-DC or PP-DC (Fig. 6, E and F). On the other hand, excess numbers of unpulsed PLN-DC did not affect the induction of 4?7 by Ag-pulsed PP-DC, but tended to decrease somewhat the induction of CCR9 (Fig. 6, G and H). Furthermore, unpulsed PP-DC increased the induction of 4?7 by peptide-pulsed PLN-DC or PP-DC in a dose-dependent manner, and they increased the expression of CCR9 on CD8 T cells activated with Ag-pulsed PLN-DC. Importantly, when live PP-DC and PLN-DC were present in cocultures at equivalent concentrations, the resulting homing receptor repertoire on effector T cells was always skewed toward a gut-homing phenotype, irrespective of which DC population presented the cognate Ag. Tissue-tropic CD8 T cells preserve their susceptibility to further imprinting signals An important question is whether the tissue-specific homing potential of effector–memory T cells is a stable property or if it can be modulated when T cells are reactivated in a different tissue context. We addressed this question by reactivating either CD8 T cells activated with PLN-DC or PP-DC (4–5 d after their primary activation) with the same or the opposite DC, and then analyzing the reactivated cells 4–5 d later. When CD8 T cells activated with PLN-DC were reactivated with PLN-DC, they maintained high levels of selectin ligands, whereas restimulation with PP-DC induced a dramatic loss of selectin ligands to levels equivalent to those found on CD8 T cells activated exclusively with PP-DC (Fig. 7, A and B). Conversely, CD8 T cells activated with PP-DC and reactivated with PLN-DC rapidly gained high levels of selectin ligands similar to those found on CD8 T cells activated with PLN-DC. Restimulation had essentially the same effect on T cell expression of 4?7 (Fig. 7 C) and CCR9 (Fig. 7 D). In each case, the last DC population encountered by CD8 T cells determined the homing phenotype, irrespective of the first imprinting event. As reported previously (17), prolonged stimulation with PLN-DC or PP-DC increased the levels of 4?7 on both CD8 T cells activated with PLN-DC or PP-DC above those on na?ve T cells (Fig. 7 C). Nevertheless, the increase was always significantly higher when cells were stimulated by PP-DC as compared with PLN-DC. Having discovered that recently activated ex vivo–generated CD8 T cells can change their homing phenotype when they are reactivated in a new context, we investigated whether endogenously generated effector–memory T cells retain a similar plasticity. To address this question, we isolated CD44High effector–memory CD8+ T cells from pooled lymphoid organs of adult donors and sorted them based on their ability to bind P-selectin (Fig. 8 A) or to express CCR9 (Fig. 8 B), which served as surrogate markers for skin- and gut-homing potential, respectively. These markers were chosen because control experiments showed that 50% of CD8+CD44HighCCR9+ cells expressed 4?7 but few cells expressed selectin ligands. On the other hand, a minority of 4?7+ memory cells coexpressed CCR9 (see Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20041645/DC1), which is necessary for homing to the small intestine (6, 8, 9). Therefore, sorting of CD8+CD44HighCCR9+ T cells generated the most enriched memory population with the potential to home to the small bowel. Conversely, CD8+ CD44HighP-lig+ cells were mostly CCR9Neg4?7Neg and thus likely to contain a sizeable fraction of skin-tropic memory cells (11). Consistent with our in vitro–generated effector CD8 T cells, FACS-sorted endogenous effector–memory CD8 T cells that were activated with anti-CD3 plus anti-CD28 expressed more selectin ligands and less 4?7 when cocultured with PLN-DC, whereas PP-DC enhanced the expression of 4?7 and suppressed selectin ligands irrespective of the phenotype of the starting effector–memory T cells. The same trend was also observed with CCR9, although the differential effects of DC restimulation on this molecule did not always reach statistical significance. Discussion DC from gut-associated lymphoid tissues induce on activated CD8 T cells the expression of the gut-homing molecules 4?7 (9, 17–19) and CCR9 (9, 17), as well as the capacity to migrate to the small bowel (17). We now provide evidence that na?ve CD8 T cells activated by DC from skin-draining LN assume a skin-homing phenotype, i.e., they express significantly more selectin ligands and mRNA for CCR4, undergo more frequent and prolonged adhesive interactions with skin venules, and home more efficiently to inflamed skin as compared with CD8 T cells activated with intestinal DC. IVM observations strongly suggest that the preferential migration of CD8 T cells activated with PLN-DC to inflamed skin in competitive homing experiments was due to their superior ability to adhere to dermal microvessels compared with CD8 T cells activated with PP-DC. Not only did CD8 T cells activated with PLN-DC roll more frequently and at a lower velocity, but also, unlike those activated with PP-DC, CD8 T cells activated with PLN-DC underwent spontaneous arrest in noninflamed skin-venules. In the mouse ear, postcapillary venules constitutively express both P- and E-selectin, which mediate exclusively the tethering and rolling step (23). Thus, T cells must express selectin ligands at a sufficient density to marginate and to scan the endothelial surface for chemoattractants (10, 13). (1,3)-fucosylation of sialomucins by FucT-VII is essential for selectin ligand biosynthesis in leukocytes (20, 21, 23). Consistent with this concept, CD8 T cells activated with PLN-DC contained more FucT-VII mRNA than those activated with PP-DC as early as 2 d after stimulation, i.e., immediately preceding the up-regulation and ultimately high surface expression of selectin ligands. This time course is in good agreement with in vivo studies of selectin ligand induction on CD4 T cells (16) and faster than the kinetics of selective 4?7 up-regulation on CD8 T cells activated with PP-DC, which was already apparent at day 3, but 4?7High T cells arose only after day 4–5 of coculture (17). Once a skin-homing T cell has engaged in rolling, the integrin-activating stimulus must be provided by chemokines that act either on CCR4 or CCR10 (11, 12). Agonists for both receptors are probably expressed at a low level in normal skin and either one is sufficient for T cell recruitment (14). CCL1, the agonist for CCR8 was recently detected in human skin (22), but the role of this pathway in T cell traffic to murine skin has not yet been determined. We could not detect evidence for CCR8 or CCR10 expression in CD8 T cells activated with PLN-DC or PP-DC, but those activated with PLN-DC contained higher mRNA levels for CCR4, although both populations responded similarly to CCR4 ligands in chemotaxis assays. These seemingly conflicting data could either indicate that CCR4 protein expression and/or function are not regulated at the mRNA level or that the lower levels of CCR4 on CD8 T cells activated with PP-DC might still be sufficient to allow chemotaxis but not firm arrest in venules, as has been shown for CXCR4 on na?ve CD8 T cells (32). In this context, it is interesting to note that only CD8 T cells activated with PLN-DC, but not those activated with PP-DC, underwent spontaneous firm arrest in ear venules. While it remains to be determined if this adhesion event was mediated by CCR4, it is likely that the higher selectin ligand expression on CD8 T cells activated with PLN-DC made an indirect contribution. P-selectin primarily mediates the initial tethering and determines the frequency of rolling interactions, whereas E-selectin decreases the rolling velocity (23, 24). Consistent with the high expression of E- and P-selectin ligands, CD8 T cells activated with PLN-DC rolled not only at a higher frequency than those activated with PP-DC, but also at a lower speed, which resulted in longer intravenular dwell times. The increased duration of rolling contacts might have allowed CD8 T cells activated with PLN-DC to sense and respond to chemokine(s), presumably CCR4 agonists, that may have been presented at low densities in skin microvessels, thus increasing the likelihood for triggering integrin-dependent firm arrest (24, 33). A tissue-specific multistep adhesion cascade is also responsible for T cell homing to the small intestine (2). The critical receptor–ligand pairs in this setting are 4?7–MAdCAM and CCR9–CCL25, although recent reports indicate that P-selectin may also play a role in gut homing, at least for Th1-polarized CD4+ cells (34). The constituent chains of the 4?7 heterodimer are also components of two other integrins, 4?1 and E?7. Thus, 4?7 surface expression levels could be regulated by a variety of mechanisms that affect the equilibrium between these three integrins. However, the only consistent difference observed by real-time PCR between CD8 T cells activated with PLN-DC and those activated with PP-DC was in mRNA levels for 4, but not for ?7, ?1, or E. These observations are consistent with previous observations that activation by PP-DC or PLN-DC up-regulates several integrin chains on the surface of CD8 T cells, including 4, E, L, ?1, and ?7, but only the surface expression of 4 (and the 4?7 heterodimer), but not the ?7 chain is selectively increased on CD8 T cells activated with PP-DC (17). Therefore, it seems plausible that the differential induction of 4?7 on CD8 T cells activated with PLN-DC versus those activated with PP-DC is at least in part controlled at the level of the 4 chain. However, this does not exclude the possibility that 4?7 expression is subject to additional posttranslational regulation that might affect the stoichiometry of integrin chain pairing. Our current observations suggest that the regulation of CCR9 is similarly complex. CCR9 is highly expressed on na?ve CD8+ T cells (35). Our present kinetic analysis indicates that at day 2 of coculture, activated T cells uniformly down-regulate CCR9 mRNA and surface protein levels irrespective of the activating DC, but CCR9 was selectively reexpressed in CD8 T cells activated with PP-DC starting on day 3. Thus, imprinting signal(s) provided by PP-DC promote reexpression of this receptor after initial activation-induced down-regulation. It remains to be determined how PP-DC and PLN-DC imprint gut- and skin-tropism in T cells, respectively. Compared with PLN-DC, PP-DC express higher levels of several surface markers, including the integrin E?7 and the "nonclassical" costimulatory molecules B7-H1/PDL1 and B7-DC/PDL2, but blocking mAbs to these molecules did not affect their imprinting capacity (unpublished data). Likewise, a survey of several other candidate pathways (OX-40L, Qa2, MAdCAM, IL-10R, TGF?, CCL25, and pertussis toxin treatment) was unrevealing. However, the induction of 4?7 on CD8 T cells activated with PP-DC was very sensitive to inhibitors of T cell activation, such as anti-CD18, anti-CD11a, anti-CD80, or loading of DC with a low peptide concentration (unpublished data). Therefore, efficient T cell activation seems to be necessary but not sufficient for the induction of gut-homing T cells. Since PLN and PP differ in DC subset composition (17, 26, 27), and different DC subsets induce distinct T cell responses (25), it seemed reasonable to explore the role of individual DC subsets in tissue imprinting. Irrespective of the DC population examined and despite some subtle differences between subsets (particularly with respect to CD11bhigh PLN DC), spleen- and PLN-derived DC induced preferentially skin-homing receptors, while their PP-derived counterparts always induced more gut-homing molecules, indicating that the ability to acquire tissue-specific imprinting mechanism(s) is a common property of all DC in the same lymphoid tissue. Of note, a recent report found that Langerhans cells from skin explants induce higher E-selectin ligands on CD8 T cells than mesenteric LN DC (19). This suggests that the ability of DC to induce a skin-homing phenotype in T cells is not only acquired in PLN. Indeed, we observed that T cell activation by Ag-presenting DC from spleen or by anti-CD3 plus anti-CD28 also induced selectin ligands (but not 4?7 or CCR9) on CD8 T cells, although at somewhat lower levels than in response to PLN-DC (Fig. S2; unpublished data). These findings are in good agreement with reports showing that signaling through the TCR is sufficient for the induction of FucT-VII and E-selectin ligands on T cells independent of IL-12/STAT-4 signaling (36). This is consistent with our observation that anti–IL-12 did not interfere with the induction of selectin ligands by PLN-DC. these results raise the possibility that the up-regulation of selectin ligands on CD8 T cells reflects a hard-wired response to activation in general, and that this default differentiation pathway is actively suppressed by gut-derived DC. Several experimental observations are consistent with this notion. First, fixed PP-DC were incapable of inducing gut-homing receptors. Second, CD8 T cells that were activated by fixed PP-DC or unfixed PLN-DC equivalently up-regulated E- and P-selectin ligands, and fixed PLN-DC tended to super-induce these traffic molecules. Third, the presence of unfixed, but not fixed, PP-DC in cocultures with fixed PLN-DC drove T cells toward a gut-homing phenotype rather than allowing the acquisition of skin-homing receptors, indicating that the fixation procedure destroyed one or more pathways in PP-DC that are not only critical for intestinal imprinting per se, but also for their ability to suppress the induction of cutaneous homing receptors. Fourth, unfixed PP-DC did not need to present Ag to suppress the induction of selectin ligands by Ag-presenting fixed or unfixed PLN-DC; the mere presence of PP-DC was sufficient to confer "in trans" the preferential expression of gut-homing molecules. Fifth, both the suppression of skin-homing receptors and the induction of gut tropism by PP-DC were clearly apparent in cocultures with a 1:1 ratio of PLN-DC:PP-DC. Thus, T cells assume a gut-homing phenotype when afforded with equal opportunity to interact with PP-DC and PLN-DC, indicating that the former override putative imprinting signals from the latter. Finally, sixth, T cells in cocultures with high ratios of unpulsed PLN-DC to antigen-pulsed PP-DC expressed increased levels of selectin ligands, but did not lose the expression of gut-homing receptors. Thus, while PP-DC can efficiently suppress the induction of skin tropism by cutaneous DC, PLN-DC appear incapable of antagonizing intestinal imprinting signals generated by gut-derived DC. While selectin ligands may be more readily induced in CD8 T cells than gut-homing receptors, it is likely that the magnitude of homing receptor expression is subject to regulation. For example, it has been reported that the cutaneous lymphocyte Ag (the principal E-selectin ligand on human skin-homing T cells) is reversibly up- or down-regulated on activated CD4 T cells upon exposure to different cytokines (37). Moreover, we have observed that a large fraction of TCR?+CD8?+ T cells in the small intestine express little or no 4?7, even though 4?7 is presumably required for their homing to the intestine (reference 17; unpublished data). These observations suggested that tissue-tropism remains malleable, even after a T cell has been imprinted in a specific tissue context. Consistent with this idea, we observed remarkable plasticity in the response of tissue-committed CD8 T cells activated with PLN-DC or PP-DC to restimulation by PP-DC and PLN-DC, respectively. However, it could be argued that short-term differentiation, as performed in our ex vivo assay, is insufficient to establish an irreversible homing phenotype. A case in point is the differentiation of polarized Th1 cells, which can be reprogrammed to produce Th2 cytokines after short-term differentiation, but lose this plasticity once they become fully committed (38). To address this possibility, we also tested whether PP-DC and PLN-DC induce differential tissue-tropism upon restimulation of endogenously generated effector–memory CD8 T cells from adult donor mice that expressed either P-selectin ligands or CCR9, presumably in response to prior encounters with environmental Ag in skin- or gut-associated lymphoid tissues, respectively. The results from these experiments were consistent with those obtained with in vitro–differentiated effector cells and further support the intriguing possibility that tissue specificity is a dynamic property of effector–memory T cells, which can be modified if T cells are reactivated in a different anatomic context. Under what circumstances might tissue-committed effector–memory cells be exposed to DC from another organ system? One mechanism may be the progressive reexpression of homing receptors for LN on long-lived effector memory cells (39). Thus, when effector memory cells in the skin or gut reexpress CCR7, they should be able to enter draining lymphatics and return to the blood stream (40). Indeed, it has been shown that the majority of 4?7High and CLA+ memory T cells in human blood coexpress the essential LN homing receptors CCR7 and L-selectin (41). These cells may then be able to home to both MLN and PLN where they could encounter a recall Ag that arises in the gut or skin, respectively. Thus, the acquisition and maintenance of migratory specificity in memory T cells might be a much more dynamic process than has been assumed. This question also has practical implications, because if memory T cells can be readily reeducated with regard to their tissue specificity, then it should be feasible to modulate effector–memory cell trafficking for therapeutic purposes. MATERIALS AND Methods Mice and reagents. C57BL/6 and C57BL/6 Thy-1.1 mice were obtained from Jackson ImmunoResearch Laboratories. P14xT-GFP double transgenic mice have been described (42). P14xTCR–/– mice were obtained from Taconic Farms (43). Mice were housed in an SPF/VAF facility and used in accordance with CBR1 and Harvard Medical School animal committees' guidelines. mAbs to the murine 4?7 heterodimer (DATK32), 4 (R1-2), ?7 (M293), E (M290), and ?1 (HM?1-1) integrin chains, as well as P-selectin-Fc and all mAbs for T cell and DC purification were from BD Biosciences. E-selectin-Fc chimera and cytokines were from R&D Systems. Anti-mouse CCR9 (clone 5F2) was generated at Millennium Pharmaceuticals, Inc. LCMVgp33-41 peptide was from Biosource International. isolation and cocultures. C57BL/6 mice were injected s.c. with B16 cells secreting Flt3 ligand as previously described (17). After 12–14 d, mice were killed and PLN and PP were digested using 250 μg/ml Liberase CI plus 50 μg/ml DNase-I (Roche) in IMDM for 40 min at 37°C with mild agitation. Cell separation was performed at 4°C in PBS containing 2 mM EDTA and 2% FBS (GIBCO BRL). For negative selection, cells were incubated with mAbs to CD3, CD19, Pan-NK, Ter-119, and Thy-1, incubated with anti-rat IgG microbeads and purified on BS columns (Miltenyi Biotec). DC (85–90% CD11c+) were resuspended to 107/ml, pulsed for 2 h at 37°C with 10 μg/ml LCMVgp33-41 in IMDM plus 10% FBS, 50 μg/ml gentamycin, 0.5 μg/ml fungizone, 50 μM 2-ME, and standard supplements, washed twice, and used immediately in cocultures. In some experiments, peptide-pulsed DC were fixed with 0.05% glutaraldehyde (Sigma-Aldrich). For some experiments, negatively selected DC were sorted into different DC subpopulations (FACS Vantage; BD Biosciences). Na?ve CD8+ T cells were purified from splenocytes by negative selection as previously described (17), and in some experiments effector–memory T cells were isolated by negative selection and FACS sorting. For cocultures, 106 na?ve T cells were added at a 1:1 ratio to peptide-pulsed DC in complete IMDM using 12-well plates, or for some experiments, in flat bottom 96-well plates (105 na?ve CD8 T cells and DC). In some experiments, cocultures were supplemented from day 0 with 10 ng/ml rmIL-12, 25 ng/ml rmIL-4, and/or 20 μg/ml of blocking mAbs (from BD Biosciences) to IL-12p40/p70 (C17.8), IFN- (XMG1.2), or IL-4 (11B11). For fluorescent labeling, T cells were stained with 5 μM CFSE (Molecular Probes) in DMEM + 1% FBS + 20 mM Hepes. For homing experiments (see below), T cells were also labeled with TRITC (Molecular Probes). Cells were resuspended at 2 x 107/ml, incubated with 2.5 μg/ml TRITC for 20 min at 37°C, and washed with an FBS gradient. Reverse transcription and real-time PCR. Real-time PCR was performed as previously described (17) on CD8 T cells activated with PLN-DC or PP-DC, using the following primers: FucT-VII forward, ACTGATGTTGAAACCAAAGAGC, and reverse, GCCCAGTCTTCTCCTTATATCC; CCR4 forward, GGTACCTAGACTACGCCATCC, and reverse, ATGTACTTGCGGAATTTCTCC; 4 integrin chain forward, AAACACTGGGATTAGCATGG, and reverse, ATTGCCCTGTAGTTGTCTGG; CCR9 forward, AGGTTAGTCAGCCAATGTACAGC, and reverse, ATCCTTTCCTAGTTTGTGCTTGC; GAPDH forward, CAACTTTGTCAAGCTCATTTCC and reverse, GGTCCAGGGTTTCTTACTCC. mRNA levels were expressed relative to GAPDH mRNA for each sample. Homing assays. Thy1.2+ CD8 T cells activated with PLN-DC or PP-DC were differentially labeled with TRITC and CFSE (Molecular Probes) as previously described (17). 2–3 x 107 cells from each population were mixed and injected into recipient C57BL/6 Thy1.1+ mice with preexisting cutaneous inflammation in both ears induced by a standard delayed-type hypersensitivity protocol (13). In brief, shaved abdomens of Thy-1.1+ mice were painted with 25 μl 0.1% 2,4-dinitro-1-fluorobenzene (DNFB; Sigma-Aldrich) in acetone on days 0 and 2. On day 5, mice were challenged with 25 μl 0.25% DNFB on both ears, and used on the next day as recipients for competitive homing experiments. Recipients were killed after 18 h, and single cell suspensions were generated from blood, spleen, PLN, MLN, and both ears after digestion with collagenase-D (Roche). Cell samples were incubated with anti-CD8 and anti–Thy-1.2 and analyzed on a FACScalibur (BD Biosciences) by gating on viable CD8+Thy-1.2+ cells. The homing index (HI) was calculated as HI = [CD8 T cells activated with PLN-DC]tissue/[CD8 T cells activated with PP-DC]tissue: [CD8 T cells activated with PLN-DC]input/[CD8 T cells activated with PP-DC]input. Intravital microscopy (IVM). C57BL/6 mice were anesthetized by i.p. injection of physiologic saline (10 ml/kg) containing ketamine HCL (5 mg/ml) and xylazine (1 mg/ml). The right carotid artery was catheterized, T cells were labeled with Calcein AM (Molecular Probes), administered intra-arterially in small boluses and visualized by video-triggered stroboscopic epi-illumination. Samples of CD8 T cells activated with PLN-DC or PP-DC were injected consecutively and analyzed in the same venules as previously described (44). Rolling (cells interacting visibly with venules and traveling at a slower velocity than the blood stream) and noninteracting T cells were counted in each venule. The rolling fraction was calculated as percentage of rolling cells among the total number of T cells that entered a venule. The sticking fraction was determined as percentage of T cells becoming firmly adherent for 30 s among all T cells that rolled in a venule during the same time interval. Velocity analysis was performed using a customized image analysis system as previously described (44). Online supplemental material. The supplemental material (Figs. S1–S3) is available at http://www.jem.org/cgi/content/full/jem.20041645/DC1. Fig. S1 shows mRNA expression, integrin chain expression, and chemotactic responsiveness of CD8 T cells activated with PLN-DC or PP-DC. Fig. S2 depicts the effect of sorted DC subsets in the acquisition of selectin ligands and 4?7 on CD8 T cells. Fig. S3 shows the correlation betwen the expression of the homing molecules 4?7, CCR9, and ligands for E- and P- selectin on endogenous effector–memory T cells used for FACS sorting. Acknowledgments We are grateful to N. Barteneva for assistance with the FACS sorting. We thank I. Mazo for technical suggestions and J. Moore for editorial assistance. J.R. Mora is indebted to Ingrid Ramos for constant support. Supported by a Pew Fellowship to J.R. Mora and National Institutes of Health grants HL62524, HL54936, HL56949, and AI-061663 to U.H. von Andrian. he authors have no conflicting financial interests. Note added in proof: While this paper was under review, Iwata et al. (Iwata, M., A. Hirakiyama, Y. Eshima, H. Kagechika, C. Kato, and S.Y. Song. 2004. Immunity. 21:527–538) reported that intestinal DC, but not DC from other tissues, produce retinoic acid, which was necessary and sufficient to imprint gut-tropism and suppress skin-homing molecules on T cells. Submitted: 16 August 2004 Accepted: 14 December 2004 References Stein, J.V., G. Cheng, B.M. Stockton, B.P. Fors, E.C. Butcher, and U.H. von Andrian. 1999. L-selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity. J. Exp. Med. 189:37–50....查看详细 (45735字节)
☉ 11120211:Positive selection of the peripheral B cell repert
Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express VHn allotype immunoglobulin (Ig)M. Within weeks, the number of VHn B cells decreases, whereas VHa allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from VHn to VHa B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the VHn to VHa repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of VHa B cells. By comparing amino acid sequences of VHn and VHa Ig, we identified a putative VH ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with VHa B cells results in their selective expansion. Abbreviations used: BCR, B cell receptor; FR, framework region; GALT, gut-associated lymphoid tissue; LigApx, ligated appendix. K.-J. Rhee and P.J. Jasper contributed equally to this work. Vertebrates have developed two general strategies for generating a diverse primary B cell repertoire. In humans and mice, the primary B cell repertoire is generated by rearrangement of multiple V, D, and J gene segments in the bone marrow throughout the life of the animal. In other species, such as the rabbit (1–3), chicken (4, 5), and sheep (6, 7), this repertoire initially develops by rearrangement of a limited number of V genes in primary lymphoid tissue and further diversifies in gut-associated lymphoid tissues (GALTs). In rabbits, the D proximal VH gene, VH1, is preferentially rearranged during B cell development in the fetal liver and bone marrow (8). The VDJ genes undergo somatic diversification via somatic hypermutation and gene conversion in GALT in response to intestinal microflora (9). In the absence of appropriate intestinal microflora, GALT develops poorly, and both the number of B cells and the diversification of VH genes are greatly inhibited (9). Most (80–90%) rabbit serum Ig molecules express VHa allotypic markers that are encoded by the predominantly rearranged gene, VH1 (1). The following three alleles of VH1 are found in laboratory rabbits: VH1-a1, VH1-a2, and VH1-a3; they encode the VHa1, VHa2, and VHa3 allotypes, respectively. These VHa allotypes differ in amino acid residues in framework region (FR)1 and FR3 (10). 10–20% of serum Ig does not react with anti-VHa1, anti-VHa2, or anti-VHa3 allotypic antibodies and is referred to as VHn (VHa-negative) Ig. Kelus and Weiss (11) identified rabbits with a variant VHa2 allotype-encoding allele, ali, which has a 10-kb deletion of DNA encompassing VH1 (Fig. 1 and reference 1). In contrast with wild-type rabbits, nearly all Ig in young ali/ali rabbits (designated Alicia) is VHn. VHn Ig is encoded predominantly by VHx, VHy, and VHz (12, 13), which reside >50 kb upstream of VH1 (1). In adult Alicia rabbits, high levels of serum Ig with the VHa (a2) allotype are found (11). This increase in VHa Ig is a result of increased numbers of VHa B cells that use VH4, VH7, and VH9, gene segments that encode several of the VHa (a2) allotype-associated amino acids (14, 15). Pospisil et al. (16) found that, in the appendix, more VHa B cells were proliferating and fewer were dying compared with VHn B cells. The molecular basis underlying the repertoire shift from VHn to VHa is unknown and is the subject of the current paper. A shift in the B cell repertoire could arise from VH gene replacement or from secondary Ig gene rearrangements on the unexpressed Ig allele. Although it is generally believed that these events occur primarily in the bone marrow (17–19), there is evidence that VH gene replacement and secondary Ig gene rearrangement occur in peripheral tissues (20, 21). Another possible explanation for the B cell repertoire shift is that VHa B cells are positively selected in the periphery. Positive selection of B cells in the periphery has been demonstrated in several transgenic mouse models (22–26). In rabbit, intestinal microflora interacts with GALT to promote development of follicles containing proliferating B cells and to generate the primary B cell repertoire (9). We hypothesized that, in GALT of Alicia rabbits, interactions between GALT and the intestinal microflora also promote the increased proliferation of VHa B cells compared with VHn B cells and lead to the repertoire shift from VHn to VHa B cells. To test this possibility, we surgically disrupted the GALT–bacterial interaction in Alicia rabbits and tested whether the repertoire shift from VHn to VHa B cells was abrogated. Results Kinetics of the B cell repertoire shift The repertoire shift in Alicia rabbits, from the predominant expression of VHn allotype early in life to the predominant expression of VHa allotype later in life, was originally shown by Kelus and Weiss (11), who analyzed Ig allotypes in serum. Pospisil et al. (16) showed that a similar shift occurred in B cells in the appendix. By using antibodies to both VHn and VHa allotypes, we found that, in 9-wk-old rabbits, VHa B cells represented 35–50% of the B cells in spleen, mesenteric lymph nodes, appendix, and PBLs (Fig. 2). We analyzed cells of various tissues from newborn to 2-yr-old Alicia rabbits to follow the appearance and disappearance of VHa and VHn B cells, respectively, throughout life. We found that, although 10–25% of B cells at birth are VHa, at 3 wk of age essentially all B cells (95%) in spleen, appendix, and PBLs were VHn (Fig. 3 and not depicted). Subsequently, the percentage of VHn cells steadily declined, so that by 2 yr of age, 75% were VHa. These data demonstrate that VHa B cells accumulate throughout life, with a sharp increase between 4 and 10 wk of age. The VHa B cells accumulate faster in the appendix than in spleen, suggesting that the B cell repertoire shift from VHn to VHa B cells may occur primarily in GALT. B cell repertoire shift and GALT Because GALT development and somatic diversification of Ig genes both require interaction between GALT and intestinal microflora (9, 27), we hypothesized that the repertoire shift in Alicia rabbits also requires this interaction. To investigate this possibility, we generated ligated appendix (LigApx) rabbits by surgically removing the Peyer's patches and the sacculus rotundus and ligating the lumen of the appendix to prevent bacterial colonization (9). If interactions between GALT and intestinal microflora are required for the repertoire shift from VHn to VHa B cells, we expected that the peripheral blood B cells in LigApx rabbits would be predominantly VHn. In each of three 12-wk-old LigApx Alicia rabbits (94S, 353X2, 353X4), we found that the percentage of B cells was approximately eightfold less than in unmanipulated rabbits of that age and that almost all B cells (90–96%) were VHn (Fig. 4). As expected, the percentage of VHn B cells in unmanipulated Alicia rabbits was 50%. We examined one of the LigApx Alicia rabbits (94S) at 8 mo of age and found that >90% of the B cells were still VHn, showing that the B cells remained predominantly VHn for many months. These results indicate that, without interactions between GALT and intestinal flora, the shift from VHn to VHa did not occur. To confirm that the repertoire shift from VHn to VHa B cells was abrogated in LigApx rabbits, we examined the nucleotide sequences of VDJ genes cloned from peripheral blood of 12-wk-old LigApx Alicia rabbits. We expected that the VH genes used in the VDJ gene rearrangements would be primarily genes that encode VHn molecules rather than VHa molecules. From one LigApx Alicia rabbit (94S), shown in Fig. 4, and from three additional LigApx rabbits (32P2, 144T, 199T1) for which flow cytometry data are not available, we analyzed a total of 80 VDJ gene sequences. As predicted, most (76%) of the VDJ genes used VHn gene segments (Table I), whereas almost none (4%) of the VDJ genes from control (unmanipulated) Alicia rabbits of the same age used VHn gene segments. We think the PCR analysis underestimated the expression of VHn genes because, by FACS analysis, 50% of the peripheral B cells from 12-wk-old control Alicia rabbits were VHn, whereas only 4% of the PCR-amplified VDJ genes were VHn. To determine whether the low percentage of VHn-encoding genes (Table I) resulted from preferential amplification of VHa cDNA, we conducted two independent experiments in which VDJ genes were PCR amplified from cDNA prepared from a pool of cells containing equivalent numbers of FACS-sorted VHa and VHn B cells from peripheral blood. In the two experiments, 67% (14 out of 21) and 81% (17 out of 21) of PCR-amplified VDJ genes used VHa gene segments. The reduced number of VHn PCR products was also observed with another 5' VH primer, VHldr (5'-GGCTTCTCCTGGTCGCTG-3'), which anneals to a different target site. The preferential amplification of VHa cDNA with independent primers suggests that, even though VHa and VHn B cells appear to express equivalent amounts of surface IgM (Fig. 4), VHa B cells might produce higher levels of IgM mRNA, possibly as a result of their stimulation in GALT (16). Although we do not understand the molecular basis for the PCR skewing toward VHa-encoding VDJ genes, the data confirm the FACS analysis, which showed that most of the B cells of LigApx Alicia rabbits were VHn instead of VHa. We conclude that the repertoire shift from VHn to VHa B cells required interactions between GALT and the microflora and that expansion of VHa B cells requires such interactions. Rearrangement status of IgH alleles in VHa B cells The repertoire shift from VHn to VHa B cells in the periphery could occur by replacement of VHn-using VDJ genes with VHa gene segments (20), by rearrangement of a VHa-encoding VHa gene segment on the second IgH allele (17, 21), or by selective expansion of VHa B cells (16, 24). We think that VH gene replacement is unlikely to explain the repertoire shift because the VHn genes (VHx, VHy, and VHz) used in VDJ gene rearrangements in VHn B cells reside upstream of the VHa genes (VH4, VH7, and VH9) used in VDJ gene rearrangements in peripheral VHa B cells of Alicia rabbits (15). Accordingly, the rearrangement of VHn genes during VDJ gene rearrangements would likely result in deletion of the VHa genes (Fig. 1). If the repertoire shift from VHn to VHa B cells is caused by gene rearrangements of VHa gene segments on the second IgH allele in VHn B cells, we expected to find VDJ gene rearrangements on both IgH alleles in VHa B cells. To test this possibility, we sorted VHa B cells from an adult Alicia rabbit and assessed the status of VDJ gene rearrangements by single cell PCR. We used PCR primers that would detect rearranged VDJ genes and germline JH genes (Fig. 5 a). Of 26 single cells from which a rearranged VDJ PCR product was obtained, all but one had a product of the expected size for an unrearranged (second) IgH allele (Fig. 5 b). This result showed that essentially all VHa B cells rearranged only one IgH allele, indicating that the B cell repertoire shift from VHn to VHa B cells in Alicia rabbits is not due to secondary IgH gene rearrangements on the other allele. Instead, we propose that the B cell repertoire shift occurs through positive selection due to preferential expansion of VHa B cells. B cell receptor (BCR) signaling in VHa and VHn B cells One possible explanation for the preferential expansion of VHa B cells and the concomitant decrease in VHn B cells in Alicia rabbits is that VHa B cells are more responsive to BCR stimulation than VHn B cells. To test this possibility, we assessed the release of intracellular calcium after BCR cross-linking on VHa and VHn B cells from 12-wk-old Alicia rabbits. The Alicia rabbits had the b5 chain allotype; therefore, we incubated PBLs with anti-b5 antibody and measured the release of intracellular calcium, as described in Materials and Methods. We found that the VHn B cells responded to anti-b5 antibody as well as the VHa B cells when the differences in baseline stimulation were taken into account (Fig. 6). Anti-b4 chain allotype antibody served as a negative control. Although we cannot explain the different baseline stimulation of VHa and VHn B cells, we conclude that the inherent signaling capacity of VHa and VHn B cells is similar and, therefore, does not explain the selective expansion of VHn B cells. Discussion The intestinal microflora are important in regulating many immune functions, including development of GALT (27), induction of oral tolerance (28), and induction of mucosal immunity (29). In rabbits, intestinal microflora are required not only for GALT to develop but also to generate a diverse primary B cell repertoire (9). Previously, we found that surgical disruption of GALT–bacterial interactions prevented GALT development, B cell expansion, and somatic diversification of the B cell repertoire (9). In the current paper, we found that the repertoire shift from VHn to VHa B cells in Alicia rabbits also depends on GALT–bacterial interactions. At birth, 10–25% of B cells in peripheral tissues of Alicia rabbits were VHa, and these B cells subsequently declined to nearly undetectable levels by week 3. Although these VHa B cells could represent maternal B cells, we think the percentages are much higher than would be expected for maternal B cells. We also do not think these cells are VHn B cells with maternal VHa Ig bound through Fc receptors because, in this case, we would expect all B cells, rather than a subset, to be VHa. Instead, we think the decline in the percentage of VHa B cells may be due to a dramatic increase in VHn B cells from a second wave of B lymphopoiesis in the bone marrow. We recently identified a burst of both pre–B cells and B cells in bone marrow at 3 wk of age and we suggest that in Alicia rabbits, the newly generated B cells may be primarily VHn (30). The shift from VHn to VHa B cells after 3 wk of age likely occurs in GALT rather than in the bone marrow because the shift requires GALT–bacterial interactions. Therefore, we favor the idea that this shift is due to selective expansion of VHa B cells as proposed by Pospisil et al. (16), who showed that more VHa B cells proliferate and fewer die than VHn B cells in the appendices of Alicia rabbits. We suggest that VHa B cells are preferentially stimulated by interaction with a bacterial ligand or a bacterially induced GALT-derived ligand. Such preferential stimulation of VHa B cells could be due to differences between VHa and VHn B cells in BCR density (31, 32), in localization of BCR in lipid rafts (33), or in BCR structure leading to differential stimulation and subsequent proliferation. We found no difference in surface IgM levels between VHa and VHn B cells, suggesting that differences in BCR density in VHa and VHn B cells do not contribute to the differential stimulation. Although we have not studied the localization of VHa and VHn BCR in lipid rafts, we suggest that VHa and VHn B cells are differentially stimulated by bacteria because of structural differences between the VH regions of VHa and VHn BCR. Differential stimulation of VHa and VHn B cells by bacteria will be investigated in future studies. When we compared amino acid sequences encoded by VHa and VHn gene segments, we found many differences in FR1 and FR3. These differences include VHa2 allotype-associated amino acids, which Pospisil et al. (16, 34) proposed may interact with a ligand, leading to expansion of VHa2 B cells. However, because the VHa2 allotype-associated amino acids are not present in allelically encoded VHa1 and VHa3 allotypes (10), and because VHa1 and VHa3 B cells in a1/a1 and a3/a3 rabbits, respectively, also proliferate in GALT, we suggest that the a2 allotype-associated amino acids are not critical for preferential expansion of VHa B cells. Instead, we suggest that the nonallotype-associated amino acids present in VHa molecules, but absent in VHn molecules, are responsible for preferential expansion of VHa B cells. We examined the amino acid sequences encoded by VHa and VHn gene segments and found six positions in FR1 and FR3 (3, 19, 21, 23, 78, 82A) in which the same amino acids were encoded by all six VH gene segments known to encode VHa molecules, but not by the three VH gene segments known to encode VHn molecules (Fig. 7 a). In addition, we found that, at positions 79 and 82 (FR3), the same amino acids were encoded by five out of six VHa gene segments, but not by the VHn gene segments (Fig. 7 a). If selective expansion of VHa B cells results from interaction of a ligand with VH molecules, the contacting amino acids are likely to be present on the exterior surface of the VH region. By three-dimensional modeling, we found that of these eight amino acids, five (19, 21, 23, 79, 82A) are clustered on the external face of the VH domain with their side chains exposed for potential interaction with a ligand (Fig. 7 b). Two out of the eight amino acids (78 and 82) are nonpolar and, thus, their side chains are not likely to be exposed to solvent. Another conserved amino acid (position 3) is located at a flexible region, making it difficult to predict whether this amino acid will participate in a ligand interaction. We propose that the five amino acids (19, 21, 23, 79, 82A) clustered on the exterior face of the VHa molecules are part of a binding site for a bacterial ligand or a bacterially induced GALT-derived ligand. Closer examination of the putative binding site reveals two additional amino acids (at positions 77 and 81) that may contribute to ligand binding, even though they are present in both VHa and VHn molecules. We propose that a combination of seven VH amino acids at positions 19, 21, 23, 77, 79, 81, and 82A constitutes a ligand binding site and, furthermore, that the ligand interacts more strongly with VHa than with VHn molecules, leading to the differential stimulation and subsequent expansion of VHa B cells. The putative VH ligand binding site is on the exterior surface of the VH region, similar to the VH binding site of Staphylococcus aureus protein A in human VH Ig molecules (35). Protein A binds to and preferentially stimulates B cells that use VH gene segments of the VH3 family (36). Similarly, we think that a putative bacterial B cell superantigen (37) or a bacterially induced GALT-derived superantigen (38) preferentially binds to and stimulates VHa B cells. If a B cell superantigen promotes positive selection of VHa B cells in GALT, the interaction between such a B cell superantigen and the rabbit VH region would be expected to stimulate the B cells in an antigen-nonspecific, polyclonal manner. Consistent with this idea, Sehgal et al. (39) found that the nature of somatic mutation in VDJ genes in the appendix of young rabbits differed from that which occurs in response to specific antigens in the spleen. Furthermore, Casola et al. (40) demonstrated that anti-HEL transgenic mice had normal-sized Peyer's patches, indicating that B cell expansion in GALT is specific-antigen independent. However, we cannot rule out the possibility that the microflora stimulate B cells in a non-BCR–dependent manner, rather than through interaction with the VH region (40). Using IgH-transgenic mice, it has been shown that peritoneal B-1 cells undergo antigen-specific B cell–positive selection (23). Evidence for positive selection of conventional B cells (B-2), whether dependent or independent of specific antigen, is more circumstantial (24). Here, we demonstrated in a nontransgenic model that B cells can be positively selected in the GALT during generation of the primary B cell repertoire, likely in an antigen-independent manner (37, 39). Furthermore, this occurs as a result of interactions between GALT and the intestinal microflora. These data demonstrate the potential for commensal intestinal microflora to shape the B cell repertoire. The extent to which commensal microflora play a role in modifying the B cell repertoire in other species remains to be elucidated. Materials and Methods Rabbits and antiallotype antibodies Ali/ali rabbits (designated Alicia; reference 1), which are homozygous for the b5 -chain allotype (b5/b5), were maintained in the Comparative Medicine Facility at Loyola University Chicago, Maywood, IL. All experiments were performed following the guidelines of the Loyola University Chicago Institutional Animal Care and Use Committee. The anti-b4 and anti-b5 anti– chain allotype antisera were as described previously (41). Anti-VHn antibody directed against VHx and VHy allotypes was produced by immunizing a homozygous a1x–y– (IgH haplotype A/A) rabbit (L76-3) with IgG from a homozygous a2-suppressed a2x32y33 rabbit (42). Ig fractions of the anti-VHn and anti-VHa2 antisera (41) obtained by precipitation with 40% saturated ammonium sulfate were biotinylated for use in immunofluorescence analysis and in Ca2+ mobilization assays. By immunofluorescence, the anti-VHn antibody reacted with <5% of peripheral B cells in adult homozygous a2x32y33 rabbits, as expected (unpublished data). To confirm that the anti-VHn allotype antibodies reacted with VHx and VHy Ig, we analyzed PCR-amplified VDJ genes from FACS-sorted splenic VHn B cells from Alicia rabbits, using a 5' conserved VH leader primer and a 3' primer specific for JH. Nearly all of the VDJ genes (32 out of 34) encoded amino acids characteristic of the VHn molecules encoded by the VHx and VHy gene segments (references 10, 13 and unpublished data). We also analyzed 12 VDJ genes PCR-amplified from splenic B cells that did not react with anti-VHn antibodies and found that, as expected, all 12 genes encoded amino acids characteristic of those encoded by the VHa gene segments VH4, VH7, and VH9 (10, 12). Immunofluorescence and flow cytometry 106 PBLs were prepared from buffy coat and stained with biotinylated rabbit anti-VHn or biotinylated rabbit anti-VHa2 allotype antibodies followed by streptavidin-PE as a secondary reagent (Molecular Probes). CD4+ T cells were stained with FITC-conjugated anti-CD4 mAb (clone KEN4; reference 43). B cells were detected using biotinylated affinity-purified goat anti-IgL chain antibodies and streptavidin-PE or FITC-conjugated anti-IgM mAb (clone 367; reference 3). Cells within the side- and forward-scatter lymphocyte gate were analyzed using a FACSCalibur flow cytometer (BD Biosciences) in the FACS core facility at Loyola University Chicago. PCR analysis to determine rearrangement status of the IgH locus Single VHa B cells were FACS sorted into 96-well V-bottom plates containing 1x lysis buffer as described previously (30). VDJ genes were PCR amplified using nested primers as follows: the 5' primers were 5'-T[G/C]-GATAT[T/G]AAGGG[T/C]ACACA-3' (sense-outside primer) and 5'-CATAAAAATTCA[T/C]ATGATC-3' (sense-inside primer), taken from conserved sequences 5' of VH promoter regions; the 3' primers were 5'-AGTTGAGTAGGAGAGAGA-3' (antisense-outside primer) and 5'-GAGTTGGCAAGGACTCAC-3' (antisense-inside primer), taken from conserved sequences 3' of JH4 (JH4 is used in 80–90% of VDJ gene rearrangements) and JH2. To determine whether rearrangements in the JH region had occurred, nested PCR amplification was performed by using the 5' primers 5'-TGAGTGCTGTTGGACTGGCT-3' (sense-outside primer) and 5'-CAGAGCTGGAGCTGTGCTAT-3' (sense-inside primer), taken from a region 5' of the JH locus; the antisense primers were the same as those used for VDJ gene rearrangements. Development of rabbits with a LigApx The LigApx rabbits were developed as described previously (9). In brief, we removed the sacculus rotundus from newborn rabbits and ligated the lumen of the appendix to prevent bacterial colonization. The vasculature to the appendix was left intact. Peyer's patches were removed at 4 wk of age, when they became macroscopically visible. Cloning and nucleotide sequence analysis of VDJ cDNA VDJ genes were PCR amplified from splenic- and PBL-derived cDNA (44). For the PCR, we used a 5' conserved VH leader primer (VHRPS; reference 45) and a 3' primer specific for exon 1 of Cμ (primer CH1-μ; reference 46). The PCR products were cloned into pGEM-T Easy (Promega), and the nucleotide sequences were determined using an automated ABI Prism 310 sequencer with Big Dye–labeled terminators (PerkinElmer and Applied Biosystems). The VH gene segments used in the VDJ genes were identified by comparing the nucleotide sequences to those of known germline VH gene segments. The germline VH gene segment sequences most similar to those of the VDJ genes were designated as the used genes. All VH gene sequences were submitted to GenBank/EMBL/DDBJ and are available under the following accession nos.: rabbit, no. 32P2 (AY676759, AY676760, AY676761, AY676762, AY676763, AY676764, AY676765, AY676766, AY676767, AY676768, AY676769, AY676770, AY676771, AY676772, AY676773, AY676774, AY676775, AY676776, AY676777, AY676778, AY676779, AY676780, AY676781); no. 144T (AY676782, AY676783, AY676784, AY676785, AY676786, AY676787, AY676788, AY676789, AY676790, AY676791, AY676792, AY676793, AY676794, AY676795, AY676796, AY676797, AY676798, AY676799, AY676800, AY676801, AY676802); no. 94S (12 wk) (AY676803, AY676804, AY676805, AY676806, AY676807, AY676808, AY676809, AY676810, AY676811, AY676812, AY676813, AY676814, AY676815, AY676816, AY676817, AY676818, AY676819, AY676820, AY676821, AY676822, AY676823); no. 199T1 (AY676824, AY676825, AY676826, AY676827, AY676828, AY676829, AY676830, AY676831, AY676832, AY676833, AY676834, AY676835, AY676836, AY676837, AY676838); no. 94S (8 mo) (AY676695, AY676696, AY676697, AY676698, AY676699, AY676700, AY676701, AY676702, AY676703, AY676704, AY676705, AY676706, AY676707, AY676708, AY676709, AY676710); no. 320W2 (AY676711, AY676712, AY676713, AY676714, AY676715, AY676716, AY676717, AY676718, AY676719, AY676720, AY676721, AY676722, AY676723, AY676724, AY676725); no. 127W1 (AY676726, AY676727, AY676728, AY676729, AY676730, AY676731, AY676732, AY676733, AY676734, AY676735, AY676736); no. 199T3 (AY676737, AY676738, AY676739, AY676740, AY676741, AY676742, AY676743, AY676744, AY676745, AY676746, AY676747, AY676748); and no. 127W2 (AY676749, AY676750, AY676751, AY676752, AY676753, AY676754, AY676755, AY676756, AY676757, AY676758). Ca2+ mobilization PBLs isolated with LSMR (ICN Biomedicals) were stained with anti–rabbit T cell mAb (clone KEN5; reference 43) and with biotinylated anti-VHn or anti-VHa allotype antibodies. Secondary reagents were biotinylated Fab goat anti–mouse IgG (Jackson ImmunoResearch Laboratories) and streptavidin-APC (BD Biosciences). The stained cells were suspended in phenol red-free HBSS containing Ca2+ and Mg2+ (GIBCO BRL) and were incubated with rotation for 45 min at room temperature in 10 μM Fura-red, 5 μM Fluo-3 (prepared as 1 mM stocks in 100% DMSO; Molecular Probes), and 2.8 μl 20% pluronic F-127 (Molecular Probes). VHa or VHn B cells were electronically gated as follows: VHn B cells were those cells in the lymphocyte gate that did not react with anti-VHa or anti–T cell antibodies, and the VHa B cells were cells that did not react with anti-VHn or anti–T cell antibodies. The electronically gated VHn and VHa B cells were FACS sorted and, upon reanalysis by FACSCalibur, were shown to be at least 90% pure. The calcium flux of the VHn and VHa B cells in response to anti-b4 and anti-b5 -chain allotype antisera was measured essentially as described previously (47). The fluorescence of Fluo-3 and Fura-red was measured over time, in a linear format. The baseline was determined from data collected 30 s before the addition of antiallotype antibody. The ratio of Fluo-3 to Fura-red and the corresponding mean intracellular calcium ([Ca2+]i) levels were calculated and analyzed using FlowJo software (Tree Star, Inc.). Three-dimensional modeling of rabbit VH domain The crystal structure of a Fab fragment of a human IgM antibody-encoding IgM rheumatoid factor (VH3-30/1.9III; reference 35) was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) and used as a modeling template for the rabbit VH region. Modeling was performed using DeepView/Swiss-PdbViewer v3.7 (http://www.expasy.org/spdbv), and images were rendered using POV-Ray for Windows v3.5 (http://www.povray.org). Acknowledgments We thank Dr. A. Edmunson for help with the three-dimensional analysis of rabbit VH regions and identification of a potential ligand binding site. This work was supported by National Institutes of Health grant no. AI150260. The authors have no conflicting financial interests. Submitted: 7 September 2004 Accepted: 19 November 2004 References Crane, M.A., M. Kingzette, and K.L. Knight. 1996. Evidence for limited B-lymphopoiesis in adult rabbits. J. Exp. Med. 183:2119–2127. Sun, T., M.R. Clark, and U. Storb. 2002. A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling. Immunity. 16:245–255. Kabat, E.A., T.T. Wu, H.M. Perry, K.S. Gottesman, and C. Foeller. 1991. Sequences of Proteins of Immunological Interest, fifth edition. United States Department of Health and Human Services, Washington D.C. 2597 pp....查看详细 (30066字节)
☉ 11120212:Phosphorylation-dependent translocation of sphingo
Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal–regulated kinase 1/2–mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK1 is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK1 to the plasma membrane, rather than from enhanced catalytic activity of this enzyme. B. Wattenberg's present address is James Graham Brown Cancer Center, Louisville, KY 40202. Sphingosine kinases (SKs) catalyze the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1, 2). Some of these cellular processes are mediated by five S1P-specific G protein–coupled receptors, whereas other effects appear controlled by intracellular S1P, through as yet unidentified intracellular targets (2, 3). Two human SK isoforms exist (SK1 and SK2), which differ in their tissue distribution, developmental expression, catalytic properties, and somewhat in their substrate specificity (4, 5). Although these two enzymes appear to have differing roles (6), several studies have shown the effects of SK1 in enhancing cell proliferation and supressing apoptosis (7–9). Furthermore, we have shown that overexpression of human SK (hSK) 1 in NIH3T3 fibroblasts results in acquisition of the transformed phenotype and the ability to form tumors in mice, demonstrating the oncogenic potential of this enzyme (8). Recent work has also demonstrated the involvement of hSK1 in estrogen-dependent regulation of breast tumor cell growth and survival (10, 11), whereas other studies have shown elevated hSK1 mRNA in a variety of human solid tumors and inhibition of tumor growth in vivo by SK inhibitors (12). Thus, the involvement of hSK1 in cell growth, survival, and tumorigenesis is well established. Less clear, however, are the mechanisms whereby hSK1 brings about these effects. Recent studies have indicated it is independent of G protein–coupled receptors (13), being mediated solely by intracellular S1P generated by SK activity. Although the direct targets of intracellular S1P are unknown, hSK1 has been implicated in several proproliferative and prosurvival pathways, such as activation of extracellular signal–regulated kinase (ERK) 1/2 (14), phosphatidylinositol-3-kinase (15), NF-B (16), and inhibition of caspase activation (9). We have shown recently that activation of hSK1 occurs, at least in response to TNF and phorbol esters, as a direct consequence of phosphorylation at Ser225 by ERK1/2 (17). The effects of this single phosphorylation are unusual since it not only directly increases the catalytic activity of hSK1 but is also necessary for agonist-induced translocation of this protein from the cytosol to the plasma membrane. Notably, hSK1 is phosphorylated to a moderate extent when overexpressed in cells even in the absence of external stimuli. In the current study, we report that the ability of overexpressed hSK1 to support enhanced proliferation, survival, and transformation is blocked by mutation of the phosphorylation site. This indicates that it is the phosphorylated form of hSK1 that is responsible for these biological effects. Furthermore, we have shown through constitutive localization of hSK1 to the plasma membrane that these biological effects are brought about through the phosphorylation-dependent translocation of hSK1 to the plasma membrane rather than from the observed phosphorylation-induced increases in hSK1 catalytic activity. Results and Discussion Phosphorylation of hSK1 is required for enhanced cell proliferation and survival Overexpression of wild-type hSK1 is known to significantly enhance cell proliferation and survival (7–11, 13), although the precise molecular mechanisms whereby this occurs are unknown. These effects are dependent on the catalytic activity of hSK1 being blocked by the SK inhibitor, N,N-dimethylsphingosine (7–9), and appear independent of G protein–coupled S1P receptors (7, 13). Thus, the enhanced growth and survival appear due to the action of S1P on as yet unidentified intracellular targets. To date, it has been considered that an increase in total S1P levels in the cell, as a consequence of the high intrinsic catalytic activity of the overexpressed hSK1 (4), was sufficient to induce these biological effects (7–11). However, we have noted that even without external stimulation, overexpressed hSK1 is phosphorylated to a moderate extent (Fig. 1 and reference 17). Therefore, we tested whether this phosphorylation was important for the biological consequences of hSK1 overexpression. To test whether phosphorylation of hSK1 is important in proliferation and survival, we examined the ability of the nonphosphorylatable hSK1S225A mutant to promote these processes. Consistent with our previous findings (8), overexpression of wild-type hSK1 not only markedly enhanced the growth of NIH3T3 cells in media containing either 1 or 5% serum but also conferred to these cells the ability to survive and grow in the absence of serum (Fig. 1, A–C). In contrast, however, cells overexpressing hSK1S225A displayed no such enhanced growth or serum independence (Fig. 1, A–C). Notably both wild type and hSK1S225A were expressed at similar level, and extracts of the cells possessed comparable overall SK activities (Fig. 1 D). Previous studies have shown that the increased growth rates from overexpression of wild-type hSK1 result from a combination of both increased cellular proliferation and reduced apoptosis (7, 9, 13). Consistent with these studies, assays for cellular proliferation by bromodeoxyuridine (BrdU) incorporation into nascent DNA showed overexpression of wild-type hSK1 had a significant effect in increasing cell proliferation (Fig. 1 E). In contrast, however, cells overexpressing hSK1S225A displayed no such enhanced proliferation, showing comparable incorporation of BrdU as control cells (Fig. 1 E). Similarly, overexpression of wild-type hSK1 dramatically reduced serum deprivation–induced apoptosis, as measured by condensation and fragmentation of nuclei (Fig. 1 F). Again, however, overexpression of hSK1S225A showed markedly different results, providing cells with no such protection against apoptosis (Fig. 1 F). Therefore, in stark contrast to wild-type hSK1 overexpression of the nonphosphorylatable hSK1S225A mutant neither increases proliferation, nor protects against apoptosis despite both transfected proteins generating similar cellular SK activities. Thus, phosphorylation of hSK1 imparts a qualitative change in the enzyme that is critical for the signaling processes leading to enhanced proliferation and survival. It has been well established that hSK1 translocates from the cytosol to the plasma membrane upon exposure of cells to certain agonists (17, 18–21). Although the molecular mechanism whereby translocation of hSK1 occurs is unknown, we have shown recently that it is dependent on phosphorylation of hSK1 at Ser225 (17). Therefore, it was important to test whether translocation was the phosphorylation-dependent process required for hSK1 signaling. We predicted that if this was true, then artificially directing hSK1S225A to the plasma membrane would restore its ability to promote proliferation and transformation. Plasma membrane–localized hSK1 proteins were created through addition of the 10–amino acid Lck tyrosine kinase myristoylation/palmitylation motif to the N terminus of wild-type hSK1 and hSK1S225A, generating mp-hSK1 and mp-hSK1S225A, respectively. Overexpression of these proteins in NIH3T3 cells produced slightly lower cellular SK activities than that observed with overexpression of hSK1 and hSK1S225A (Fig. 1 D). Consistent with previous studies that have shown this motif is sufficient to target proteins to the plasma membrane (22), we observed a substantial localization of mp-hSK1 and mp-hSK1S225A to the membrane fraction (Fig. 2 A). Further immunofluorescence analysis (Fig. 2 B) showed clear localization of mp-hSK1 and mp-hSK1S225A to the plasma membrane, whereas hSK1 and hSK1S225A were mainly present in the cytoplasm. Interestingly, localization of wild-type hSK1 to the plasma membrane in this manner substantially reduced its basal phosphorylation (Fig. 1 D). This is presumably due to partial sequestration of hSK1 away from cytosolic ERK1/2, its upstream activating protein kinases (17). Strikingly, and in stark contrast to hSK1S225A, overexpression of mp-hSK1S225A in NIH3T3 cells conferred an enhancement of growth and survival in serum-deprived conditions (Fig. 1, A–C). As expected, mp-hSK1 also had these effects (Fig. 1, A–C). Further examination of these cells showed that, like wild-type hSK1, both mp-hSK1 and mp-hSK1S225A increased cell growth through enhancing cellular proliferation and reducing serum deprivation–induced apoptosis (Fig. 1, E and F). Similar results were also observed in HEK293 cells (not depicted). Therefore, localization of hSK1 to the plasma membrane is sufficient to enhance cellular proliferation and protect against apoptosis irrespective of the phosphorylation status of the enzyme. This strongly suggests that phosphorylation of hSK1 mediates these observed biological effects through inducing translocation of hSK1 to the plasma membrane rather than as a result of the associated increase in catalytic activity. Phosphorylation-induced plasma membrane localization of hSK1 mediates oncogenic signaling Since we had established that Ser225 phosphorylation of hSK1 was essential for its effects in enhancing cell proliferation and survival, we further investigated its effects on cell transformation. As described previously (8), wild-type hSK1 exhibited considerable transforming activity when overexpressed in NIH3T3 cells, as assayed by colony formation in soft agar (Fig. 3). In contrast, however, overexpression of similar levels and catalytic activity of hSK1S225A resulted in remarkably less transformation of these cells (Fig. 3). Notably, these cells expressing hSK1S225A had considerably higher SK activity than what we have previously shown necessary for transformation of NIH3T3 cells by wild-type hSK1 (8). Therefore, like the situation for enhanced proliferation and survival, these experiments demonstrate that it is not simply elevated levels of SK activity that are responsible for cell transformation but instead indicate that another aspect of the phosphorylated, activated state of the protein is responsible for these effects. We have demonstrated previously that transformation of NIH3T3 cells by oncogenic H-Ras (V12-Ras) is partially blocked by a catalytically inactive, dominant-negative form of hSK1, indicating that hSK1 is critically involved in Ras-induced cell transformation (8). Strikingly, here we find that the nonphosphorylatable hSK1S225A also reduced Ras-induced cell transformation, further confirming the requirement of hSK1 activation in this pathway (Fig. 3). Therefore, in this context hSK1S225A is apparently acting as a dominant-negative form of the protein, despite possessing full catalytic activity. Since plasma membrane localisation of hSK1 mediated the enhanced proliferation and survival resulting from hSK1 overexpression, we next examined its effects on cell transformation. Like wild-type hSK1, the overexpression of both mp-hSK1 and mp-hSK1S225A in NIH3T3 cells resulted in the formation of vigorous colonies in soft agar (Fig. 3). Although some background colonies where observed in empty vector control cells, mp-hSK1 and mp-hSK1S225A–overexpressing cells produced 20–30-fold greater colonies which were considerably larger in size. Indeed, the colonies generated by mp-hSK1 and mp-hSK1S225A overexpression were also larger and more numerous than those observed in cells overexpressing wild-type hSK1 (Fig. 3). Thus, like the situation for enhanced proliferation and survival, localization of hSK1 to the plasma membrane is sufficient to enhance cell transformation irrespective of the phosphorylation status of the enzyme. To examine the oncogenic effects of hSK1 phosphorylation and translocation in animals, NIH3T3 cells stably expressing the various forms of hSK1 were subcutaneously injected into NOD/SCID mice. Unlike wild-type hSK1-transfected cells, which we have previously reported generate tumors in these mice (8), no tumors formed in six mice injected with cells expressing hSK1S225A. In contrast, and consistent with the in vitro experiments, tumors were observed in mice injected with cells expressing mp-hSK1 (five out of six) and mp-hSK1S225A (four out of six). Phosphorylation and plasma membrane localization of hSK1 enhances S1P generation Sphingosine, the substrate of hSK1, is concentrated in the plasma membrane (23). Therefore, one possible mechanism for the observed dramatic biological effects of hSK1 localization to the plasma membrane is in enhancing S1P generation by directing hSK1 to its substrate. Therefore, we measured the effect of plasma membrane translocation of hSK1 on levels of its product, S1P. Overexpression of both mp-hSK1 and mp-hSK1S225A resulted in similar increases in intracellular S1P and enhanced S1P release into the media. These increases were substantially greater than that observed with either wild-type hSK1 or hSK1S225A (Fig. 4). These findings are consistent with our previous reports that Ser225 phosphorylation of hSK1 results in increased cellular S1P and enhanced S1P release from cells (17). This, again, suggests that phosphorylation-induced translocation of hSK1 to the plasma membrane is critical for enhanced S1P generation and the observed subsequent biological effects. Conclusions and implications of this study The involvement of hSK1 in tumorigenesis is becoming increasingly evident, with several studies suggesting that elevated hSK1 levels may be important in this process (8–12). In this study, however, we have shown that rather than total hSK1 levels, phosphorylation and subsequent translocation of hSK1 to the plasma membrane are the critical factors involved in the oncogenic effects of this protein. We have proposed previously that the substantial basal activity of hSK1 may reflect an essential housekeeping role that is distinct from its signaling role (4, 14, 17). Agonist-dependent translocation may accomplish the differentiation of these two roles of hSK1. Thus, our studies indicate that disregulation of hSK1 phosphorylation and localization may be key elements in the acquisition of malignant phenotypes through provoking the enhancement of proliferation and, perhaps most importantly, protection from the apoptotic mechanisms that normally target mutated cells for destruction. This suggests that targeting the mechanisms driving hSK1 phosphorylation and/or translocation may provide precise avenues for future anticancer therapies. Materials and Methods Cell culture, transfection, and cell fractionation. Human embryonic kidney (HEK293T) cells and NIH3T3 fibroblasts were cultured and harvested as described previously (14). Stable and transient transfections were performed using the calcium phosphate precipitation method for HEK293T cells and Lipofectamine 2000 (Invitrogen) for NIH3T3 cells. Stable transfectants were selected for G418 resistance and pooled to avoid the phenotypic artifacts that may arise from the selection and propagation of individual clones from single transfected cells. For subcellular fractionation, postnuclear supernatants of cell lysates were separated into cytosol and membrane fractions as described previously (17). Antibodies. The M2 anti-FLAG antibody was from Sigma-Aldrich, anti–H-Ras polyclonal antibody was from Santa Cruz Biotechnology, and HRP-conjugated anti–mouse and anti–rabbit IgG were from Pierce Chemical Co. Anti-hSK1 and anti–phospho-hSK1 antibodies have been described previously (17). SK assays and S1P levels. SK activity was determined as previously described (14). S1P levels were determined following metabolic labeling of cells with [32P]orthophosphate as outlined previously (17). Generation of plasma membrane–localized hSK1 constructs. A plasma membrane–targeted hSK1 construct (mp-hSK1) containing the myristoylation/palmitylation motif of the Lck tyrosine kinase (MGCGCSSHPE) (22) was generated by PCR with oligonucleotide primers 5'-TAGAATTCGCCACCATGGGCTGTGGCTGCAGCTCACACCCGGAAGATCCAGCGGGCGGCC-3' and SP6 using pcDNA3-hSK1 (4) as template DNA. The resultant product was then cloned into pcDNA3 (Invitrogen) by digestion with EcoRI. The orientation was determined by restriction analysis, and sequencing verified the integrity of the FLAG-tagged mp-hSK1 cDNA sequence. To generate FLAG-tagged mp-hSK1S225A, the KpnI–PmlI fragment representing the wild-type 5' end of pcDNA3-hSK1S225A (17) was replaced with the 150-bp KpnI–PmlI pcDNA3-mp-hSK1 DNA fragment that contains the Lck myristoylation/palmitylation motif. Immunofluorescence. Cells were plated onto fibronectin-coated 8-well glass chamber slides at 104 cells/well and incubated for 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.1% Triton X-100 in PBS, and incubated with M2 anti-FLAG antibody in PBS containing 3% BSA and 0.1% Triton X-100 for 1 h. The immunocomplexes were then detected with FITC-conjugated anti–mouse IgG. Fluorescence microscopy was performed on an Olympus BX-51 microscope equipped with a fluorescein excitation filter (494 nm) acquired to a Cool Snap FX charge-coupled device camera (Photometrics). Cell growth, bromodeoxyuridine incorporation, staining of apoptotic nuclei, and tumorigenesis assays. Assays for cell growth were performed by incubating cells in 48-well plates (2,500 cells per well) in medium containing 5 or 1% FCS, or serum free medium (containing 0.1% BSA) as described previously (8). Cell numbers were determined at the indicated times using the MTS assay (Promega). BrdU incorporation into nascent DNA were used as a measure of cell proliferation. Cells were plated onto 8-well glass chamber slides (Nalge Nunc) coated with fibronectin at 104 cells/well and grown for 24 h in DMEM with 2% FCS. Cells were then incubated with 10 μM BrdU for 3 h, and then fixed and stained for its incorporation using an anti–BrdU-FLUOS antibody (Roche) following the manufacturer's protocol. Cells positive for BrdU incorporation were visualized with an Olympus BX-51 fluorescence microscope with at least 300 cells scored per point. Apoptosis was assessed by staining cells with 1 μg/ml DAPI in methanol for 15 min at room temperature. Apoptotic cells were identified by condensation and fragmentation of nuclei using fluorescence microscopy and expressed as a percentage of the total cells counted. A minimum of 300 cells were scored per point. Colony formation in soft agar and tumorigenesis assays in mice were performed as detailed previously (8) with approval from the Institute of Medical and Veterinary Science Animal Ethics Committee. Acknowledgments This work was supported by a R. Douglas Wright Biomedical Research Fellowship and a grant from the National Health and Medical Research Council of Australia to S.M. Pitson. The authors have no conflicting financial interests. Submitted: 23 March 2004 Accepted: 22 November 2004 References Johnson, K.R., K.P. Becker, M.M. Facchinetti, Y.A. Hannun, and L.M. Obeid. 2002. PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA). J. Biol. Chem. 277:35257–35262. Melendez, A.J., and A.K. Khaw. 2002. Dichotomy of Ca2+ signals triggered by different phospholipid pathways in antigen stimulation of human mast cells. J. Biol. Chem. 277:17255–17262. Young, K.W., J.M. Willets, M.J. Parkinson, P. Bartlett, S. Spiegel, S.R. Nahorski, and R.A.J. Challiss. 2003. Ca2+/calmodulin-dependent translocation of sphingosine kinase: role in plasma membrane translocation but not activation. Cell Calcium. 33:119–128. Zlatkine, P., B. Mehul, and A.I. Magee. 1997. Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J. Cell Sci. 110:673–679. Slife, C.W., E. Wang, R. Hunter, S. Wang, C. Burgess, D.C. Liotta, and A.H. Merrill. 1989. Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum. J. Biol. Chem. 264:10371–10377....查看详细 (21901字节)

☉ 11120213:Ro/SSA autoantibodies directly bind cardiomyocytes
Many autoimmune conditions are associated with increased risk of pregnancy complications and fetal loss. Complete congenital atrioventricular (AV) heart block develops in the fetus in 2–5% of Ro/SSA autoantibody-positive pregnancies of rheumatic women, usually between 18 and 24 wk of gestation (1, 2). Initiated as a first-degree AV block (3), the condition progresses to a complete third-degree AV block after mononuclear cell infiltration, fibrosis, and calcification of the cardiac tissue (4, 5). The Ro/SSA antigen is intracellular and contains Ro52 and Ro60 protein components to which autoantibodies are induced in the mother (6). Systematic analyses have been undertaken to identify the subpopulation and specificity of Ro/SSA antibodies that correlate with congenital heart block (7–9). Recent studies indicate that antibodies recognizing the Ro52 protein of the Ro/SSA complex are pathogenic (3, 9), and more specifically, our studies have demonstrated that antibodies to amino acids 200–239 (p200) of the Ro52 protein were detected in the mothers of children with complete heart block (9). However, the fine specificity and the mechanism by which p200-specific antibodies mediate heart block have not been elucidated. We and others have shown that early treatment of an incomplete block with high dose fluorinated steroids prevents progression of, or even reverts, the block, decreasing fetal morbidity and mortality (3, 10, 11). However, a complete third-degree block is permanent (11), making it relevant also from a clinical point of view to define the specific antibody-mediating heart block. A marker with high predictability could identify high risk pregnancies and allow initiation of treatment at the critical stage to prevent irreversible heart block in the fetus. In this paper, we show that not all, but Ro52 autoantibodies with a particular specificity for the p200 sequence of the Ro52 protein correlate with AV time prolongation in the fetus, bind the surface of cardiomyocytes, and induce Ca2+ dysregulation and ultimately apoptosis in affected cells. Results AND Discussion Maternal anti-p200 antibody levels correlate with neonatal AV conduction time To evaluate the role of Ro52 antibodies in development of congenital heart block, we followed 25 pregnant Ro52 autoantibody-positive women prospectively with weekly fetal echocardiographic examinations between 18 and 24 wk of gestation. Maternal autoantibodies to different parts of the Ro52 protein (Fig. 1 A) were investigated by ELISA. Fetal AV time was defined using two different Doppler techniques (Fig. 1, B and C), and development of heart block was correlated with antibody specificity. 9 of the 25 (36%) fetuses had signs of first-degree AV block by both methods. One of these nine developed a second-degree and another a complete AV block (Videos 1 and 2, available at http://www.jem.org/cgi/content/full/jem.20041859/DC1). We found a significant correlation between prolongation of AV time and levels of antibodies to amino acids 200–239 (p200) of Ro52 (P 95% confidence interval limits for normal fetuses. The method is described in detail in the Supplemental Materials and Methods section, which is available at http://www.jem.org/cgi/content/full/jem.20041859/DC1. Immunization and ECG recordings in rats 6-wk-old female DA rats (B&K) were immunized with p200 or virally derived control JB4 peptide (amino acid sequence GIWGCSGKLICTTAVPWNAS; reference 15). Rats were mated 2–4 wk after the last booster. The Stockholm North Ethics Committee approved the study. On the day of delivery, three lead ECGs were recorded from conscious pups using four silver microelectrodes attached to a body clip (16). The ECG was digitalized and files were recorded for at least 3 min for each pup and analyzed with Pharmlab (AstraZeneca). AV block I was defined as PR intervals in control animals +2 SD. Expression of scFv antibodies Expression and purification of scFv antibody fragments were performed as described previously (12). The purified antibodies were dialyzed against several changes of PBS and filtered for sterility before use. Preparation of primary cardiomyocyte cell cultures from rat pups Cultures of cardiomyocytes were prepared using a kit (Worthington Biochemical Corporation). Hearts from 1-d-old DA rats were dissected and prepared according to the manufacturer's instructions. The cardiomyocytes were cultured in DMEM/F12 supplemented with 10% FCS, 1 μg/ml gentamicin, 2.5 μg/ml insulin, 2.5 μg/ml transferrin, 2.5 ng/ml selenin, 30 μg/ml BrdU, and 15 mM Hepes at 37°C with 5% CO2. Immunohistochemical staining For cell surface staining, cardiomyocytes were cultured for 4–5 d on glass slides coated with collagen type I (BD Biosciences). After this, all steps until fixation were performed at 4°C. Slides were incubated with S3A8 or M4H1 antibody, followed by anti-VSV (1:2,500; Boehringer) and TRITC-conjugated goat anti–mouse antibodies (Jackson ImmunoResearch Laboratories). Cells were fixed in 4% paraformaldehyde and stained with Hoechst 33258 (Farbwerke Hoechst) before analysis in a confocal microscope (Eclipse TE300; Nikon). Caspase-3 and TUNEL stainings were performed on cells fixed in 2% formaldehyde with a polyclonal rabbit anti–caspase-3 antibody (0.3 μg/ml; AF835; R&D Systems) and a cell death detection kit (POD; Roche). Calcium level measurements Cardiomyocytes prepared as described above were cultured on polylysine-covered glass slips (VWR) for 5 d. Cells were loaded with the Ca2+ indicator fluo-4 acetoxymethylester (fluo-4 a.m.; Molecular Probes) by incubation for 50 min at 37°C, 5% CO2, in conditioned medium containing 2 mM fluo-4 a.m. mixed with pluronic acid (final concentration: 0.2%). This was followed by 20 min of deesterification before measurements began. The coverslips were mounted in a chamber with conditioned medium (37°C) and analyzed with an inverted confocal microscope (TCS SP; Leica). 20 min of consecutive images collected every 1.705 s (and in some cases every 0.7 s) were recorded for each experiment. Cells were returned to the incubator and reexamined 60 min and 24 h after drug application. Images were processed with the ImageJ software (NIH) and imported into Microcal Origin 7.5 (Originlab.com) for further analysis. Statistical analysis The Mann-Whitney U test was used to compare autoantibody levels between pregnant women with and without fetal AV block. A p-value of <0.05 was considered significant. Online supplemental material Video 1 illustrates echocardiographic recordings from a fetal heart with signs of first degree AV block, and in Video 2, the complete third-degree AV block after progression in the same patient is shown. Fig. S1 contains explanatory anatomical labels for Video 1 and 2. Video 3 contains a film recorded in the confocal microscope of flou-4–loaded cardiomyocytes before and after the addition of S3A8 antibody, and in Fig. S2 a single cell tracing of [Ca2+]i from this experiment is shown. Table S1 contains information on the patients. Videos 1–3, Figs. S1 and S2, and Table S1 are available at http://www.jem.org/cgi/content/full/jem. 20041859/DC1. Acknowledgments We gratefully acknowledge Ms. Gabriella Dombos for skillful technical assistance and Dr. Pierre Eftekhari for help with ECG registration equipment. The study was supported by the Swedish Research Council, the Heart-Lung Foundation, and the Swedish Rheumatism Association. The authors have no conflicting financial interests. Submitted: 8 September 2004 Accepted: 19 November 2004 References Andelfinger, G., J.C. Fouron, S.E. Sonesson, and F. Proulx. 2001. Reference values for time intervals between atrial and ventricular contractions of the fetal heart measured by two Doppler techniques. Am. J. Cardiol. 88:1433–1436 Chiodi, F., T. Mathiesen, J. Albert, E. Parks, E. Norrby, and B. Wahren. 1989. IgG subclass responses to a transmembrane protein (gp41) peptide in HIV infection. J. Immunol. 142:3809–3814Eftekhari, P., J.C. Roegel, F. Lezoualc'h, R. Fischmeister, J.L. Imbs, and J. Hoebeke. 2001. Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor. Eur. J. Immunol. 31:573–579 Related Article Heart-stopping antibodies Heather L. Van Epps J. Exp. Med. 2005 201: 2. [Full Text]...查看详细 (18784字节)
☉ 11120214:The Plasmodium circumsporozoite protein is proteol
Malaria infection is initiated when an infected Anopheline mosquito injects sporozoites during a blood meal. After injection, sporozoites enter the bloodstream and go to the liver, where they invade hepatocytes and develop into exoerythrocytic forms. The circumsporozoite protein (CSP) is the major surface protein of the sporozoite and forms a dense coat on the parasite's surface. Studies have shown that CSP mediates sporozoite adhesion to target cells (for review see reference 1) and that it is required for sporozoite development in the mosquito (2). In addition, CSP has been extensively studied as a vaccine candidate and, thus far, is the only Plasmodium protein shown to confer protection to immunized individuals (for review see reference 1). Comparison of the deduced amino acid sequences of CS proteins from all species of Plasmodium shows that they have a similar overall structure (see Fig. 1 A and reference 1). They all contain a central repeat region whose amino acid sequence is species specific and two conserved regions: a five amino acid sequence called region I, immediately before the repeats, and a known cell-adhesive sequence with similarity to the type I thrombospondin repeat (TSR; reference 3). CSP has a canonical glycosylphosphatidyl inositol (GPI) anchor addition sequence in its COOH terminus; however, the presence of a GPI anchor has not been demonstrated. It was noted 20 yr ago that CSP immunoprecipitated from sporozoite lysates consists of one to two high MW bands (that differ by 1 kD) and a low MW band that is 8–10 kD smaller (4, 5). Biosynthetic studies showed that the initial label is incorporated into the top bands and the lower MW band appears later as a processed product (4, 5). The precise nature of this processing, as well as its functional significance, have remained unknown. In this report, we have determined the structural basis for this conserved feature of CSPs and have explored its role during sporozoite invasion of hepatocytes. Results and Discussion The NH2-terminal portion of CSP is proteolytically cleaved by a cysteine protease To study the structure of the high and low MW CSP forms, we made polyclonal antisera to peptides representing the entire NH2-terminal and COOH-terminal thirds of CSP from Plasmodium berghei, a rodent malaria parasite. These antisera recognized the appropriate full-length peptides (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20040989/DC1) and did not recognize peptides representing the central repeat domain (Fig. S1 B). In addition, the NH2-terminal antiserum did not recognize the COOH-terminal peptide and the COOH-terminal antiserum did not recognize the NH2-terminal peptide (unpublished data). Western blot analysis of a P. berghei sporozoite lysate showed that the NH2-terminal antiserum recognized only the high MW CSP form, indicating that all or part of the NH2 terminus is proteolytically cleaved to generate the low MW CSP form (Fig. 1 B). In contrast, mAb 3D11 (which recognizes the repeat region) and the COOH-terminal antiserum recognized both CSP forms. To determine what class of protease was responsible for cleavage, we performed pulse-chase metabolic labeling experiments in the presence of different protease inhibitors. We labeled sporozoites with [35S]Cys/Met and chased with cold medium containing the indicated inhibitor (Fig. 1 C). In the absence of protease inhibitors, 80% of labeled CSP was cleaved after 2 h. In the presence of the metalloprotease inhibitor 1,10 phenanthroline or the aspartyl-protease inhibitor pepstatin, there was no effect on CSP processing. In addition, EDTA had no effect on CSP processing, indicating that divalent cations are not required. L-transepoxysuccinyl-leucylamide-[4-guanido]-butane (E-64), a highly specific cysteine protease inhibitor, and PMSF, a serine protease inhibitor, inhibited CSP processing. Leupeptin and TLCK, inhibitors of both cysteine and serine proteases, also inhibited processing. Although PMSF has been reported to have inhibitory activity against some papain family cysteine proteases (6), it is a prototypical serine protease inhibitor. To further examine the role of serine proteases, we assayed two other serine protease inhibitors, aprotinin and 3,4 dichloroisocoumarin (3,4 DCI). Aprotinin inhibits most classes of serine proteases and would be predicted to inhibit the serine proteases of Plasmodium, which are subtilisin-like (7). 3,4 DCI is a serine protease inhibitor that has some activity against cysteine proteases but does not react with papain-like cysteine proteases (8). Neither compound had an effect on CSP processing. We also performed pulse-chase metabolic labeling experiments with the human malaria parasite, Plasmodium falciparum, and found that E-64 inhibited CSP processing in this species (Fig. 1 D). These data suggest that CSP cleavage occurs by a similar mechanism in both rodent and human Plasmodium species. To ensure that the protease inhibitors were not toxic to sporozoites, we incubated sporozoites with the different inhibitors and added propidium iodide, a dye that is excluded by viable cells but penetrates membranes of dying cells. The percentage of sporozoites that took up the dye in the presence of any of the protease inhibitors was no different from controls (unpublished data). In addition, we tested whether sporozoites incubated with protease inhibitors were less metabolically active. Analysis of CSP synthesis after sporozoites had been incubated with individual inhibitors for 2 h showed that it was not affected by E-64, leupeptin, or PMSF (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20040989/DC1). Our data suggest that the processing enzyme is a cysteine protease. The cysteine proteases found in parasites are members of two clans, CA (papain-like) and CD (legumin-like) (for review see reference 9), which can be distinguished by their sensitivity to E-64. The protease that cleaves CSP is inhibited by E-64 and, therefore, is a Clan CA, papain family cysteine protease. However, we found that PMSF, a serine protease inhibitor, also inhibited processing. As stated before, PMSF has been reported to have activity against papain family cysteine proteases and this could explain its inhibitory activity in our processing assay. Nonetheless, it is also possible that CSP cleavage is a complex multistep process involving distinct proteases. Region I likely contains the cleavage site To determine where CSP is cleaved, we mapped the epitopes recognized by the NH2-terminal antiserum using overlapping peptides. As shown in Fig. 2 A, the NH2-terminal antiserum recognized peptides interspersed throughout the NH2-terminal third of the protein, suggesting that the processed form lacks this entire region. These data raised the intriguing possibility that region I, found at the end of the NH2 terminus, contained the cleavage site. To test this, we used a recombinant P. berghei parasite in which the last 21 amino acids of the NH2 terminus and the entire repeat region had been replaced by the orthologous region from P. falciparum CSP [Pf/Pb sporozoites; Fig. 2 B and reference 10). A Western blot of Pf/Pb sporozoites shows that both CSP forms are present, suggesting it is processed (Fig. 2 C). We performed pulse-chase metabolic labeling experiments with Pf/Pb sporozoites and found that after a 4-h chase, 50–80% of the high MW CSP is processed to the low MW form (unpublished data). When we tested whether E-64 could inhibit processing of the hybrid CSP, we found that it did (Fig. 2 D), indicating that the same protease cleaves both the native and hybrid CS proteins. These data suggest that the cleavage site is found within region I because this sequence remains unchanged in the hybrid protein. Although it is possible that the cleavage site is outside of the swapped region, this is unlikely because the NH2-terminal antiserum, which recognized peptides throughout the NH2-terminal third of CSP, did not recognize the low MW CSP form. Previous studies have shown that the difference in size, by SDS-PAGE, between the high and low MW forms is 8–10 kD (4, 5, 11–13). The NH2-terminal portion of CSP, beginning after the signal sequence and ending just before the repeat region, is predicted to be this size. CSP cleavage occurs extracellularly by a sporozoite protease We investigated the cellular location of CSP processing. Immunofluorescence experiments with live sporozoites showed that they were recognized by the NH2-terminal antiserum, demonstrating that full-length CSP was on the surface (Fig. 3 A). To confirm this, we biotinylated sporozoites expressing GFP with a reagent that does not enter cells. As shown in Fig. 3 B, the high MW CSP form is biotinylated, indicating that it is on the surface. As a control, we immunoprecipitated GFP, an intracellular protein, and found that it was not labeled (Fig. 3 C). These findings are in agreement with a previous paper that showed that high MW CSP was on the surface of Plasmodium vivax sporozoites (12) and suggest that processing occurs on the sporozoite surface. In contrast with our findings, other investigators found that the majority of CSP on the surface was the low MW form, and concluded that processing occurred intracellularly (4, 5). In these studies, CSP was immunoprecipitated from sporozoites that were metabolically labeled and trypsinized. When compared with controls, trypsin-treated sporozoites were primarily missing the low MW CS band, indicating that the high MW CSP form was intracellular. However, in these experiments, trypsin was added immediately after labeling, which may not have allowed sufficient time for export of all the labeled CSP to the sporozoite surface. To investigate whether this was the case, we repeated this experiment and incorporated a chase into the experimental design. Sporozoites were metabolically labeled and kept on ice or chased in the presence of cyclohexamide to prevent further protein synthesis. Next, they were treated with pronase or pronase plus an inhibitor cocktail. As shown in Fig. 3 D, if the parasites were kept on ice after labeling, the high MW CSP was not digested by pronase. However, if sporozoites were chased before pronase treatment, both CSP forms were digested, indicating that both forms were found on the sporozoite's surface, making this the likely location of processing. Sporozoites isolated from salivary glands of infected mosquitoes are invariably contaminated with mosquito debris, raising the possibility that the protease that cleaves CSP is of mosquito origin. To address this question, we dissected and purified sporozoites in the presence of E-64, and then metabolically labeled them in medium without E-64. Cysteine proteases of mosquito origin would be extracellular and, therefore, irreversibly inhibited by the E-64 present during sporozoite isolation. However, we found that CSP was processed with the same kinetics regardless of whether sporozoites were purified in the presence or absence of E-64. These data suggest that the protease was synthesized (or secreted) after the removal of E-64 and, therefore, was of sporozoite origin (Fig. 3 E). CSP cleavage is required for cell invasion Proteolytic cleavage of cell surface and secreted proteins occurs during invasion of erythrocytes by the merozoite stage of Plasmodium (for review see reference 14). To determine whether CSP cleavage was required for sporozoite entry into cells, a variety of protease inhibitors were tested for their ability to inhibit sporozoite invasion of a hepatocyte cell line. As shown in Fig. 4 A, E-64 inhibited invasion by 90% and PMSF and leupeptin also had inhibitory activity. Pepstatin had no effect on invasion and the serine protease inhibitors aprotinin and DCI, which do not have activity against the papain family cysteine proteases, also did not have inhibitory activity on invasion. Importantly, pretreatment of target cells with E-64 had no inhibitory effect on sporozoite invasion. The ability of E-64 to inhibit invasion was not restricted to P. berghei sporozoites, as invasion by both P. yoelii and P. falciparum sporozoites was also inhibited by E-64. Notably, the number of extracellular sporozoites was always enhanced in the presence of E-64, suggesting that there was an accumulation of attached sporozoites that were prevented from entering (Fig. 4 B). Because attachment to cells is a distinct stage of sporozoite invasion (15), these results suggest that E-64 specifically blocks invasion and that attachment to cells does not require proteolytic cleavage of CSP. These data suggest that CSP is cleaved during cell invasion. Therefore, we predicted that intracellular sporozoites would lose their reactivity to the NH2-terminal antiserum, which recognizes only full-length CSP. However, we found that the majority of sporozoites associated with cells lost their reactivity to the NH2-terminal antiserum regardless of whether they were intracellular or extracellular (unpublished data). In the absence of cells, 80–90% of sporozoites stained with this antiserum (unpublished data), suggesting that cell contact was the trigger for CSP cleavage. To test this, sporozoites were preincubated with cytochalasin D (CD), an inhibitor of sporozoite invasion but not attachment to cells (15), in the presence or absence of E-64 and added to cells. As shown in Table I, sporozoites incubated with CD plus E-64 stained with the NH2-terminal antiserum, whereas those incubated with CD alone did not. mAb 3D11, directed against the repeat region of CSP, bound to both E-64–treated and untreated sporozoites. Controls in which sporozoites were incubated without cells showed that neither elevated temperature nor serum alone had a significant effect on CSP cleavage (Table I). In this assay, sporozoites were incubated with cells for only 2 min before being fixed and stained. The rapid loss of reactivity to the NH2-terminal antiserum indicates that there is a dramatic increase in the kinetics of CSP cleavage when parasites are added to cells. In the absence of cells, the half life of newly synthesized CSP is 1 h (Fig. 1 and references 4, 5). These data indicate that the secretion of the protease that cleaves CSP is regulated. It is likely that the low level cleavage observed in the absence of cells is due to leaky secretion from apical organelles, whereas exocytosis of larger amounts of protease is mediated by specific signals that are transduced upon contact with target cells. CSP cleavage is not required for migration through cells It has been shown that sporozoites interact with cells in two distinct ways: they either rupture the plasma membrane and migrate through a cell or they enter with a vacuole and productively invade the cell (16). To study whether CSP processing was preferentially associated with one of these processes, we tested whether E-64 inhibited sporozoite migration through cells. Migration can be quantified by including a high MW fluorescent tracer in the medium because it will enter cells that are wounded by sporozoites as they pass through. As shown in Fig. 4 C, E-64 had no effect on sporozoite migration through cells. These data indicate that CSP cleavage is associated with productive invasion of cells and suggests that sporozoites differentially recognize cells that they will invade; a finding that makes sense given that, in vivo, they travel through several cell barriers to reach their target, the hepatocyte. One question raised by these findings is how do sporozoites recognize hepatocytes? Previous work has shown that CSP binds to heparan sulfate proteoglycans (HSPGs) found on hepatocytes, making these molecules likely candidates for target cell recognition (for review see reference 1). We are currently investigating whether binding of CSP to HSPGs triggers cleavage and initiates the cascade of events leading to productive invasion of cells. Inhibition of cysteine proteases prevents malaria infection Lastly, we tested E-64 as an inhibitor of malaria infection in vivo using a rodent model of the disease. Using a quantitative PCR assay, we compared the amounts of parasite rRNA in the livers of mice pretreated with E-64 or buffer and infected with Plasmodium sporozoites. We found that mice injected with E-64 were completely protected from malaria infection (Fig. 4 D). Although inhibitors of cysteine and serine proteases have not yet been used for the treatment of human disease, animal studies have shown the feasibility of using these inhibitors as drugs in the treatment of parasitic infections (for review see references 17, 18). Our finding that we can completely prevent malaria infection by targeting the cysteine proteases of the sporozoite stage could lead to the development of new prophylactic agents for malaria. In conclusion, we have shown that the high MW CSP form is proteolytically cleaved by a papain family cysteine protease of parasite origin. Several lines of evidence support a role for CSP cleavage during cell invasion. First, under conditions in which CSP cleavage is inhibited, cell invasion is similarly inhibited. Second, rapid and complete CSP cleavage occurs when sporozoites contact target cells, indicating that cleavage is temporally associated with invasion. And lastly, the conservation of this process across the genus indicates that it is of importance to the parasite. These data are part of a growing body of work demonstrating that proteolytic processing of secreted and surface proteins is required for cell invasion by Plasmodium and other Apicomplexan parasites such as Toxoplasma (14, 19, 20). One of the most well-studied examples is MSP-1, the major surface protein of Plasmodium merozoites, the infective form of the erythrocytic stage (for review see reference 14). Interestingly, both CSP and MSP-1 have known cell-adhesive domains in their COOH termini, raising the possibility that cleavage controls the exposure of these domains. In CSP, the COOH terminus contains the TSR, a known cell-adhesive sequence that has been shown to bind with high affinity to HSPGs (for review see reference 1). Previous studies have shown that the NH2-terminal portion of CSP also binds to HSPGs (21). Our data suggest a model for CSP cleavage that explains why this protein has two heparin-binding domains. Our hypothesis is that an initial interaction between cell surface HSPGs and the NH2-terminal portion of CSP cross-links the protein and provides the signal for cleavage. In turn, cleavage exposes the cell-adhesive TSR, which binds with high affinity to HSPGs, initiating a cascade of events that ultimately lead to cell entry. Materials and Methods Antibodies and peptides mAb 3D11 is directed against the repeat region of P. berghei CSP (22); mAb NYS1 is directed against the repeat region of P. yoelii CSP (23); and mAb 2A10 is directed against the repeat region of P. falciparum CSP (11). For immunoprecipitations, mAbs 3D11 and 2A10 were conjugated to sepharose as outlined previously (24). Antisera to the NH2- and COOH-terminal thirds of P. berghei CSP were generated in rabbits using peptides that were provided by G. Corradin and M. Roggero (Institute of Biochemistry, Lausanne, Switzerland). The sequences of the NH2- and COOH-terminal peptides were GYGQNKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYIRNTVNRLLADAPEGKKNEKKNKIERNNKLK and NDDSYIPSAEKILEFVKQIRDSITEEWSQCNVTCGSGIRVRKRKGSNKKAEDLTLEDIDTEICKMDKCS, respectively. Overlapping peptides and repeat peptides were synthesized and purified by Midwest Bio-Tech. Sporozoites. P. yoelii, P. berghei, P. berghei–expressing GFP (25), and recombinant P. berghei sporozoites expressing a hybrid P. berghei–P. falciparum CSP (Pf/Pb sporozoites; reference 10) were grown in Anopheles stephensi mosquitoes. P. falciparum–infected mosquitoes were obtained from D. Carucci (Naval Medical Research Center Malaria Program, Silver Spring, MD). Where indicated, sporozoites were purified by passage through two 3-μm polycarbonate membranes (Whatman). ELISAs Peptides were coated onto wells of Immunlon 2HB microtiter plates (ThermoLabsystems) and blocked, and antisera were added at the indicated dilutions. Binding was revealed with anti–mouse or anti–rabbit Ig-conjugated to alkaline phosphatase followed by the fluorescent substrate, 4-methylumbelliferyl phosphate and fluorescence was read in a Fluoroskan II plate reader. Metabolic labeling. P. berghei or where indicated, P. falciparum or Pf/Pb sporozoites, were metabolically labeled in DMEM without Cys/Met, 1% BSA, and 400 μCi/ml L-[35S]Cys/Met for 1 h at 28°C and chased in DMEM with Cys/Met and 1% BSA at 28°C in the presence or absence of the indicated protease inhibitor. For the pronase experiment, sporozoites were metabolically labeled in medium without BSA for 45 min at 28°C, washed, and resuspended in DMEM with Cys/Met and 100 μg/ml cycloheximide for 10 min and kept on ice or chased at 28°C for 1 h. Sporozoites were resuspended in 100 μg/ml pronase, ± pronase inhibitor cocktail (500 μg/ml antipain, 30 μg/ml aprotinin, 600 μg/ml chymostatin, 5 mg/ml EDTA, 5 μg/ml leupeptin, 10 mg/ml AEBSF, 7 μg/ml pepstatin, and 2 mM PMSF; reference 26) for 1 h at 4°C, washed, and lysed in lysis buffer with pronase inhibitor cocktail and 1% BSA; CSP was immunoprecipitated. Immunoprecipitation and SDS-PAGE analysis. Metabolically labeled sporozoites were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0) with protease inhibitors for 1 h at 4°C, and lysates were incubated with mAb 3D11 agarose overnight at 4°C and washed with lysis buffer and lysis buffer with 500 mM NaCl and preelution buffer (0.5% Triton X-100, 10 mM Tris-HCl, pH 6.8). CSP was eluted with 1% SDS in 0.1 M glycine, pH 1.8, neutralized with Tris-HCl, pH 8.8, and run on a 7.5% SDS–polyacrylamide gel under nonreducing conditions. For experiments with P. falciparum or Pf/Pb sporozoites, a 10% SDS–polyacrylamide gel was used. Gels were fixed, enhanced with Amplify (Amersham Biosciences), dried, and exposed to film. Immunoblot of sporozoite lysates. Sporozoite lysates were separated by SDS-PAGE, transferred to PVDF membrane, and incubated with either 4 μg/ml mAb 3D11, NH2-terminal antiserum (1:3,000), COOH-terminal antiserum (1:3,000), or 4 μg/ml mAb 2A10 followed by anti–mouse or anti–rabbit Ig conjugated to horseradish peroxidase (HRP; 1:100,000). Bound antibodies were visualized using the enhanced chemiluminescence detection system (ECL). Biotinylation of sporozoites P. berghei transgenic for GFP was biotinylated using sulfo-succinimidyl-6'-(biotinamido) hexanoate according to the manufacturer's instructions (Pierce Chemical Co.). Lysates of biotinylated sporozoites were immunoprecipitated with either mAb 3D11 or polyclonal antibodies to GFP (1:200; Molecular Probes) followed by protein A coupled to agarose beads, loaded onto a 4–12% Tris-Glycine gel, transferred to PVDF, and incubated with either mAb 3D11 followed by anti–mouse Ig HRP, anti-GFP Ig (1:500) followed by anti–rabbit Ig HRP, or streptavidin–HRP (1:100,000). Bound antibodies were visualized using ECL. Immunofluorescence assay. Live P. berghei sporozoites were incubated with NH2-terminal antiserum (1:500 in DMEM/BSA) at 4°C for 2 h, washed at 4°C, and allowed to air dry on slides at 4°C. They were incubated with anti–rabbit Ig-FITC, washed, and mounted. Sporozoite invasion assay. Invasion assays were performed as described previously (15), with some modifications. For assays with P. berghei and P. yoelii, Hepa 1–6 cells (CRL-1830; American Type Culture Collection) were used, and for assays with P. falciparum, HepG2 cells (HB-8065; American Type Culture Collection) were used. Sporozoites were preincubated with the indicated protease inhibitor for 2 h at 28°C and plated on cells in the continued presence of the inhibitor for 1 h at 37°C. In a control, Hepa 1–6 cells were incubated with 10 μM E-64 for 2 h at 37°C, the medium was removed, and untreated P. berghei sporozoites were added. After incubation with sporozoites, cells were washed and fixed, and sporozoites were stained with a double-staining assay that distinguishes between extracellular and intracellular sporozoites. Cell contact assay. P. berghei sporozoites were incubated in DMEM ± 10 μM E-64 at 4°C for 2 h and added to Hepa 1–6 cells on glass coverslips. 30 min before sporozoites were added to coverslips, CD was added to all samples (final concentration, 1 μM). Sporozoites were centrifuged onto coverslips (1,250 g) for 5 min at 4°C. Coverslips were brought to 37°C for 2 min, fixed with 4% paraformaldehyde, and stained with either mAb 3D11 followed by anti–mouse Ig FITC or the NH2-terminal antiserum followed by anti–rabbit Ig FITC. When P. berghei sporozoites expressing GFP were used, the cells were only stained with the NH2-terminal antiserum. As a control, sporozoites were spun onto coverslips without cells using the aforementioned protocol. Sporozoite migration assay. Sporozoites were preincubated ±10 μM E-64 for 2 h at 28°C and added to Hepa 1–6 cells in the continued presence of inhibitor with 1 mg/ml rhodamine-dextran. After 1 h at 37°C, the cells were washed and fixed, and rhodamine-positive cells were counted as outlined previously (16). Assay for sporozoite infectivity in vivo. Swiss/Webster mice were given three i.p. injections of DMEM ± E-64 (50 mg/kg/injection) at 16, 2.5, and 1 h before i.v. injection of 15,000 P. yoelii sporozoites. 40 h later, livers were harvested, total RNA was isolated, and malaria infection was quantified using reverse transcription followed by real-time PCR using primers that recognize P. yoelii–specific sequences within the 18S rRNA as outlined previously (27). 10-fold dilutions of a plasmid construct containing the P. yoelii 18S rRNA gene were used to create a standard curve. Online supplemental material Fig. S1 shows the specificity of the NH2- and COOH-terminal antisera as determined by ELISA. Fig. S2 shows that the protease inhibitors that inhibited CSP processing are not toxic to sporozoites. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20040989/DC1. Acknowledgments The authors would like to thank G. Corradin and M. Roggero for their generous gift of the long NH2- and COOH-terminal peptides; D. Carucci and P. de la Vega for providing P. falciparum sporozoites; E. Nardin and G. Oliveira for providing recombinant Pf/Pb sporozoites; M. Blackman and K. Kim for helpful discussions; V. Nussenzweig, D. Eichinger, and M. Calvo-Calle for their critical reading of the manuscript; and D. Bernal and J. Noonan for their expert assistance with mosquito rearing and infection. This work was supported by National Institutes of Health (NIH) grant no. R01 AI44470 (to P. Sinnis) and NIH training grant no. 5T32 AI07180 (to A. Coppi). The authors have no conflicting financial interests. Submitted: 19 May 2004 Accepted: 19 November 2004 References Wieckowski, E.U., J.F. Kokai-Kun, and B.A. McClane. 1998. Characterization of membrane-associated Clostridium perfringens enterotoxin following pronase treatment. Infect. Immun. 66:5897–5905. Bruna-Romero, O., J.C. Hafalla, G. Gonzalez-Aseguinolaza, G. Sano, M. Tsuji, and F. Zavala. 2001. Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR. Int. J. Parasitol. 31:1499–1502 Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heigne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1–6...查看详细 (28002字节)
☉ 11120215:The Pim kinases control rapamycin-resistant T cell
Although Pim-1 or Pim-2 can contribute to lymphoid transformation when overexpressed, the physiologic role of these kinases in the immune response is uncertain. We now report that T cells from Pim-1–/–Pim-2–/– animals display an unexpected sensitivity to the immunosuppressant rapamycin. Cytokine-induced Pim-1 and Pim-2 promote the rapamycin-resistant survival of lymphocytes. The endogenous function of the Pim kinases was not restricted to the regulation of cell survival. Like the rapamycin target TOR, the Pim kinases also contribute to the regulation of lymphocyte growth and proliferation. Although rapamycin has a minimal effect on wild-type T cell expansion in vitro and in vivo, it completely suppresses the response of Pim-1–/–Pim-2–/– cells. Thus, endogenous levels of the Pim kinases are required for T cells to mount an immune response in the presence of rapamycin. The existence of a rapamycin-insensitive pathway that regulates T cell growth and survival has important implications for understanding how rapamycin functions as an immunomodulatory drug and for the development of complementary immunotherapeutics. Abbreviations used: BrdU, bromo-deoxyuridine; PI, propidium iodide; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription; TSST, toxic shock syndrome toxin. C.J. Fox and P.S. Hammerman contributed equally to this work. The murine immune system maintains a pool of lymphocytes that respond to antigenic challenge with rapid growth and proliferation. Naive T cells compete in vivo for exogenous factors including the cytokines IL-4 and IL-7 and for MHC-dependent proliferative signals (1, 2). Once T cells encounter antigen, their size increases dramatically (blastogenesis) as they prepare for clonal expansion and the acquisition of an effector phenotype (3). The ligation of cytokine or antigen receptors promotes T cell growth and survival in part by activating the effector enzymes of the PI3K pathway, the kinases Akt and TOR (4). Mice expressing an activated Akt transgene have increased numbers of peripheral T cells that manifest enhanced resistance to apoptotic stimuli in vitro and this effect correlates with the Akt-dependent activation of TOR (5–7). T cells from Akt transgenic mice are larger and show enhanced proliferation in reponse to mitogens (8, 9). However, Akt-deficient animals (10, 11) and mice treated with the TOR inhibitor rapamycin (12) mount a normal primary immune response. These data suggest that alternate pathways exist that can promote lymphocyte growth and survival in a PI3K/Akt/TOR-independent manner. IL-4, IL-7, IL-2, and TCR ligation activate members of the signal transducers and activators of transcription (STAT) family to promote expression of prosurvival molecules including the Pim family of oncogenic serine/threonine kinases. Pim-1, Pim-2, and Pim-3 are novel components of the transcriptional response to cytokine or antigen receptor ligation (13) and their function is regulated primarily at the level of expression (14). Pim-1 and pim-2 are expressed in most hematopoietic cells whereas pim-3 expression is highest in brain, kidney, and mammary tissue (15). Several Pim targets have been identified and include the proapoptotic protein Bad (14, 16), members of the suppressor of cytokine signaling (SOCS) family (17, 18), the translational repressor eIF-4E binding protein 1 (4E-BP1; 14) and the transcription factor Myb (19). Pim-1 and Pim-2 transgenes can promote growth and survival of hematopoietic cell lines (14, 17, 20). We now report that Pim-1 and Pim-2 are essential components of an endogenous pathway that regulates T cell growth and survival. Pim-1 up-regulation occurred rapidly after cytokine treatment or mitogenic stimulation and high levels of Pim-2 were observed several hours later. T cells from Pim-1–/–Pim-2–/– mice responded to cytokine- or antigen receptor–ligation comparably to cells from wild-type littermates. However, rapamycin treatment eliminated the ability of IL-4 and IL-7 to promote the survival of Pim-1–/– Pim-2–/– but not wild-type T cells. This correlated with the failure of Pim-1–/–Pim-2–/– T cells to maintain the phosphorylation-dependent inactivation of the Bcl-2–related protein Bad in the presence of rapamycin. Rapamycin also blocked the mitogen-induced activation of Pim-1–/–Pim-2–/– T cells at an early stage of blastogenesis before the up-regulation of surface activation markers and cell cycle entry. Pim deficiency enhanced the effect of rapamycin in vivo and prevented superantigen-induced T cell activation and expansion. The identification of Pim-1 and Pim-2 as required components of a rapamycin-insensitive pathway that regulates lymphocyte growth and survival suggests that the Pim kinases might serve as attractive targets for the development of novel immunotherapeutic regimens. Results Pim-1 and Pim-2 are induced by prosurvival signals In contrast to most kinases implicated in T cell responses, Pim-1 and Pim-2 were undetectable in nonstimulated T cells. Pim-1 and Pim-2 expression was not observed in murine T cells ex vivo or after 12 h of culture without added cytokine (Fig. 1 A). However, when the T cells were cultured in the presence of IL-4 or IL-7, Pim-1 protein was detected at 3 h and Pim-2 by 12 h. The dissimilarity in the kinetics of their expression suggested that Pim-1 and Pim-2 might play independent or sequential roles in the response to prosurvival or proliferative signals. However, mice deficient in Pim-1 (hereafter referred to as Pim-1–/–2+/+), Pim-2 (Pim-1+/+2–/–), or both kinases (Pim-1–/–2–/–) showed no obvious differences when compared with Pim-1+/+2+/+ littermates with respect to thymus size or thymocyte or peripheral T cell distribution (unpublished data) as described previously by Mikkers et al. (15). im transgenes can promote cell survival, the response of T cells from Pim-1–/– 2–/– animals to the prosurvival cytokines IL-4 and IL-7 was examined. Splenic T cells from Pim-1–/–2–/– or Pim-1+/+2+/+ littermates cultured in vitro in the absence of added cytokines showed identical survival kinetics (Fig. 1 B). IL-4 and IL-7 addition strongly promoted T cell survival in both Pim-1+/+2+/+ and Pim-1–/–2–/– T cells. Thus, either endogenous Pim-1 or Pim-2 play no role in the regulation of T cell survival or alternate survival pathways are activated by cytokine treatment. IL-4 and IL-7 treatment of naive T cells also activated the central antiapoptotic effector of the PI3K pathway, Akt, as measured by the induction of Akt phosphorylation at Serine 473 (Fig. 2 A). TOR is one key mediator of Akt-dependent signal transduction (21). The TOR inhibitor rapamycin can suppress Akt-dependent cell survival (14, 22). Therefore, we examined the ability of rapamycin to affect T cell viability after cytokine treatment. As one readout of TOR activity, the phosphorylation status of the TOR substrate p70 S6 kinase (p70S6K) and its primary target, the S6 ribosomal subunit, was first assessed. IL-4 or IL-7 addition increased total p70S6K and S6 expression and phosphorylation relative to nonstimulated cells (Fig. 2 B). The addition of rapamycin prevented cytokine-dependent p70S6K and S6 phosphorylation (Fig. 2 B). Despite this observation, rapamycin had no effect on the ability of these cytokines to promote T cell viability (Fig. 2 C), suggesting that TOR might also be dispensable for the T cell response to prosurvival cytokines. The cytokine-dependent induction of Pim-1 and Pim-2 by IL-4 or IL-7 was not affected by rapamycin treatment (Fig. 2 D). Endogenous levels of Pim-2 confer rapamycin-resistant T cell survival IL-4 and IL-7 activate the Pim kinases and Akt/TOR but neither pathway is absolutely required for cytokine-dependent survival. Reasoning that the disruption of both pathways might have a more significant effect, we assessed cytokine-induced survival in wild-type, Pim-1–/–2+/+, Pim-1+/+2–/–, and Pim-1–/–2–/– T cells treated with rapamycin. Spontaneous apoptosis of T cells from all four genotypes was suppressed by IL-4 or IL-7 (Fig. 3 A). Although rapamycin had no effect on cytokine-dependent survival of wild-type and Pim-1–/–2+/+ T cells, rapamycin treatment suppressed the ability of T cells from Pim-1+/+2–/– or Pim-1–/–2–/– mice to survive in response to IL-4 (Fig. 3 A). Pim-1+/+2–/– and Pim-1–/–2–/– T cells also failed to survive in response to IL-7 in the presence of rapamycin. These phenotypes could not be explained by decreased IL-4 or IL-7 receptor expression or increased levels of the death receptor Fas in rapamycin-treated T cells relative to control Pim-1+/+2–/– or Pim-1–/–2–/– T cells (unpublished data). Cytokines promote cell survival in part by affecting the expression and function of members of the Bcl-2 protein family. Survival can be potentiated by enhanced Bcl-2 and Bcl-xL expression and their antiapoptotic function can be suppressed by association with the BH3-only, proapoptotic proteins Bim and Bad (2). High levels of Bcl-2, Bcl-xL, Bim, and Bad were evident in IL-7–treated Pim-1+/+2+/+ and Pim-1–/–2–/– T cells in the presence and absence of rapamycin (Fig. 3 B). The proapoptotic function of Bad can be suppressed by its phosphorylation at multiple residues including serines 112 (Ser112) and Ser136 (23). The pattern of Bad phosphorylation at Ser136 in response to IL-7 and rapamycin treatment was identical when Pim-1–/–2–/– were compared with Pim-1+/+2+/+ T cells (Fig. 3 B). A dissimilar pattern of Ser112 phosphorylation was observed. Whereas IL-7–induced Ser136 phosphorylation was detected after 30 min of cytokine treatment, Bad phosphorylation at Ser112 was not observed until 12 h. This pattern overlapped with that of Pim-2 induction in response to IL-7 (Figs. 1 A and 2 D). Further, IL-7–induced Ser112 phosphorylation was resistant to rapamycin in Pim-1+/+2+/+ T cells but not in Pim-1–/–2–/– cells (Fig. 3 B). Thus, the Pim kinases contribute to the regulation of cell survival in part through regulating the phosphorylation of Bad. The Pim kinases are required for rapamycin-resistant T cell activation In addition to its role in Akt-dependent cell survival (22), TOR is considered a critical regulator of protein translation and cell growth (24). Since CD28 ligation has been reported to activate Akt in T cells and might therefore promote TOR-dependent translation (25), we examined the relative contributions of endogenous Pim-1, Pim-2 and TOR to T cell growth after mitogenic stimulation with anti-CD3/anti-CD28 antibodies (CD3/CD28). CD3/CD28 treatment induced a pronounced increase in p70S6K and S6 phosphorylation that could be suppressed by rapamycin (Fig. 4 B). CD3/CD28 treatment up-regulated Pim-1 expression in T cells within 30 min and Pim-2 by 12 h (Fig. 4 A). Activation-induced Pim kinase expression was rapamycin resistant (Fig. 4 C). T cells increase in size before their entry into the S phase of the cell cycle. Consistent with this, the mean forward scatter of T cells had more than doubled 2 d after CD3/CD28 treatment (Fig. 4 D). Blastogenesis was accompanied by increased surface expression of the activation markers CD69 and CD25 and CD62L down-regulation. All of these parameters were nearly identical when CD3/CD28-treated T cells from Pim-1+/+2+/+, Pim-1–/–2+/+, Pim-1–/ – 2–/–, or Pim-1+/+2–/– mice were compared (Fig. 4 D). Rapamycin had little effect on CD3/CD28-induced blastogenesis and CD69 or CD62L expression in Pim-1+/+2+/+ or Pim-1–/–2+/+ T cells (Fig. 4 D) even when added at 200 nM (unpublished data). In contrast, rapamycin treatment suppressed CD3/CD28-induced blastogenesis in Pim-1+/+ 2–/– T cells and completely prevented activation-induced blastogenesis in Pim-1–/–2–/– T cells. Pim-1–/–2–/– T cells also failed to up-regulate surface CD69 and CD25 expression and decrease CD62L when stimulated by CD3/CD28 in the presence of rapamycin. For both Pim-1–/– 2–/– and Pim-1+/+2–/– T cells, the addition of IL-2 had no effect on the rapamycin-dependent suppression of cell growth (unpublished data). In the absence of stimulation, >98% of T cells were in the G0/G1 phase of the cell cycle as assessed by bromo-deoxyuridine (BrdU) incorporation (Fig. 4 E) and did not proliferate in culture (Fig. 4 F). After 2 d of CD3/CD28 treatment, Pim-1+/+2+/+ T cells were distributed across the cell cycle with approximately one-third of the population in each of G1, S, or G2M phases. At 4 d, all input Pim-1+/+2+/+ T cells had divided at least once and 45% of the population had undergone three or more rounds of division (Fig. 4 F). Both rapamycin treatment and Pim deficiency alone had modest effects on cell cycle entry and proliferation after CD3/CD28 treatment relative to control. However, the combined effect of suppressing both pathways was striking. No T cells derived from Pim-1–/–2–/– mice had entered the S phase of the cell cycle 2 d after CD3/CD28 treatment in the presence of rapamycin (Fig. 4 E). Close to 90% of input Pim-1–/–2–/– T cells failed to divide in the presence of rapamycin even 4 d after CD3/CD28 stimulation (Fig. 4 F). Pim kinase deficiency enhances rapamycin action in vivo The deletion of Pim-1 and Pim-2 sensitized T cells to TOR inhibition in vitro. This raised the possibility that the in vivo immunosuppressive action of rapamycin might be enhanced in Pim-deficient animals. Superantigens such as the toxic shock syndrome toxin (TSST) have been widely used to study activation and proliferation of T cells in vivo. Both Pim-1+/+2+/+ and Pim-1–/–2–/– animals treated with TSST for 2 d contained sixfold more CD25+ T cells (Fig. 5 A) and a higher proportion of TSST-reactive V?3+ cells (Fig. 5 B) relative to vehicle-treated littermates. In Pim-1+/+ 2+/+ animals, rapamycin did not affect these parameters. In contrast, rapamycin treatment of Pim-1–/–2–/– mice suppressed TSST-induced T cell activation (Fig. 5 A). Further, V?3+ T cell expansion was completely suppressed by rapamycin in TSST-treated Pim-1–/–2–/– mice (Fig. 5 B). Discussion We report that Pim-1 and Pim-2 are regulators of rapamycin sensitivity in T cells. Although mature T cells developed normally in Pim-deficient animals, T cells from Pim-1+/+ 2–/– and Pim-1–/–2–/– mice were unable to respond to prosurvival cytokines in the presence of the drug. The data suggest that endogenous Pim-2 is the primary mediator of rapamycin-resistant cell survival and that Pim-1 expression alone is insufficient to promote prolonged rapamycin-resistant survival. Rapamycin also blocked CD3/CD28-induced T cell activation at early blastogenesis before cell cycle entry in Pim-1–/–2–/– but not wild-type T cells. Despite its clinical use in delaying transplant rejection, the precise basis by which rapamycin functions as an immunosuppressant is unclear. This report suggests that rapamycin does not inhibit T cell growth, proliferation or survival because endogenous Pim-1 and Pim-2 can maintain the survival and growth of rapamycin-treated T cells. Therefore, despite the established clinical efficacy of rapamycin, it remains at best a partial immunosuppressant in vivo and in vitro. Complete immunosuppression requires the concomitant inhibition of Pim kinase activity. Our work suggests that endogenous Pim kinase expression is an important determinant of rapamycin sensitivity. The data help to explain some of the confusing results that have been reported concerning the effect of rapamycin treatment in lymphocyte activation and survival. Although rapamycin was originally described as an inhibitor of cell proliferation that acts by inducing arrest at the G1 phase of the cell cycle (26), subsequent data suggest that its antiproliferative effects may be stimulus or cell type specific. For example, rapamycin treatment can cause thymic atrophy in mice and rats (27–29). However, rapamycin has only a modest effect on the survival of peripheral T cells in vivo (29). In activated T cells, rapamycin has been reported to inhibit IL-2R up-regulation and IL-2–dependent proliferation in some studies (26, 30–32) but not others (33–35). Recent work suggests that while some CD4-dependent immune responses are rapamycin sensitive (36, 37), CD8+ T cell function is largely rapamycin resistant (29, 36, 38, 39). It is tempting to speculate that the Pim kinases might account for the residual immune function observed in the presence of rapamycin in these studies. Pim-1 and Pim-2 do not contribute equally to the phenotypes we have observed. Pim-2 was induced much later than Pim-1 and appeared to play a more substantial role in the regulation of cytokine and mitogen-induced survival and growth. Nevertheless, endogenous Pim-1 does appear to partially compensate for Pim-2 deficiency in mitogen-induced T cell growth. Because the combined deficiency of Pim-1 and Pim-2 eliminated cytokine-dependent cell survival and CD3/CD28- and superantigen-induced T cell activation in the presence of rapamycin, Pim-3 seems unlikely to contribute to the rapamycin-insensitive growth of T lymphocytes. Recently, animals deficient in all three Pim kinases have been reported. Although these mice show reduced body size, their lymphocytes display only mild defects in cytokine-induced proliferation (15). The molecular basis of Pim kinase–dependent lymphocyte survival is explained in part through regulation of the proapoptotic Bcl-2–related protein Bad. Pim-2–deficient T cells die subsequent to prosurvival cytokine treatment in the presence of rapamycin despite continued Bcl-2 and Bcl-xL expression. It is well appreciated that Bcl-2 and Bcl-xL function are antagonized in part by their heterodimerization with Bad and that this interaction is controlled by Bad phosphorylation at multiple residues, including Ser112, Ser136, and Ser155 (23). Phosphorylated Bad is retained in the cytoplasm by the 14-3-3 proteins (23). The relative roles played by each of these residues in regulating the ability of Bad to antagonize Bcl-2 and Bcl-xL function remains controversial. Many studies suggest that Akt is one of the primary kinases that phosphorylates Bad at Ser136 (40, 41), in part because Ser136 phosphorylation is sensitive to PI3K inhibitors (41, 42). Another group has reported that the kinetics of Ser112 and Ser136 phosphorylation in response to IL-7 treatment differ and that only the former occurs in a PI3K-independent manner (42). Recently, Chiang et al. showed that the PP2A-dependent dephosphorylation of Bad at Ser112 is required for Ser136 dephosphorylation, the dissociation of Bad from 14–3-3, and apoptosis (43). The authors proposed that Ser112 served as the "gatekeeper" of Bad function. Although kinases in addition to Pim-2 can phosphorylate Bad at Ser112 in vitro (41, 44, 45), the present data suggests that Pim-2 plays a primary role in regulating Bad phosphorylation at Ser112 in T cells. In summary, Pim kinase deficiency greatly enhances the immunosuppressive action of rapamycin in vivo and in vitro. Thus, inhibitors of the Pim kinases in combination with rapamycin would be expected to induce a greater degree of immunosuppression as compared with either drug alone. Analogous to the rapamycin-dependent inhibition of TOR, the pharmacological inhibition of the Pim kinases might have immunomodulatory benefits on its own. The observation that the germline deletion of Pim-1 and Pim-2 has a modest phenotype outside of the hematopoietic system and that, aside from its effects on T cells, rapamycin is tolerated well in Pim-1–/–2–/– animals, suggests that these possibilities should be explored. Materials and Methods Mice. Pim-1+/+2+/+, Pim-1–/–2+/+, Pim-1+/+2–/–, and Pim-1–/–2–/– animals were generated from Pim-1+/–Pim-2+/– mice, a gift of Paul Rothman, Columbia University, New York, NY) (17). Genotyping by PCR with tail snip DNA was performed as described previously (17). C57BL/6 mice were purchased from Jackson ImmunoReasearch Laboratories. Unless otherwise indicated, all experiments were performed on male littermates at 4 to 10 wk of age. In all experiments, mice were injected IP. Mice were bred and maintained at the University of Pennsylvania in accordance with the Institutional Animal Care and Use Committee guidelines. Antibodies and Western blots. Mouse anti–Pim-2 1D12, mouse anti–Pim-1 19F7, and goat anti–actin I-19, and goat anti–mouse IgG, bovine anti–goat IgG, and goat anti–rabbit IgG coupled to horseradish peroxidase were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc.). Hamster anti–mouse Bcl-2 3F11, rabbit anti–mouse Bim, and anti-hamster IgG–HRP were purchased from BD Biosciences. Rabbit anti–mouse phospho-serine 371 p70S6K, rabbit anti–mouse phospho-Ser235/Ser236 S6 ribosomal protein, rabbit anti-phospho Ser473 Akt, rabbit anti-phospho Ser136 Bad, rabbit anti-phospho Ser112 Bad, rabbit anti-phospho Ser155 Bad, rabbit anti-S6 ribosomal protein, rabbit anti-Akt, and rabbit anti-Bad were purchased from Cell Signaling. Preparation and quantitation of whole cell extracts lysed in PBS + 1% NP-40 supplemented with protease (Roche) and phosphatase inhibitor cocktails I and II (both from Sigma-Aldrich) and Western blotting as described previously in detail (14). 50–100 μg of protein was resolved on a 4–12% or 10% NuPage bis-tris polyacrylamide gels and transferred to nitrocellulose as directed (Invitrogen). Blots were blocked in PBS containing 10% milk and 0.2% Tween-20 (both from GIBCO BRL), incubated in primary antibody in 5% milk overnight at 4°C and washed several times for 15 min each in PBS + 0.2% Tween. Target protein was visualized by incubation with a species appropriate HRP-conjugated secondary antibody at room temperature for 1 h and ECL-Advance chemiluminescent reagent (Amersham Bioscience). For serial Western blotting, membranes were stripped by incubating for 30 min at 65°C in PBS + 1% SDS and 100 μM ?-mercaptoethanol followed by six washes in PBS + 0.2% Tween-20. Thymocyte and T cell preparation, activation, and flow cytometric analyses. Thymuses and spleens were pooled from three to five animals. Live thymocytes were enriched by Ficoll centrifugation as directed by the manufacturer (Amersham Biosciences) and then resuspended at 106/ml in DMEM supplemented with 10% FCS, penicillin/streptomycin/fungizone, 100 μM ?-mercaptoethanol, 2 mM L-glutamine, 100 μM nonessential amino acids, and 10 mM Hepes buffer (all from GIBCO BRL). Flow cytometric analysis of thymocytes was performed using a FACSCalibur (BD Biosciences) and FITC rat anti–mouse Thy1.2 53–2.1 or FITC anti–rat Thy1.1 OX-7, PE anti–mouse CD4 L3T4 and APC anti–mouse CD8a 53.6–7 antibodies (all from BD Biosciences). Total thymocyte numbers were calculated by Trypan blue exclusion. Splenic T cells were enriched to 95% purity (as assessed by staining with PE rat anti–mouse Thy1.2 30-H12 or PE anti–rat Thy1.1 OX-7 antibodies) using the StemStep T Cell Purification kit as directed by the manufacturer (Stem Cell Technologies Inc.). Lysates were then immediately prepared from a fraction of each thymocyte or T cell preparation (time 0). In some Western blotting experiments, half of each cell preparation was preincubated in 50 nM of rapamycin (Sigma-Aldrich) dissolved in methanol, or the vehicle control, for 1 h at 37°C before addition of cytokines or in vitro activation. For some flow cytometric analyses, T cells were preincubated in 25 nM rapamycin or vehicle in an identical manner and fresh rapamycin was added at day 2 of the experiment. For cytokine treatment of naive T cells, cells were cultured at 5 x 106/ml in vitro with the addition of 10 ng/ml of recombinant mouse IL-4 or 10 ng/ml of recombinant mouse IL-7 (both from BD Biosciences) for the times indicated. Viability was assessed daily by flow cytometric analysis of the exclusion of propidium iodide (PI). Flow cytometric analysis was also performed with FITC Thy1.1 or Thy1.2, biotin hamster anti–mouse Fas Jo.2, PE rat anti–mouse IL-7R SB/199, rat anti–mouse IL-4R mIL4R-M1, and FITC rat anti–mouse IgG (all from BD Biosciences). Another set of cells were activated in one of two ways, either by the addition of 10 ng/ml PMA and 250 ng/ml ionomycin (both from Sigma-Aldrich) or by antibody-mediated TCR and CD28 cross-linking as follows: 24 well plates were preincubated at 4°C overnight with 1 μg/ml of both anti-Armenian and anti-Syrian Hamster IgG antibodies (Jackson ImmunoResearch Laboratories, Inc.) in PBS, washed three times in PBS and then incubated with 1 μg/ml hamster anti–mouse CD3 145-2C11 and 2 μg/ml hamster anti–mouse CD28 37.51 in PBS at 37°C for 1 h and cells added at 5 x 106/ml. Control cells were cultured in an identical manner except anti-CD3 was omitted from the culture preparation. For T cell activation studies, live cells were enriched by Ficoll centrifugation after 2 d of culture and activation status was assessed via analysis of mean forward scatter and using FITC anti–mouse CD69 H1.2F3, APC rat anti–mouse CD25 PC61, and PE anti–mouse CD62L MEL-14 (all antibodies from BD Biosciences). A fraction of each preparation was also resuspended in medium and incubated for 2 h at 37°C with 10 μM BrdU (Calbiochem). Cells were then fixed in cold 70% ethanol and stained with mouse anti-BrdU 3D4 and FITC rat anti–mouse IgG (both antibodies from BD Biosciences) according to the manufacturer's protocol and resuspended in PI + 1 μg/ml RNase A before FACS analysis. In a series of parallel experiments, T cells were suspended in PBS and labeled after a 5-min incubation with 5 μM 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (Molecular Probes) as directed by the manufacturer and then activated by TCR and CD28 cross-linking in the presence of 50 nM rapamycin. Fresh rapamycin or vehicle was added at day 2 of culture. In some cases, recombinant murine IL-2 (BD Biosciences) was added. In vivo rapamycin and superantigen treatment For assessment of total thymocyte numbers, animals were given daily doses for 3 d of 3 mg/kg rapamycin in water. Animals were then killed and total thymocytes counted by Trypan blue exclusion. Control animals were given water only. For the superantigen-induced activation experiments, animals were treated with 3 mg/kg rapamycin daily for 3 d. On day 2, mice were given 50 mg of D-galactosamine hydrochloride (Sigma-Aldrich) in PBS, followed by 100 μg of TSST (Sigma-Aldrich) in PBS 30 to 60 min later. On day 4, spleen and LN cells from individual animals were pooled, live lymphocytes enriched by Ficoll centrifugation, and then assessed by flow cytometric analysis with PE anti-Thy1, APC anti-CD25, biotinylated anti–mouse V?3 T cell receptor KJ25, anti–mouse V?4 KT4, anti–mouse V?7 TR10, and anti–mouse V?11 RR3-15 antibodies, and then FITC–streptavidin (all antibodies from BD Biosciences). Acknowledgments We would like to thank Robert Woodland, Steven Reiner, and members of the Thompson Laboratory for their critical assessment of these data. This work was supported in part by grants from the National Institutes of Health. C.J. Fox received a Chapter Grant from the Arthritis Foundation and a Special Fellowship from the Leukemia and Lymphoma Society. P.S. Hammerman received a Training Grant from the Cancer Research Institute. The authors have no conflicting financial interests. Submitted: 30 September 2004 Accepted: 8 December 2004 References Hayakawa, J., M. Ohmichi, H. Kurachi, Y. Kanda, K. Hisamoto, Y. Nishio, K. Adachi, K. Tasaka, T. Kanzaki, and Y. Murata. 2000. Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res. 60:5988–5994Li, W.Q., Q. Jiang, A.R. Khaled, J.R. Keller, and S.K. Durum. 2004. Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival. J Biol Chem. 279:29160–29166Chiang, C.W., C. Kanies, K.W. Kim, W.B. Fang, C. Parkhurst, M. Xie, T. Henry, and E. Yang. 2003. Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BAD-mediated apoptosis. Mol. Cell. Biol. 23:6350–6362 She, Q.B., W.Y. Ma, S. Zhong, and Z. Dong. 2002. Activation of JNK1, RSK2, and MSK1 is involved in serine 112 phosphorylation of Bad by ultraviolet B radiation. J. Biol. Chem. 277:24039–24048] Fang, X., S. Yu, A. Eder, M. Mao, R.C. Bast Jr., D. Boyd, and G.B. Mills. 1999. Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway. Oncogene. 18:6635–6640...查看详细 (28910字节)
☉ 11120216:TACI and BAFF-R mediate isotype switching in B cel
Class switch recombination (CSR) in B cells requires two signals (1). The first is normally delivered by cytokines, which target specific CH genes for transcription; the second is delivered in the case of T-dependent (TD) antigens by interaction of CD40 on B cells with its ligand CD40L on activated T cells. CSR is severely impaired in patients and mice deficient in CD40L or CD40 (2, 3), although low levels of IgG and variable levels of IgA are still detected in serum. Exposure to LPS derived from Gram-negative bacteria may account for some of this residual CSR in mice, but not in humans since LPS does not activate CSR in human B cells. EBV infection triggers CSR in human B cells independently of CD40L and CD40 (4) and may contribute to residual CSR in humans with CD40L and CD40 deficiency. B cell–activating factor of the TNF family (BAFF) and A proliferation–inducing ligand (APRIL) are two TNF family members that have been shown to activate CSR in human B cells (5) and hence may contribute to residual CSR in CD40L and CD40 deficiency. BAFF is expressed mainly by monocytes and dendritic cells. APRIL is expressed in a large variety of tissues that include monocytes/macrophages, dendritic cells, and activated T cells. APRIL and BAFF both bind to two receptors, B cell maturation antigen (BCMA) and transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI), which are members of the TNF receptor family. BCMA is exclusively expressed on B cells, whereas TACI is expressed on B cells and activated T cells. A third receptor, BAFF receptor (BAFF-R), that is unique for BAFF is expressed mainly on B cells but also on T cells (6). To identify the receptors that are involved in the induction of Ig class switching by BAFF and APRIL, we ascertained that these ligands activate CSR in mouse B cells and then examined their activity on B cells from TACI-, BCMA-, and BAFF-R–deficient mice. Results and Discussion BAFF and APRIL activate IgG1, IgA, and IgE isotype switching in mouse B cells We examined the capacity of BAFF and APRIL to induce IgG1, IgA, and IgE switching in mice. Splenic B cells from CD40–/– mice were negatively sorted and consisted of >96% sIgM+sIgD+, 3–6% CD11b+, and undetectable CD3+ cells. APRIL and BAFF induced IgG1, IgA but no detectable IgE synthesis in these cells (Fig. 1 A). IL-4 enhanced the induction of IgG1 synthesis by BAFF and APRIL and synergized with these two ligands to induce IgE synthesis. As expected, B cells synthesized large amounts of IgG1 and IgE in response to LPS + IL-4, and TGF? synergized with LPS to induce IgA switching. Neutralization of TGF? had no effect on IgA secretion in response to BAFF and APRIL (unpublished data). Failure to block induction of IgA secretion by TGF? suggests that BAFF and APRIL induce germ line transcripts (GLTs) independently of TGF?, or they induce TGF?, but not all of it is accessible to neutralization by the antibody. IL-6 neutralization had no effect on IgG1 or IgA induction by BAFF or APRIL (unpublished data). IL-10 neutralization partially inhibited IgG1 secretion by BAFF (40%) and APRIL (60%) and IgA secretion by these ligands (10 and 30%, respectively). As another measure of CSR, we examined the induction of expression of surface IgG1. There were virtually no sIgG1+ cells in the negatively sorted B cells (Fig. 1 B). APRIL and BAFF alone and with IL-4 induced IgG1 surface expression in these B cells. Together these results suggest that APRIL and BAFF activate CSR in murine B cells. CSR has been linked to cell division (7). APRIL- and BAFF-induced proliferation of negatively sorted B cells in a [3H]thymidine uptake assay and of splenic B220+ B cells in a 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution assay (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20032000/DC1). Induction of CSR by APRIL and BAFF was not due to contamination with endotoxin because the preparations used contained <1 endotoxin U/μg protein; and polymyxin B, which inhibits LPS activation (8), failed to inhibit induction of IgG1 synthesis by APRIL and BAFF (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20032000/DC1). Molecular events involved in CSR include expression of GLTs, expression of the gene for activation-induced deaminase (AID), followed by deletional switch recombination and expression of Iμ-CH transcripts. APRIL and BAFF induced 1GLT and GLT, but no detectable GLT, in negatively sorted B cells from CD40–/– mice (Fig. 1 C). APRIL, and to a lesser extent, BAFF induced AID gene expression. APRIL and BAFF synergized with IL-4 in inducing GLT. Digestion circularization (DC)–PCR analysis revealed that APRIL and BAFF induced SμS1 and SμS but not SμS deletional switch recombination (Fig. 1 D). IL-4 synergized with APRIL and BAFF to induce SμS deletional switch recombination and to up-regulate SμS1 recombination. Consistent with the results of Ig synthesis, APRIL and BAFF induced Iμ-C1 and Iμ-C but not Iμ-C transcripts, unless IL-4 was added (Fig. 1 C). Since we used negatively sorted B cells from CD40–/– mice, these results indicate that APRIL and BAFF induce CSR in naive B cells. Our findings extend previous results on human B cells positively sorted for IgD expression (5). Our observation that BAFF and APRIL activate CSR in B cells from CD40–/– mice definitively establishes that CSR mediated by these ligands is independent of CD40L–CD40 interactions (Fig. 1). In the case of human B cells, BAFF/APRIL induction of secretion of the switched isotypes requires additional signals that include cross-liking of the B cell receptor and the cytokines, such as IL-10 and IL-15 (5). One possibility is that mouse B cells endogenously produce cytokines that support secretion of the switched Ig. This is supported by our observation that neutralizing IL-10 antibody inhibited BAFF- and APRIL-driven IgG1 and IgA secretion by mouse B cells. Another possibility is that mouse B cells survive better in culture to the stage where they are able to secrete Igs. TACI mediates class switching by APRIL We next examined negatively sorted splenic B cells from mice that lack BCMA or TACI. B cells from WT mice were used as controls with results similar to those obtained with CD40–/– B cells. BCMA–/– B cells synthesized IgG1, IgA, and IgE in response to APRIL and BAFF in amounts that were not significantly different from those secreted by WT B cells (Fig. 2, A and B). Intact CSR in BCMA–/– B cells was confirmed by examination of molecular events involved in CSR to IgG1, IgA, and IgE (Fig. 3). TACI–/– B cells virtually failed to synthesize IgG1, IgA, and IgE in response to APRIL (Fig. 2 A). This was not due to an intrinsic defect in CSR because they synthesized IgG1 and IgE in response to LPS + IL-4 and CD40 + IL-4 and IgA in response to LPS + TGF? (Fig. 2 C). Examination of molecular events confirmed the inability of APRIL to activate CSR in TACI–/– B cells (Fig. 3). In some experiments, CHGLT and AID were faintly detected in unstimulated B cells from TACI–/– mice. This may be related to the B cell activation observed in these mice in vivo (9). However, these faint CHGLT and AID transcripts were not up-regulated by APRIL. These results suggest that APRIL induction of CSR is mediated by TACI. Both TACI and BAFF-R mediate class switching by BAFF In contrast to their total inability to class switch in response to APRIL, TACI–/– B cells synthesized IgG1 and IgE in response to BAFF + IL-4 in amounts that were not significantly different from those secreted by normal B cells (Fig. 2 B). This was confirmed by the presence of 1 and GLTs, AID, and mature Iμ-C1 and Iμ-C (Fig. 3) and was observed in the presence of polymyxin B (unpublished data). BCMA engagement by BAFF cannot account for the ability of BAFF to induce CSR in TACI–/– B cells because these cells are unable to undergo CSR in response to APRIL, which has a higher affinity for BCMA in mice than BAFF (6). Thus, induction of CSR by BAFF in TACI–/– B cells indicates that BAFF-R engagement can activate CSR and suggests that BAFF may use both TACI and BAFF-R to induce CSR. We cannot rule out the possibility that BCMA synergizes with BAFF-R in mediating BAFF-induced CSR in TACI–/– B cells. Negatively sorted TACI–/– B cells stimulated with BAFF consistently failed to secrete IgA (Fig. 2 B), and we were unable to detect in these B cells induction of molecular events involved in IgA switching, including expression of GLT (Fig. 3). Since intracellular signaling by TACI differs from that triggered by BAFF-R (6), it is possible that TACI signals are indispensable for the activation of the I promoter and induction of IgA switching. APRIL induction of CSR is independent of BAFF–BAFF-R interaction Given the observation that BAFF-R engagement can activate CSR, it was important to rule out the possibility that APRIL-mediated switching involved, in addition to TACI, engagement of BAFF-R by BAFF which may be endogenously made by B cells. Unstimulated B cells expressed small amounts of BAFF mRNA as assessed by RT-PCR (Fig. S3, available at http://www.jem.org/cgi/content/full/jem. 20032000/DC1). Stimulation with APRIL or APRIL + IL-4 caused no detectable increase in BAFF mRNA expression. More importantly, we examined the ability of APRIL to induce isotype switching in B cells from A/WySnJ mice, which carry a mutation in BAFF-R (10). These mice have very few peripheral B cells with a decreased proportion of mature CD23+ B cells. To examine CSR under culture conditions similar to those used for WT, BCMA–/–, and TACI–/– B cells (i.e., same cell number and density), we examined B cells from pooled splenocytes of 4 A/WySnJ mice. APRIL and BAFF induced IgG1 and IgA secretion in B cells from these mice (Fig. S3). These results suggest that APRIL-mediated CSR does not involve autocrine BAFF–BAFF-R interactions and that TACI engagement is sufficient to induce CSR. We cannot rule out the possibility that TACI synergizes with a putative APRIL-specific receptor to cause CSR. Binding of TRAF2 and/or TRAF3 is essential for CD40-mediated CSR, whereas TRAF6 is important in plasma cell differentiation (11, 12). TACI, like CD40, recruits TRAF 2, 5, and 6 (6). This may explain its ability to activate CSR. BAFF-R binds TRAF3 but no other TRAF protein (6). TRAF3 may be important for CSR induced by the EBV protein LMP-1 (13). It is possible that CSR induced by BAFF in TACI–/– B cells involves a cooperative interaction between BAFF-R and BCMA, which recruits TRAF1, 2, and 3 proteins (6). The fact that BCMA fails to activate CSR may be due to the fact that the majority of BCMA has a low surface density and most of it is intracellular (6). Alternatively, non-TRAF signals may be important for CSR but may not be delivered by BCMA. A clue to the physiological role of APRIL- and BAFF-mediated CSR is provided by results obtained on mice deficient in these ligands and their receptors. BAFF–/– and BAFF-R–/– mice are severely deficient in B cells (6) and not informative. BCMA–/– mice have normal serum Ig levels and normal antibody responses (14). This is consistent with our data that B cells from these mice switch normally in response to BAFF and APRIL. TACI–/– mice have low serum IgA and deficient antibody responses to immunization with type II T-independent antigens (15, 16). This is consistent with the failure of B cells from these mice to secrete IgA in response to BAFF and APRIL. We have shown that APRIL–/– mice have a selective IgA deficiency and decreased serum IgA antibody responses to mucosal immunization with TD antigen (17). This suggests that APRIL and BAFF play nonredundant roles in IgA switching in vivo. Since serum IgA levels are normal in CD40–/– mice (11, 18), APRIL–BAFF–TACI interactions play an important role in physiologic IgA switching and could be manipulated therapeutically to enhance antibody responses to oral vaccines. MATERIALS AND METHODS Mice CD40–/–, BCMA–/–, and TACI–/– mice were described previously (14, 16, 19). A/WySnJ mice that carry a mutation in BAFF-R were purchased from Jackson Laboratories. All mice were kept in a specific pathogen-free animal facility. In vitro isotype switching Spleen cells from CD40–/–, BCMA–/–, and TACI–/– mice were labeled with a cocktail of biotin-conjugated mAbs to CD43, CD11b, Thy1.2, CD138, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE and negatively sorted with Streptavidin magnetic beads (Dynal). B cells were cultured at 106/ml in RPMI containing 10% FCS, L-glutamine, and 50 μM 2-ME (complete medium). For Ig synthesis, B cells were cultured in complete medium alone or in the presence of 1 μg/ml sAPRIL (R&D Systems), 1 μg/ml sBAFF (Alexis), IL-4 (50 μg/ml; R&D Systems), TGF? (R&D Systems), 10 μg/ml LPS (Sigma-Aldrich), or 1 μg/ml CD40 (BD Biosciences). Neutralizing antibodies IL-6, IL-10, and TGF? (R&D Systems) were used as suggested by the manufacturer. After 6 d, supernatants were assayed for IgA, IgE, and IgG1 by ELISA (11), and genomic DNA was prepared for DC-PCR. IgG1 surface expression B cells stimulated for 6 d as above were stained with B220-FITC and IgG1 biotin–conjugated mAbs followed by staining with PE-conjugated Streptavidin and FACS analyis. RT-PCR for GLT, AID, and post switch transcripts (Iμ-CH) RNA was extracted from 4-d-cultured B cells using TRIzol (Invitrogen) and was reverse transcribed by Supercript II RT (Invitrogen). PCR primers used for 1, , and GLT, Iμ-C1, Iμ-C, Iμ-C, AID, and ?2-microglobulin were as described previously (11, 20). All PCR reactions were performed on three dilutions of cDNA (1:1, 1:3, and 1:9) for semiquantitative evaluation. Amplified products were separated on agarose gel and stained with ethidium bromide. DC-PCR Genomic DNA isolated from cultured B cells on day 6 was digested with EcoRI, circularized, and used as template for PCR using primers as reported previously for Sμ-S1, Sμ-S, Sμ-S, and the nicotinic acetylcholine receptor ? unit (11, 21). All PCR reactions were performed on three dilutions of circularized DNA (1:1, 1:3, and 1:9) for semiquantitative evaluation. Online supplemental material Figs. S1–S3 show additional analysis of BAFF- or APRIL-stimulated B cells. Supplemental Materials and methods describe CFSE staining, [3H]thymidine incorporation assay, polymycin B treatment, RT-PCR for BAFF mRNA, and isolation of Igm+ and IgD+ B cells. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20032000/DC1. Acknowledgments This work was supported by National Institutes of Health grants AI31136 and AI31541, the March of Dimes, and the Wallace Fund. The authors have no conflicting financial interests. Submitted: 19 November 2003 Accepted: 19 November 2004 References Castigli, E., S. Scott, F. Dedeoglu, P. Bryce, H. Jabara, A.K. Bhan, E. Mizoguchi, and R.S. Geha. 2004. Impaired IgA class switching in APRIL-deficient mice. Proc. Natl. Acad. Sci. USA. 101:3903–3908.[Abstract/Free Full Text] Castigli, E., F.W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A.K. Bhan, and R.S. Geha. 1994. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA. 91:12135–12139.[Abstract/Free Full Text] Hollander, G.A., E. Castigli, R. Kulbacki, M. Su, S.J. Burakoff, J.C. Gutierrez-Ramos, and R.S. Geha. 1996. Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice. Proc. Natl. Acad. Sci. USA. 93:4994–4998.[Abstract/Free Full Text] Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 102:553–563.[Medline] Zelazowski P, Carrasco D, Rosas FR, Moorman MA, Bravo R, and S. CM. 1997. B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching. J Immunol. 159:3133-3139.[Abstract]...查看详细 (16335字节)

☉ 11120217:Synergy of IL-21 and IL-15 in regulating CD8+ T ce
Abbreviations used: CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester; rFPVhgp100, recombinant fowlpox virus encoding hgp100; vPE16, vaccinia virus–expressing HIV gp160. The common cytokine receptor -chain (c) is mutated in X-linked severe combined immunodeficiency (1), a disease with severely impaired T cell and NK cell development and diminished B cell function (2). c is a critical component of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (2), which together regulate lymphocyte development and control a broad spectrum of activities that shape innate and acquired immune responses. The IL-21/IL-21 receptor (IL-21R) system is the most recently identified of these cytokine systems (3, 4). IL-21 is produced by activated CD4+ T cells, and its receptor is expressed on T, B, and NK cells. IL-21 is most closely related to IL-2, IL-4, and IL-15, and IL-21R is most similar to the IL-2 receptor ? chain (IL-2R?), which is a component of the IL-2 and IL-15 receptors. Based on in vitro assays, IL-21 was originally implicated as a regulator of T and B cell proliferation as well as of NK cell maturation (4), whereas another group later reported that IL-21 could inhibit NK cell expansion (5). IL-21R–/– mice have normal lymphocyte compartments, including normal NK cell development (5, 6), indicating that IL-21 is not essential for the development of lymphoid lineages, but leaving open the possibility that it contributes to this process in a potentially redundant fashion. Together with IL-4, IL-21 plays a critical role in regulating Ig production (6). IL-21R–/– mice have diminished IgG1, but greatly elevated IgE levels in response to antigen challenge, whereas IL-21R–/–IL-4–/– double knockout mice exhibit a severely impaired IgG response as well as diminished IgE levels, indicating a cooperative role of these two cytokines for Ig production (6). The size of naive and memory T cell pools is tightly regulated, at least in part, by growth and survival signals conferred by c-dependent cytokines (7, 8). IL-7 is crucial for the survival and homeostatic expansion of naive CD8+ T cells and also can contribute to memory CD8+ T cell homeostasis (9, 10). IL-15 potently promotes proliferation of memory CD8+ T cells (8, 11, 12); the major subset of memory (IL-2R?highCD44high) CD8+ T cells depends on IL-15 for survival and turnover, whereas the IL-2R?lowCD44high CD8+ T cells are IL-15 independent (13). IL-15 may also contribute to the homeostatic proliferation of naive CD8+ T cells (14, 15). In contrast, IL-2 can decrease memory CD8+ T cell function by inducing regulatory T cells (16–18). Late in the proliferative phase of the T cell response, activated T cells differentiate into effector T cells that produce critical effector molecules. We have now investigated the role of IL-21 in T cell homeostasis and effector functions. Results IL-21 synergistically acts with IL-15 to expand CD8+ T cells IL-21 was initially reported to costimulate anti-CD3–activated murine thymocytes and mature murine T cells in vitro and to enhance the proliferative effects of IL-2, IL-7, and IL-15 even without the addition of anti-CD3 (4). Interestingly, however, a second group reported that the addition of IL-21 blocked the IL-15–dependent, TCR-independent expansion of CD44highCD8+ T cells (5). To further explore the actions of IL-21 and how it integrates its signals with other c-dependent cytokines, we studied the effect of a range of concentrations of IL-15 and IL-21 on normal splenocytes cultured for 7 d. As reported previously (5), IL-21 inhibited IL-15–mediated expansion of resting NK cells; we observed a dose-dependent inhibition by IL-21 that was most evident at 100 ng/ml of IL-15 (Fig. 1 A, lanes 16 and 17 vs. 5 and 15; also, Fig. 1 E, e vs. c). However, in contrast with a previous paper (5), we observed a marked increase, rather than decrease, in the number of T cells after culture with both IL-21 and IL-15 as compared with IL-15 alone (Fig. 1 B, lanes 9–11 vs. 3, 12–14 vs. 4, and 15–17 vs. 5). The majority of these expanded cells were CD8+ T cells (Fig. 1 F, e vs. c). IL-21 by itself had little effect on T cell cellularity (Fig. 1 B, lanes 6–8 vs. 2), but again there was an increase in the percentage of CD8+ T cells (Fig. 1, C, lanes 6–8 vs. 2, and F, d vs. b). Although IL-15 alone induced an increase in the percentage of CD8+ T cells (Fig. 1 F, c vs. b), the absolute number of CD8+ T cells was, if anything, slightly less than the number on day 0 (Fig. 1 C, lane 5 vs. 1). Strikingly, after 7 d of culture with IL-15 plus IL-21, the total numbers of CD8+ T cells markedly increased (Fig. 1 C, lanes 9–17). IL-15 and IL-21 had a synergistic effect on CD8+ T cell proliferation, as a combination of low concentrations of these two cytokines (10 or 50 ng/ml) was more potent than the effect of either cytokine alone at a concentration of 50 or 100 ng/ml (Fig. 1 C, lane 9 vs. 4 and 7, and lane 13 vs. 5 and 8). In contrast with the effect on CD8+ T cells, cooperative effects on CD4+ T cells were not evident (Fig. 1 D). IL-21 also synergistically acts with IL-7, but not IL-2 Because IL-21 was reported to act in concert with IL-2 or IL-7 to enhance T cell proliferation (4), next we compared the effects of IL-2, IL-7, IL-15, and IL-21 on splenocytes cultured for 3, 5, and 7 d (Fig. 2). Consistent with the results of Fig. 1, IL-21 increased the total T cell and CD8+ T cell expansion mediated by IL-15, but it had no effect on CD4+ T cells (Fig. 2, A and B, lanes 25–27 vs. 13–15, and C, h vs. d, and not depicted). The synergy of IL-15 and IL-21 on CD8+ T cell expansion was evident as early as day 3 (Fig. 2 A, lane 25 vs. 13 and 16). Interestingly, IL-21 did not significantly increase expansion of CD8+ T cells when combined with IL-2 (Fig. 2 A, lanes 19–21 vs. 7–9), even though it increased the percentage of these cells (Fig. 2 C, f vs. b); however, it cooperated with IL-7, although not as potently as it did with IL-15 (Fig. 2, A, lanes 22–24 vs. 10–12, and C, g vs. c; Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20041057/DC1). The cooperative effects of IL-15 and IL-21 were confirmed in studies using IL-15–/– and IL-21R–/– mice (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20041057/DC1). We also evaluated the effect of IL-15, IL-21, and the combination of both cytokines on cell survival by staining with annexin V. CD8+ T cells cultured without any cytokine essentially all died within 7 d; however, the addition of IL-15, IL-21, or combinations of these cytokines resulted in >94% viability in all cases (Fig. 2 D, k–m vs. j). Thus, although the cytokines increased the viability, the synergistic effect on cell expansion seen in response to IL-15 plus IL-21 as compared with either cytokine alone primarily results from increased cell proliferation rather than a synergistic effect on survival. IL-15 and IL-21 cooperatively enhance the effector function of memory-phenotype CD8+ T cells Because IL-15 is known to expand memory-phenotype CD44highCD8+ T cells, we examined if IL-21 could enhance IL-15–mediated expansion and function of these cells. IL-21 alone had little effect on CD44highCD8+ T cells, but the number and percentage of CD44lowCD8+ T cells was higher than was found in cultures without cytokines (Fig. 3 A, d vs. a and b). As expected, IL-15 expanded CD44high cells (Fig. 3 A, c vs. a and b), but the combination of IL-15 and IL-21 resulted in a striking further increase in CD44high cells (note percent and total cellularity; Fig. 3 A, e vs. c). To distinguish effector and central memory-phenotype CD8+ T cells (11), we stained cells with anti-CD62L mAb. Cells stimulated with IL-15 were primarily central memory-phenotype (CD62LhighCD44high; Fig. 3 B, c vs. a and b), whereas those stimulated with IL-21 lacked high expression of CD44 (Fig. 3 B, d). When both IL-15 and IL-21 were added, CD62LhighCD44high cells were expanded, analogous to what was seen with IL-15 (Fig. 3 B, e vs. c); in addition, a prominent CD62LlowCD44high population of cells was evident, which may represent effector memory-phenotype cells (Fig. 3 B, panel e). These data suggest that IL-21 contributes to the expansion of both subsets of memory-phenotype CD8+ T cells. To characterize the effector function of cytokine-expanded CD8+ T cells, we examined intracellular IFN- levels after stimulation with anti-CD3 and anti-CD28. The combination of IL-15 and IL-21 resulted in a marked increase in the number of CD8+ T cells, with a modest increase in the percent of IFN-–producing cells at 1, 2, and 4 h, as compared with that seen in cells expanded with IL-15 alone (Fig. 3 C, r–t vs. j–l), whereas IL-21 by itself had little effect (Fig. 3 C, n–p). As shown in Fig. 3 D, the combination of IL-15 and IL-21 greatly increased the total number of IFN-–producing CD8+ T cells. Thus, IL-21 has a cooperative effect with IL-15 on the expansion of memory-phenotype CD8+ T cells as well as on their effector function. IL-21 and IL-15 together markedly accelerate cell division of both memory-phenotype and naive-phenotype CD8+ T cells In the aforementioned in vitro culture assays, we used total splenocytes. Thus, it is possible that CD4+ T cells or other cells might contribute to the expansion of CD8+ T cells via paracrine secretion of cytokines, and that IL-15 and IL-21 might act on other cells to indirectly affect the expansion/survival of CD8+ T cells. To investigate whether IL-15 and IL-21 had a direct synergistic effect on cell cycle progression in CD8+ T cells, we isolated CD8+ T cells from splenocytes and stained them with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). These cells divided faster when cultured with both IL-15 and IL-21 as compared with either cytokine alone (Fig. 4 A, d vs. b and c, and h vs. f and g). On day 4, cells treated with both IL-15 and IL-21 exhibited one to five more divisions than cells in control cultures and the recovered cell number was significantly higher (Fig. 4 A, d vs. a–c). On day 7, these effects were even greater (Fig. 4 A, h vs. e–g). The combination of IL-7 and IL-21 had a similar albeit less potent effect to that seen with IL-15 + IL-21 (unpublished data). To further examine the effect of IL-15 and IL-21 on memory-phenotype or naive-phenotype CD8+ T cells, we purified CD44high and CD44low CD8+ T cells, respectively, and stained with CFSE. IL-15 more potently promoted the division of CD44highCD8+ T cells than did IL-21, but both cytokines together had the greatest effect (Fig. 4 A, l vs. i–k, and p vs. m–o). The cell number was four- to fivefold higher in culture with IL-15 + IL-21 than with IL-15 alone on day 4 (Fig. 4 A, l vs. j). The relative fold increase on day 7 was reproducibly lower than on day 4; this might result from a decrease in the survival rate on day 7 (Fig. 4 B, h vs. f). Neither IL-15 nor IL-21 alone had much of an effect on the division of CD44lowCD8+ T cells (Fig. 4 A, r and s vs. q, and v and w vs. u), but each improved the cell survival rate (Fig. 4 B, j and k vs. i, and n and o vs. m). Strikingly, stimulation with both IL-15 and IL-21 potently promoted cell division (Fig. 4 A, t vs. r and s, and x vs. v and w) and cell survival (Fig. 4 B, l vs. j and k, and p vs. n and o) of CD44lowCD8+ T cells. Thus, IL-21 and IL-15 synergistically affect cell division of memory-phenotype CD8+ T cells. Moreover, in contrast with IL-15 alone, IL-21 + IL-15 increased the division of naive-phenotype CD8+ T cells as well. Defective antigen-specific CD8+ T cell responses in IL-21R–/– mice To further examine the effect of IL-21 on in vivo expansion and effector function of antigen-specific CD8+ T cells, both wild type and IL-21R–/– mice were immunized with vaccinia virus expressing HIV gp160 (vPE16). The cytotoxic activity of CD8+ T cells from IL-21R–/– mice was significantly lower than CD8+ T cells from wild-type mice (Fig. 5, A and B), suggesting that IL-21R signaling contributes to the primary CD8+ cytotoxic T cell response. Correspondingly, the frequencies of tetramer-positive (Fig. 5 C) and IFN-–positive (Fig. 5 D) CD8+ T cells, as analyzed immediately ex vivo or after 1 wk of restimulation in vitro, were lower in IL-21R–/– mice than in wild-type mice. The difference in the frequency of antigen-specific cells (Fig. 5, C and D) can at least in part account for the difference in CTL activity. These results demonstrated a role for IL-21 in antigen-specific CD8+ T cell expansion and function. Cooperative effect of IL-21 and IL-15 on tumor regression, with cures of established B16 melanomas Given the effects of IL-15 and IL-21 on CD8+ T cell expansion and cytotoxicity in vitro, next we investigated the effects of these cytokines in vivo using a tumor model. IL-21 has been reported to have antitumor effects (19–22). We used a tumor immunotherapeutic strategy that allowed us to evaluate the effects of cytokines on the regression of large, established solid tumors when cytokines and CD8+ T cells specific for the gp100 self-antigen were administered concomitantly (23–25). pmel-1 transgenic mice on a C57BL/6 background express the V1V?13 TCR from a cloned T cell, which recognizes an H-2Db–restricted epitope corresponding to amino acids 25–33 of gp100 (23). More than 95% of the CD8+ T cells in pmel-1 TCR transgenic mice were V?13+, amounting to 20% of all splenocytes (23). In vitro–activated pmel-1 splenocytes were adoptively transferred into sublethally irradiated mice bearing subcutaneous B16 melanomas (an H-2Db gp100+ spontaneous murine melanoma) established for 8 or 10 d (Fig. 6, A and B). This adoptive transfer was followed by vaccination with recombinant fowlpox virus encoding hgp100 (rFPVhgp100), which encodes the hgp100-altered peptide ligand, in the presence of exogenous cytokines. IL-15 + IL-21 (without pmel-1 cells) had little if any effect on the tumor (Fig. 6 A), indicating that reconstituted endogenous lymphocytes have no significant effect on tumor regression. Pmel-1 cells with IL-15 + IL-21 also had relatively little effect on the tumor unless vaccination with rFPVhgp100 was included (Fig. 6 A). This confirms that antigen-specific cells, vaccination, and cytokine are all required for maximal tumor regression in this model (23). As shown in Fig. 6 B, treatment with either IL-15 or IL-21 induced partial tumor regression, whereas the combination of IL-15 and IL-21 was much more effective. In the experiment shown in Fig. 6 B, all of the mice died of tumor within 32 d of treatment except for those treated with the combination of both IL-15 and IL-21 (not depicted). Moreover, all five mice receiving IL-15 plus IL-21 were alive at day 32; two of these animals had complete regression of their tumors with vitiligo at the former melanoma sites, whereas the other three mice had residual tumor. In an independent experiment, treatment with 5 μg/dose of IL-15 plus 5 μg/dose of IL-21 was more effective than either cytokine alone at 10 μg/dose (unpublished data). Consistent with an enhanced effect of IL-15 plus IL-21 on CD8+ T cells, at 3 and 4 wk of treatment, the absolute number of pmel-1 TCR transgenic CD8+ T cells (V?13+ CD8+) in blood was higher in mice treated with both IL-15 and IL-21 than with either cytokine alone (Fig. 6 C, left). These findings were confirmed in a second experiment at day 20; day 28 data are not available for this experiment as it was terminated at day 23, at which point four out of six mice treated with the combination of IL-15 and IL-21 had complete regression of their tumors (unpublished data). Thus, IL-15 and IL-21 synergistically expand CD8+ T cells in vivo and this correlated with marked regression of large, established solid tumors. A subset of genes is synergistically regulated by IL-21 and IL-15 To begin to clarify the molecular mechanisms by which IL-21 cooperates with IL-15 on CD8+ T cell expansion and function, next we performed DNA microarray analyses (Affymetrix, Inc.) using mRNAs isolated from naive CD8+ T cells treated for 4 h with cytokines, and we found 300 genes that were regulated by IL-15 and/or IL-21. The overall expression pattern seen in CD8+ T cells stimulated with both IL-15 and IL-21 is more similar to that stimulated with IL-21 than IL-15 (Fig. 7 A). However, as expected, some genes were regulated by the combination of IL-15 and IL-21 in similar fashion to that seen with either cytokine alone (Fig. 7, B and C), whereas other genes, such as granzyme B and c-Jun, exhibited induction or repression that was greater with IL-15 plus IL-21 than with either cytokine alone (Fig. 7 D and see Discussion). Discussion In this paper, we demonstrate that T cells are markedly expanded by IL-21 in synergy with IL-15 or IL-7, and that this expansion is most potent for CD8+ T cells. IL-15 and/or high doses of IL-7 are known to be required for memory (CD44high) CD8+ T cell survival and proliferation (8). Our results indicate that both memory-phenotype and naive-phenotype CD8+ T cells are expanded by IL-21 + IL-15 and that IL-21 is necessary for an optimal CD8+ T cell response to antigen. Kasaian et al. reported an enhancement of the antigen-driven CD8+ T cell response by IL-21 but surprisingly found no effect of IL-21 on IL-15–mediated, TCR-independent T cell expansion (5), which differs from our results. The concentration of IL-21 in their paper is not defined in nanograms per milliliter as they used conditioned medium from transfected COS cells as a source of IL-21 and, thus, we speculate that the amount of IL-21 used in their work may not have been sufficient to achieve the synergistic effect that we observed. At higher levels of IL-15, we observed marked synergy even with 10 ng/ml of IL-21, and importantly this synergistic effect of IL-15 and IL-21 on expansion in vitro of purified CD8+ T cells is consistent with the effect that we observe in vivo. How do IL-15 and IL-21 regulate CD8 T cell expansion and effector functions? Our results indicate that IL-15 and IL-21 together accelerate cell division of isolated CD8+ T cells. Although more work is needed to clarify which genes mediate the synergistic actions of IL-15 and IL-21, it is interesting that granzyme B, which plays an important role for cytotoxicity, and c-Jun, which is important for optimal proliferation, are both preferentially induced by the combination of IL-15 and IL-21. Homeostatic control of CD8+ T cells is essential for defense against infectious pathogens. IL-15 is known to be a critical regulator of memory CD8+ T cell homeostasis and might also contribute to naive CD8+ T cell survival. IL-7 is required for naive CD8+ T cell survival and homeostatic proliferation but also contributes to memory CD8+ T cell homeostasis. We have now identified IL-21 as a new regulator of these cells, suggesting that it is yet another c-dependent cytokine that critically regulates T cell homeostasis. Although IL-21 alone showed little effect on CD8+ T cells, it synergistically promoted the proliferation and survival of both memory and naive CD8+ T cells. Our data are consistent with a cooperative effect of IL-15 and IL-21 on the generation and expansion of cytotoxic T cells, as would occur, for example, after viral infection. Although IL-15 was previously shown to cooperate with IL-21 in preventing the establishment of a murine lymphoma, our data demonstrate that synergistic actions of IL-15 and IL-21 can result in complete regression of large established B16 melanomas, with an associated expansion of tumor-specific CD8+ T cells. This supports the previously suggested role of IL-15 as an antitumor agent (26). A recent paper suggested that IL-21 contributed to the homeostatic expansion of T cells, but that it could not support their survival (27). Our experiments collectively indicate that IL-21 has both proliferative and cell survival effects for CD8+ T cells and that its effects on expansion are greatly augmented when IL-15 is also added. Although CD8+ T cells expanded in vitro using IL-2 or IL-15 can be reintroduced in vivo to augment the killing of tumor cells (28, 29), our results indicate that the combination of IL-21 with IL-15 may be a more powerful method for expanding CD8+ T cells, both in vitro and in vivo, and enhancing CD8+ T cell function. Materials and Methods Mice. WT mice (C57BL/6 or Balb/c) were obtained from the National Cancer Institute, IL-15–/– mice were purchased from Taconic Laboratory, and IL-21R–/– mice were described previously (6). Mice were analyzed at 8–16 wk of age. All experiments were performed under protocols approved by the appropriate Animal Use and Care Committees and followed the National Institutes of Health (NIH) guidelines entitled "Using Animals in Intramural Research." In vitro cell culture and survival assay. Single cell suspensions of spleen were prepared by gently pressing the tissues through fine nylon screen. Erythrocytes were depleted with ACK lysis buffer (BioFluids). CD8+ T cells were prepared as described in the next paragraph. Cells were plated at 5 x 105/ml in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 50 μM ?-mercaptoethanol (RPMI 1640 complete medium) with human IL-2 (Roche), murine IL-7 (PeproTech), human IL-15 (PeproTech), or murine IL-21 (R&D Systems) as indicated. Cells were cultured at 37°C for 3, 4, 5, or 7 d, and a second dose of cytokines was added on day 4. Cells were counted and analyzed by flow cytometry on the indicated day. The cell survival assay was performed using annexin V–FITC or annexin V–Biotin Apoptosis Detection kit according to the manufacturer's protocols (R&D Systems). CD8+ T cell isolation and labeling with CFSE. CD8+ T cells were positively selected using paramagnetic Microbeads conjugated to anti–mouse CD8 (Ly-2) monoclonal antibody according to the manufacturer's instructions (MACS; Miltenyi Biotec). To purify CD44high and CD44low CD8+ T cells, CD8+ T cells were negatively selected using paramagnetic Microbeads conjugated to anti–mouse CD4 (L3T4) and anti–mouse CD45R (B220) monoclonal antibodies. The resultant cells were labeled with anti-CD8–allophycocyanin, anti-CD44–CyChrome, and anti–IL-2R? (CD122)–PE and sorted for CD44high and CD44lowCD8+ T cells on a MoFlo Cell Sorter (DakoCytomation). The resulting populations were >95% pure. Isolated CD8+ T cells were labeled with 5 μM CFSE (Molecular Probes) for 15 min at 37°C. Immunization of mice with HIV-1IIIB gp160 and measurement of cytotoxicity and tetramer+ and IFN-+ CD8+ T cells. The recombinant vaccinia virus expressing the full-length HIV-1IIIB gp160 (vPE16) was described previously (30). Wild-type and IL-21R–/– mice were immunized i.p. with 5 x 106 PFU of vPE16. The immunodominant peptide epitope (RGPGRAFVTI; known as the P18-I10 peptide) within HIV-1IIIB gp160 in H-2Dd mice (31, 32) was synthesized (Multiple Peptide Systems). Splenocytes from the immunized mice were cultured at 4 x 106 cells/well in 24-well plates containing 2 ml of RPMI 1640 complete medium supplemented with 10% rat T-stim (Collaborative Biomedical). To stimulate peptide-specific CD8+ T cells in vitro, 1.0 μM or 0.001 μM P18-I10 peptide was added into the cultures. On day 7, CTL activity was measured using a 5-h 51Cr release assay. P815 cells, which were maintained in RPMI 1640 complete medium and pulsed with 1.0 μM or 0.001 μM P18-I10, were used as target cells. The percent specific lysis was calculated as 100 x (experimental release-spontaneous release)/(maximum release-spontaneous release). Maximum release was determined from supernatants of cells that were lysed by the addition of 2.5% Triton X-100. For P18-I10 H-2Dd tetramer staining, cells were incubated with FITC-labeled anti-CD8 for 30 min, PE-labeled P18-I10-H-2Dd-tetramer (provided by the NIH Tetramer Core Facility, Atlanta, GA) was added, and the cells were incubated for an additional 30 min on ice. The tetramer was used at dilutions of 1:200 or 1:300 for fresh spleen cells and 1:50 for in vitro–restimulated cells. Background staining was assessed by use of an isotype control antibody. For IFN- induction, cells were stimulated with 1.0 μM P18-I10 for 10 h in the presence of 1 μg/ml brefeldin A. Intracellular IFN- staining. Splenocytes were cultured in RPMI 1640 complete medium in 96-well plates at 2 x 105 cells/well containing no cytokine, IL-15, IL-21, or both cytokines. After 7 d, cells were stimulated for 1, 2, or 4 h with 2 μg/ml of soluble anti-CD3 and 2 μg/ml anti-CD28, and stained for cell surface markers. The cells were fixed and permeabilized using Cytofix/Cytoperm solution, followed by staining with PE-conjugated IFN- mAb (BD Biosciences) as described previously (33). Flow cytometric analyses Cells were stained and analyzed on a FACSCaliber or FACSort with CellQuest software (BD Biosciences). The following mAbs, all from BD Biosciences, were used: anti-CD4–FITC, anti-CD8–allophycocyanin, anti–mouse CD8-FITC, anti-TCR?–allophycocyanin, anti–IL-2R? (CD122)–FITC, anti–IL-2R? (CD122)–PE, anti-CD44–CyChrome, anti-B220–FITC, anti-NK1.1–PE, anti-V?13–FITC, and anti-CD44–PE. Immunotherapy of B16 melanoma Sublethally irradiated (500 rad) female C57BL/6 mice (The Jackson Laboratory) were injected subcutaneously with 3 x 105 mycoplasma-free B16-F10 melanoma cells. B16 is an H-2b gp100+ spontaneous murine melanoma and was maintained in RPMI 1640 complete medium. 8–10 d later, animals (n = 5–7 for each group) were treated by intravenous injection of in vitro–cultured splenocytes (0.5–1 x 106 V?13+CD8+ T cells) from pmel-1 TCR transgenic mice (23). For culturing, fresh splenocytes from pmel-1 mice were depleted of erythrocytes and cultured in RPMI 1640 complete medium containing 2 ng/ml of human IL-2 (Chiron Corp.) and 1 μM hgp10025-33. Cells were used for adoptive cell transfer (ACT) 6–7 d later. Where indicated, mice were also immunized with 2 x 107 PFU of rFPVhgp100 (Therion Biologics; reference 23). 5–10 μg IL-15 or IL-21 was freshly reconstituted in PBS and administered by i.p. injection twice daily, beginning the day of adoptive transfer, for 3 d. Tumors were measured in a blinded fashion using calipers, and the products of the perpendicular diameters were calculated. Tumor size (rank sum test) and survival data (Kaplan-Meier) were recorded for over 4 wk after treatment and analyzed. For quantifying pmel-1 T cells, for each group, treated mice were bled by tail vein, samples were pooled, and total lymphocyte numbers and flow cytometric profiles for V?13 and CD8 were determined by the NIH Clinical Center Clinical Immunology Laboratory. RNA purification and Affymetrix Gene Chip analysis. Total RNA was isolated (RNeasy; QIAGEN) from naive CD8+ T cells with 4 h of stimulation of cytokines (100 ng/ml each), processed to cRNA probes for gene chip analysis, and probes were hybridized to U430A GeneChips (Affymetrix, Inc.; these chips contain oligonucleotides corresponding to 22K transcripts per microarray), washed, and scanned (Hewlett Packard Gene Array scanner G2500A) according to procedures outlined by the manufacturer (Affymetrix, Inc.). Data were analyzed with open source clustering software Cluster 3.0 (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/index.html; reference 34). Online supplemental material. Fig. S1 shows that IL-21 cooperated with IL-7 but not as potently as it does with IL-15 on CD8+ T cell expansion. Fig. S2 shows that synergistic expansion of CD8+ T cells is diminished in both IL-15–/– and IL-21R–/– mice. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20041057/DC1. Acknowledgments We thank members of our laboratories for valuable advice and discussions; J. Bollenbacher, C. Robinson, P.J. Spiess, and D. Surman for technical support; National Heart, Lung, and Blood Institute Flow Cytometry Core Facility for cell sorting, and J.-X. Lin, H.–P. Kim, H.-H. Xue, K. Zhao, A. Sher, C. Feng, and C. Klebanoff for valuable discussions and/or critical comments. This work was performed at the National Institutes of Health. The authors have no conflicting financial interests. Submitted: 28 May 2004 Accepted: 23 November 2004 References Takeshita, T., H. Takahashi, S. Kozlowski, J.D. Ahlers, C.D. Pendleton, R.L. Moore, Y. Nakagawa, K. Yokomuro, B.S. Fox, D.H. Margulies, et al. 1995. Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule. J. Immunol. 154:1973–1986 Kelly, J., R. Spolski, K. Imada, J. Bollenbacher, S. Lee, and W.J. Leonard. 2003. A role for Stat5 in CD8+ T cell homeostasis. J. Immunol. 170:210–217Eisen, M.B., P.T. Spellman, P.O. Brown, and D. Botstein. 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA. 95:14863–14868...查看详细 (29209字节)
☉ 11120218:Yellow fever 17D as a vaccine vector for microbial
Abbreviations used: 17D, yellow fever vaccine 17D; CS, circumsporozoite protein; EEF, exoerythrocytic stage; MVA, modified vaccinia virus Ankara; MOI, multiplicity of infection. D. Tao and G. Barba-Spaeth contributed equally to this paper. Many human vaccines that are currently in use require multiple injections to be fully effective over the long term. This requirement is a severe hurdle for their utilization, particularly in underdeveloped countries where they are much needed. One notable exception is the yellow fever vaccine using the attenuated YF17D strain (17D). Developed in the 1930s, this live, highly attenuated viral vaccine has been given to hundreds of millions of individuals with minimal risk of severe side effects. Administration of a single dose of this genetically stable vaccine confers excellent protection that persists for 30 or more years, perhaps lifelong (1). The mechanisms of protection are not well understood, but they involve the production of neutralizing antibodies and probably the production of T cell–mediated immune mechanisms (2, 3). In recent years, the remarkable properties of 17D vaccine have been exploited to create candidate vaccines against other flavivirus-mediated diseases by exchanging the premembrane and envelope genes of 17D with those of Japanese encephalitis, dengue types 1–4, and West Nile viruses. These chimeric viruses replicate to high titers in cell culture, elicit protective immunity in rodents and monkeys (4, 5), and retain or surpass the monkey neurovirulence safety standards set for 17D (6). Phase I human trials for some of these hybrid vaccines have been initiated. However, the 17D vaccine has not been used as a vector to deliver epitopes from unrelated microbial pathogens. The only exception has been the successful insertion of the immunodominant protective B cell epitope of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, into the envelope protein of 17D. The genomic stability of that recombinant virus, named 17D/8, was confirmed by serial in vitro passages. Immunization of mice with 17D/8 led to long lasting production of antibodies to P. falciparum sporozoites. In addition, immune serum neutralized the infectivity of wild-type YF virus (7). Here, we studied the immunogenicity of a recombinant 17D virus expressing the protective H-2Kd–restricted CTL epitope of the CS protein of P. yoelii, a rodent malaria parasite (8). In this experimental model, a rapid and quantitative assessment of protective immunity is feasible upon sporozoite challenge of immunized mice. Moreover, the efficacy of different viral vaccine constructs can be compared. This experimental model has provided the basis for the development of various human malaria vaccine candidates that underwent clinical trials (9, 10), as well as some currently undergoing human trials. Results Insertion of the malaria CD8+ T cell epitope in the genome of 17D We inserted a 10-amino acid-long H-2Kd CTL epitope of the CS protein of P. yoelii (SYVPSAEQIL) in 17D polyprotein in frame between the NS2B and NS3 nonstructural proteins (Fig. 1 A). The foreign epitope sequence was flanked by viral protease recognition sites in order to release the epitope in the cytoplasm of the host cell leaving unaltered functional viral replicase proteins. This strategy and the insertion site were chosen based on a previous observation showing that an ovalbumin epitope, inserted in the same site, yielded viable recombinant virus that elicited the production of ova-specific CTL and protection in a rodent tumor model (11). The recombinant construct was designated 17D-Py and the parental 17D2B/3 (referred to as 17D in this paper). The infectivity of RNAs transcribed from linearized plasmid templates was tested by electroporation of human SW-13 cells. Both the 17D-Py and the parental 17D RNAs had similar specific infectivities of 5 x 105 PFU/μg RNA (Fig. 1 B). 17D-Py plaques were slightly larger than those of the parental virus. We compared the growth kinetics of 17D-Py and 17D after infecting SW-13 cells at either low (0.1 PFU/cell) or high (5 PFU/cell) multiplicities of infection (MOIs). Interestingly the 17D-Py recombinant exhibited faster growth kinetics at both MOIs than the 17D parent, although similar plateau titers were obtained for both viruses. To assess the stability of the inserted sequence in cell culture, transfection supernatants were used to infect naive SW-13 cells at low MOI (0.1 PFU/cell). The resulting virus-containing supernatant was harvested and analyzed for the presence of the CTL epitope by RT-PCR, restriction digestion, and sequence analysis. As shown in Fig. 1 D, the RT-PCR products were of the expected sizes and the fragment from 17D-Py was digested to completion with SspI, which is present in the inserted P. yoelii sequence. Sequence analysis of the RT-PCR products revealed the expected sequences. In vivo stability of 17D-Py Young mice are highly permissive for flavivirus infection and succumb to virus-induced paralysis. To examine the stability of the recombinant virus in vivo, we injected 105 PFU of the recombinant 17D-Py or parental 17D viruses i.p. into suckling mice. All of the mice eventually became paralyzed by 10–14 d after infection. The infectious virus could be recovered from brain homogenates of the moribund animals, with high viral titers ranging from 5 x 105 to 5 x 106 PFU per brain. Viral RNA was recovered from clarified brain homogenates and assayed for the presence of the insert as described above. As found in cell culture, the expected RT-PCR products were obtained, and the 17D-Py product was digested with SspI (Fig. 1 D). In addition, sequencing of the RT-PCR products revealed the expected input sequences. These analyses indicate that the P. yoelii insert is still present in the recombinant virus after multiple cycles of replication in vivo. A single dose of 17D-Py elicits long-lasting immunity against malaria Groups of 6–10-wk-old BALB/c (H-2Kd) mice were immunized with a single viral dose by the s.c. route, using 20-fold dilutions of viral doses, from 102 PFU to 106 PFU of 17D-Py per mouse. 2 wk later, the mice were challenged intravenously with 20,000 sporozoites. After 40–42 h, close to the near completion of the exoerythrocytic stage (EEF) cycle, the animals were killed, and the efficacy of immunization was evaluated by enumerating IFN-secreting CS-specific CD8+ T cells in the spleen, and by measuring the parasite burden in the liver, as compared with controls immunized with parental 17D (105 PFU). As shown in Fig. 2, and in several subsequent experiments, immunization with 105 PFU elicited 67% inhibition of EEF development. Notably, a considerable degree of inhibition (52%) was obtained with viral doses as low as 5 x 103 PFU (Fig. 2). Heterologous prime/boost regimens can achieve sterilizing immunity We first tried a series of homologous boost regimens with 17D-Py. No significant differences in parasite burden were seen in mice given either one dose or five consecutive daily injections of 104 or 105 PFU of 17D-Py (Fig. 4 A, groups A and B, respectively). In mice boosted with 105 PFU 3 wk after priming, the EEF burden was significantly reduced, which correlated with an increase in the number of IFN-secreting CS-specific CD8+ T cells (Fig. 4, A and B). The highest degree of protection (close to 90% reduction in parasite burden) was obtained when the smallest viral dose (103 PFU) was used for priming (Fig. 4, group E). It is well documented that the prime/heterologous boost strategy elicits very strong immune responses when using viral vaccine vectors. In previous experiments, the vaccinia virus or the modified vaccinia Ankara virus (MVA) expressing the CS of P. yoelii greatly enhanced primary responses to influenza or Sindbis virus expressing the same CD8+ T cell malaria epitope (12–15). We found that priming mice with different doses (102 –105 PFU) of 17D-Py, and boosting 2 wk later with a single intravenous dose (107 PFU) of MVA-PyCS elicited large numbers of CS-specific IFN-secreting CD8+ T cells, antibodies to sporozoites (IFA titers, 1,600-3,200), and very effective protection (Fig. 5, A and B). Close to a 100% decrease in parasite burden in the liver was seen when the challenge was given 2–16 wk after priming with 105 PFU. This protection lasted for at least 16 wk after boosting, which is when the experiment was terminated (Fig. 6, A and B). In another series of experiments, we sought to determine whether the prime/sporozoite boost regimen led to sterile immunity. The animals were challenged i.v. with 75 sporozoites, and we examined them daily for parasitemia for 2 wk. Sterile immunity was achieved in 9 out of 10 sporozoite-boosted animals. This protection lasted at least 16 wk after the sporozoite booster (Table II). In contrast, all control mice primed with either 17D-Py or vaccinated once with irradiated sporozoites became parasitemic. Mechanism of protection As aforementioned, the prime/boosting regimen led to the production of antibodies to CS as well as IFN-secreting CD8+ T cells. To determine the protective mechanisms, groups of mice were injected with antibodies to CD4+ or CD8+ to deplete these T cell compartments just before challenge. After ablation of CD8+ T cells, protection was abolished, but depletion of CD4+ T cells had no effect as determined by ELISPOT and tetramer assays (Fig. 8). Discussion Here, we use for the first time the highly attenuated YF17D as a vaccine vector to deliver a microbial T cell epitope. The YF17D virus, like other flaviviruses, is encoded by a single positive stranded RNA molecule. Translation of the viral message produces a polyprotein precursor that is cleaved by viral and cellular proteases (16). We inserted a CTL epitope of the CS protein of P. yoelii between the two nonstructural 17D viral proteins, NS2B and NS3, and generated the recombinant virus 17D-Py. The most important observation of this paper is that the injection of a single dose of 105 PFU of 17D-Py elicited a long lasting, high level of protection against challenge with highly infective sporozoites of P.yoelii. A significant degree of protection was seen with doses as small as 103 PFU. Other viral vectors bearing the same CTL epitope of CS has been used in rodent malaria models. However, like in other human vaccines currently in use, the T cell response after priming alone was of short duration. More importantly, protection against sporozoite challenge usually required one or more booster injections. Therefore, it appears that the immunogenicity in mice of T cell epitope inserted in the YF17D vaccine is distinctive and mimics the immunogenicity of the 17D vaccine in humans; i.e., it is long lasting and effective after priming. Our findings provide strong support for using 17D as a general vaccine vector, even though there is scant information on the molecular mechanisms leading to the long-term memory that it evokes in humans. It is generally believed that protection in vaccinated humans is mediated in large part by neutralizing antibodies against the envelope protein E and complement-fixing antibodies to NS1, but the functional epitopes have not been identified. Human CTL responses to the vaccine have been documented (2), but there is no evidence that these vaccine-induced T cell responses have a role in protection. However, in mice, T cell epitopes in the envelope protein E and NS3 proteins are targets of antiviral responses (3). Regardless of the protective mechanism, vaccination with 17D seems to be sufficient for triggering the programs of expansion and differentiation of long-lived memory cells. The mechanism of induction of primary CD8+ T cell responses to the malaria epitope in 17D-Py is not known but currently under investigation. The small CTL epitope is inserted between NS2B and NS3 and should be released in the cytoplasm of the infected cell. In vivo, the 17D vaccine is administrated subcutaneously and may infect professional APCs (pAPCs), such as dendritic cells, and/or parenchymal cells. However, CTL responses can only be generated by pAPCs. If the virus multiplies in a parenchymal cell, its remnants and viral antigens need to enter the pAPCs to initiate a primary response via cross-presentation. However, several independent lines of evidence show that short-lived peptides such as the malaria epitope cannot be cross-presented (17). The implication is that only the 17D-infected pAPCs participate in the initial sensitization of T cells. It will be of interest to determine the nature of the cells that are initially infected by 17D. A preference for pAPCs would provide an explanation for its unusually strong immunogenicity. In fact, dengue virus, which belongs to the same genus as YF, preferentially targets immature dendritic cells (18). Another important finding is that the resulting recombinant virus 17D-Py is not only replication competent, but also stable in vitro and in vivo, thus minimizing the concern that the heterologous insert would be eliminated or modified during viral replication. However, if 17D is to be used as a general vaccine vector for multiple CTL epitopes, it will be necessary to understand the size and sequence constraints for inserting several microbial sequences in the same or in other positions in the 17D genome. We have recently shown that three tandem repeats of the same P. yoelii epitope can be inserted between NS2B and NS3 (unpublished data). This 17D recombinant replicates with kinetics similar to the 17D parent and is also stable in vitro and in vivo. Therefore, the potential already exists for vaccination with mixtures of a few recombinant viruses, each containing strings of three different protective T cell epitopes, to elicit immune responses in a large proportion of humans. The present results are of particular relevance to the development of preerythrocytic human malaria vaccines. Their targets are the sporozoites that enter the host during the mosquito bite, and the liver stages (EEFs). P. falciparum vaccines that target CS, the main surface protein of sporozoites, are undergoing human trials. One of them named RTS,S protects 50% or more of naive volunteers (19), or individuals living in an endemic area in Africa. However, protection lasts only for a couple of months (20). The mechanism of protection involves neutralizing antibodies and effector CD4+ T cells (21). CS vaccines that elicit additional effector CD8+ T cells would be highly desirable. In the rodent models of malaria, there is compelling evidence that CTL can destroy the early EEFs by releasing IFN (22). In addition, production of specific antibodies, such as after MVA-PyCS injection, can only reduce the number of EEF in the liver. Furthermore, CS is produced in large amounts during EEF development and is processed and presented on the surface of the infected liver cells (23, 24). As shown here CS is an excellent target for CTL. Indeed, priming mice with 17D-Py that contains a single CS CTL epitope leads to the elimination of 60–70% of liver stages. This is particularly noteworthy because priming of mice using other recombinant viral vectors such as influenza or vaccinia expressing the same malaria CD8+ CTL epitope do not lead to protective responses without boosting (12, 15). In the case of Sindbis vectors, protection is optimal 12 d after priming (14), but decays quickly afterwards (Tsuji, M., personal communication). Another observation important for malaria vaccine development is the finding that the protection elicited by 17D-Py can be boosted with sporozoites and that the boosting leads to sterile immunity. It is therefore plausible that in endemic areas the immune response of vaccinated individuals will be magnified by the bite of infected mosquitoes. This is particularly encouraging considering that the vaccines actually undergoing clinical trials for malaria in endemic areas are based on a prime/boost strategy involving two priming immunizations 3 wk apart, a boost 3 wk later, and finally another boost after 12 mo (25). In contrast, YF17D is able to induce neutralizing antibodies in 90% of vaccine recipients 10 d after vaccination, and the percentage increases to 99% after 4 wk. Moreover, the fact that YF vaccination has been included since 1993 in the World Health Organization (WHO)–sponsored Expanded Program on Immunization for children in developing countries (26) could facilitate the introduction of a recombinant YF/malaria vaccine in those countries where it is most needed. Our results showing that such recombinant viruses can be viable, stable in vivo, and are able to induce an effective antimalarial immunity in the P. yoelii-mouse model are encouraging for future attempts targeting P. falciparum. Although such vaccine candidates will not be readily testable for protection against malaria short of human clinical trials, their safety, immunogenicity, and protective efficacy against virulent YF can be tested in rhesus macaques. Yellow fever 17D is a remarkably safe and effective vaccine, but adverse effects have been noted. Over 60 yr, an estimated 400 million doses have been administered worldwide with 2 deaths due to encephalitis. From 1996 to 2001, 150 million doses were administered and seven cases of vaccine-associated viscerotropic disease (including six deaths) occurred. The cause of this rare, but severe, reaction is unknown; but, it seems to involve an atypical host response rather than reversion of the vaccine virus to virulence (27–29) and the WHO guidelines regarding the vaccine have remained unchanged (30). This would certainly not contraindicate vaccination in malaria-endemic countries. Finally, one can envision modernizing the 17D vaccine or 17D recombinant vaccine candidates. Currently, a standardized seed lot inoculum is amplified in embryonated chicken eggs and a crude homogenate stored frozen after lyophilization. This vaccine is difficult to manufacture, expensive, and requires refrigeration. Recombinant plasmid DNAs can now be used as a stable repository for the vaccine with seed stocks and vaccine lots generated by amplification in cell culture. Beyond this, recent work has shown that flaviviruses can be launched directly from transfected plasmid DNA (31), making it possible to eliminate cell culture propagation completely. Such new approaches offer the advantages of the DNA vaccines, ease of manufacture, and the possibility of bypassing the cold chain. Materials and Methods Plasmids and recombinant viruses. For 17D2B/3, we modified the 17D genome (32) to introduce a BssHII and a BstEII cleavage site at the recognition site of the viral serine protease NS2B-NS3, as described by McAllister and colleagues (11). For 17D-Py, we engineered the CTL epitope of Plasmodium yoelii (SYVPSAEQIL) into the 17D genome by assembly PCR using two specific oligonucleotides containing the P.yoelii CTL sequence (forward, GGAGCGCGCAGAAGTTCTTATGTCCCAAGCGCAGAACAAATATTAGGTCACCGGAGAA; and reverse, CCGGTGACCTAATATTTGTTCTGCGCTTGGGACATAAGAACTTCTGCGCGCTCCCCTGACAT) and two 17D-specific primers to generate two PCR fragments containing the P. yoelii CTL and a portion of the NS2B or NS3 gene, respectively. 1 μl out of 50 μl of each PCR reaction was mixed together and was used as template for a PCR with 17D-specific primers lying in NS2B and in NS3. The final PCR product was digested with BssHII and a BstEII and cloned into 17D2B/3. MVA-PyCS is an MVA recombinant that expresses the entire CS protein of P.yoelii (12) and was used for heterologous boosting in some experiments. Preparation of 17D and 17D-Py virus stocks. The parental 17D and recombinant 17D-P.yoelii plasmids were purified by banding on CsCl and linearized by digestion with XhoI. The linearized template was transcribed by SP6 RNA polymerase in the presence of cap analogue, purified, and used to electroporate SW-13 cells as described previously (33). The specific infectivity of the viral RNA was measured by seeding serial dilutions of transfected cells on monolayers of untransfected SW-13 cells, overlaying with 0.6% agarose in medium, and counting viral plaques after 96 h. Virus stocks were harvested at 48 h after electroporation with typical yields of 107–108 PFU/ml, as determined by a plaque assay on SW-13 cells. Single use aliquots were stored frozen at –80°C until use. 17D . growth and stability in cell culture. SW-13 cell monolayers were infected with parental 17D or recombinant 17D-Py at low (0.1 PFU/cell) or high (5 PFU/cell) multiplicity. Supernatants were collected at various times after infection and frozen at –80°C. For plaque titration, serial 10-fold dilutions were used to infect monolayers of SW-13 cells. After 1 h at 37°C, cells were overlayed with 0.6% agarose in medium and the plaques counted at 96 h after fixation with formaldehyde and staining with crystal violet (33). To assess the stability of the inserted sequence, virus in the supernatants was concentrated by PEG precipitation (34), and the RNA was isolated by TRIzol extraction according to the manufacturer's instructions. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random hexamers and amplified by PCR using 17D-specific oligonucleotides flanking the 2B/3 insertion site. Products were sized by acrylamide gel electrophoresis, cleaved with a restriction enzyme diagnostic of the P. yoelii epitope sequence (SspI) or sequenced as a population. Animals and parasites. P. yoelii (17X NL strain) was maintained by alternate cyclic parasite passage in Anopheles stephensi and infected blood transfer to mice. Sporozoites were obtained by dissection of salivary glands of A. stephensi and collected in medium RPMI 1640 at 4°C. BALB/c and C57BL/6 mice were obtained from Charles River Laboratories. Animals were maintained in the animal facility of the Department of Medical and Molecular Parasitology. All animal experimental procedures were reviewed and approved by the Institutional Animal Review committee. Immunization, challenge, and in vivo stability of recombinant viruses. Except when stated otherwise in the figure legends, mice were primed with 5 x 105 PFU of 17D-Py injected s.c. When given a boost, this consisted of either the recombinant 17D-Py virus used at the same dose and route, MVA-PyCS (107 PFU), or irradiated sporozites injected i.v. Immunized mice were challenged i.v. with 2 x 104 sporozoites 2 wk after immunization, except when otherwise noted. Newborn C57BL/6 mice were injected in the peritoneum with 105 PFU of 17D (seven mice) or 17D-Py (five mice). All mice developed hind limb paralysis 10–14 d after infection. Virus was recovered from spleen and brain homogenates, and titers were determined by plaque assay (33). Virus in clarified homogenates was concentrated by PEG precipitation and RNA isolated by TRIzol extraction. The presence of Py insert was assessed by RT-PCR amplification, restriction digestion, and sequencing as described above. Evaluation of immunogenicity and protective immunity. In most experiments, after each vaccination regimen, we evaluated the protective immunity (inhibition of the EEF development) and malaria-specific CD8+ T cell responses. The degree of protection against sporozoite challenge elicited by the vaccination was evaluated by two criteria: the level of inhibition of development of liver stages (EEFs), and the absence of parasites in the peripheral blood of immunized and sporozoite-challenged mice (sterile immunity). To measure the number of EEFs, livers were harvested 40–42 h after i.v. challenge with 2 x 104 sporozoites. This is the time when EEFs are almost fully developed, and the newly formed merozoites are nearly ready to exit the hepatocytes, and enter the blood circulation. Quantification of the EEFs was done by real-time PCR analysis of parasite specific rRNA. In most instances, data are presented as the average parasite RNA copies in the liver ±SE in immunized mice, compared with controls vaccinated with 17D virus (35). Cellular immune responses were evaluated by enumerating IFN-producing, epitope-specific CD8+ T cells in the spleen (by ELISPOT), as described (36). Splenocytes were harvested at 40–42 h after challenge. There was no obvious difference in the spleen size in any of the experimental or control groups of mice when the animals were killed. In some experiments we determined SYVPSAEQI-CS-specific CD8+ T cells using tetramers. FACS analyses were done using FACSCalibur and CellQUEST software. To determine whether sterile immunity had been elicited, the immunized animals were challenged i.v. with 75 sporozoites. Peripheral blood smears were made daily and were examined for the presence of blood stages, starting from the third day after challenge, for a total of 2 wk. In the P. yoelii model, blood infections result from the injection of 10 or less sporozoites, and the prepatent period never exceeded 2 wk. Detection of antibodies to CS. Anti-CS antibodies were detected by an indirect immunofluorescence assay, using live or air-dried P. yoelii sporozoites as described previously (37). In vivo depletion of CD8+ or CD4+ T cells. Each vaccinated mouse received daily 0.1 mg anti-CD4 mAb (from cell line –GK1.5) or anti-CD8 mAb (from cell line –YTS 169; Harlan Bioproducts For Science) by i.p injection, for three consecutive days. The mice were challenged with P. yoelii sporozoites 2 d after receiving the last dose of mAb. The corresponding protection and CS-specific T cells were evaluated by real-time PCR and ELISPOT. Acknowledgments This work was supported by grants from the Starr Foundation and the Greenberg Medical Research Institute. The authors have no conflicting financial interests. Submitted: 2 August 2004 Accepted: 3 December 2004 References Nardin, E., R.W. Gwadz, and R.S. Nussenzweig. 1979. Characterization of sporozoite surface antigens by indirect immunofluorescence: detection of stage- and species-specific antimalarial antibodies. Bull. World Health Organ. 57:211–217...查看详细 (26419字节)
☉ 11120219:Surviving low oxygen Neutrophils no longer surv
Hypoxia-inducible factor–1 prolongs the life of oxygen-deprived neutrophils by inducing NF-B–driven survival signals, according to a study by Walmsley et al. on page 105. HIF-1, a transcription factor induced by low oxygen, is required for myeloid cell function but has never been linked to myeloid apoptosis. Walmsley et al. now show that cells lacking this protein are robbed of the ability to resist apoptosis in low-oxygen environments like those encountered in wounds and inflamed tissues. Neutrophils must function in adverse environments where oxygen and nutrient supplies are low. To do this, they turn on HIF-1, which drives the synthesis of enzymes that make ATP anaerobically. In mice whose myeloid cells lack HIF-1, the cells quickly lose their ATP supplies and thus have no energy to migrate or function in response to inflammatory stimuli. Neutrophils also delay apoptosis during hypoxia, and the authors now show HIF-1 is required for this delay. The accelerated death could be mimicked in HIF-1–positive cells by blocking NF-B, suggesting a key role for this transcription factor in prolonging survival. The authors speculate that the ability of HIF-1 to induce neutrophil survival might contribute to a delayed resolution of inflammation, making a bad situation even worse. hvanepps@rockefeller.edu Related Article Hypoxia-induced neutrophil survival is mediated by HIF-1–dependent NF-B activity Sarah R. Walmsley, Cristin Print, Neda Farahi, Carole Peyssonnaux, Randall S. Johnson, Thorsten Cramer, Anastasia Sobolewski, Alison M. Condliffe, Andrew S. Cowburn, Nicola Johnson, and Edwin R. Chilvers J. Exp. Med. 2005 201: 105-115....查看详细 (1691字节)
☉ 11120220:Trouble-making NKT cells NKT cells aggravate ar
The dark side of NKT cells is revealed in a new study showing that they contribute to autoimmune arthritis in mice. On page 41, Kim et al. find that arthritis is worse in joints that are invaded by NKT cells. NKT cells are innate immune cells that express both NK and T cell markers, and are protective in many other models of autoimmune disease. The authors tested NKT function in K/BxN mice—an established model of rheumatoid arthritis. These mice spontaneously develop a progressive inflammatory disease that is caused by the deposition of autoantibodies in joints. Disease can be transferred to healthy recipient mice by injecting them with K/BxN serum.Kim et al. now show that mice lacking NKT cells develop only mild joint inflammation when given K/BxN serum. This protection was reversed if the deficient mice were reconstituted with NKT cells. The nonarthritic mice lacking NKT cells had high levels of TGF-?1 in their joints, which dropped as soon as the NKT cells arrived on the scene. Blocking TGF-?1 in the same NKT cell–deficient mice increased joint swelling, suggesting a protective role for this cytokine. Suppression of TGF-?1 production depended on the ability of the NKT cells to produce both IL-4 and IFN-. Many questions remain unanswered. Future studies might reveal which cells produce the TGF-?1 in the joints, how TGF-?1 prevents arthritis, what attracts NKT cells to the joint, and how NKT cells suppress TGF-?1 production. hvanepps@rockefeller.edu Related Article NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor ?1 production Hye Young Kim, Hyun Jung Kim, Hye Sook Min, Sanghee Kim, Weon Seo Park, Seong Hoe Park, and Doo Hyun Chung J. Exp. Med. 2005 201: 41-47....查看详细 (1783字节)

☉ 11120221:Toll-like receptor 9 mediates innate immune activa
Malaria infection is still the major cause of disease and mortality in humans, especially in the tropical regions of the world. Due to a complex life cycle and rapid polymorphism of parasite antigens, host–parasite interactions and resulting innate immune responses to malaria parasites are still poorly understood despite the urgent necessity of effective immunotherapeutic interventions (1, 2). Robust innate immune activation, including proinflammatory cytokine production in response to malaria parasites and/or their metabolites released from ruptured infected red blood cells, has been linked to the major symptoms such as high fever (3). Recent evidence suggests that Toll-like receptors (TLRs) are involved in the innate immune responses to a variety of pathogens, including Plasmodium (4, 5). In murine malaria infection, myeloid differentiation factor 88 (MyD88), an essential adaptor molecule for cytokine induction through TLRs, was shown to be critical for IL-12 induction by Plasmodium berghei parasites that cause liver injury (6). A recent study has shown that Plasmodium falciparum blood-stage parasites activate human plasmacytoid DCs as well as murine DCs through MyD88- and TLR9-dependent pathways, whereas the responsible molecule(s) is yet unidentified (7). Hemozoin (HZ), known as a malaria pigment, is a detoxification product of heme molecules persisting in the food vacuoles of Plasmodium parasite (8, 9). Intracellular HZ is released into the circulation during schizont rupture and phagocytosed by myeloid cells, which results in the concentration of HZ in the reticulo-endothelial system (9). It has been shown that HZ purified from P. falciparum activates macrophages to produce proinflammatory cytokines, chemokines, and nitric oxide and enhances human myeloid DC maturation (10, 11). These studies prompted us to study the molecular mechanism(s) through which HZ activates the innate immune system, which may improve our understanding of malaria parasite–host interactions. We found that both in vivo and in vitro, HZ purified from P. falciparum activates murine immune cells that are mediated by TLR9 and dependent on MyD88. Importantly, such activation was inhibited by chloroquine (CQ), a common antimalarial drug. Results and Discussion Purified HZ from P. falciparum activates murine spleen and DCs through MyD88-dependent pathway To examine whether HZ purified from P. falciparum activates the murine immune system, spleen cells and DCs were stimulated in vitro with purified HZ, and proinflammatory cytokine production in the culture supernatant was measured by ELISA. Flt3 ligand–induced bone marrow–derived DCs (FL-DCs) produced large amounts of TNF, IL-12p40, monocyte chemoattractant protein (MCP)-1, and IL-6 in response to HZ in a dose-dependent manner to a similar extent to that of CpG oligodeoxynucleotide (ODN; Fig. 1 a). To investigate possible roles of TLRs in HZ-induced innate immune activation, mice lacking MyD88, an essential adaptor molecule for cytokine inductions mediated by most TLRs, were used (5). FL-DCs from MyD88–/– mice showed severely impaired levels of TNF, IL-12p40, MCP-1, and IL-6 production upon stimulation with HZ (Fig. 1 b). Similarly, MyD88–/– spleen cells showed impaired responses to produce MCP-1, IL-6, TNF, IL-12p40, and IFN-inducible protein 10 in response to HZ (Fig. 1 c and not depicted). Additionally, HZ stimulated both CD11c+ B220+ plasmacytoid and CD11c+ B220– myeloid DC subsets of FL-DCs to up-regulate the expression of CD40 and CD86, which were abrogated in both FL-DC subsets in MyD88–/– mice (Fig. 1 d). HZ activation of innate immune responses is Toll/IL-1 receptor domain–containing adaptor-inducing IFN? (TRIF) independent To confirm that HZ-induced innate immune activation is solely dependent on MyD88, mice lacking TRIF, an essential adaptor molecule for the MyD88-independent pathway, were used (5). In contrast to MyD88–/– mice, FL-DCs from TRIF–/– mice responded to HZ and produced TNF and IL-12p40 (P 0.05; Fig. 2). LPS-induced TNF and IL-12p40 was impaired in TRIF–/– FL-DCs, suggesting that extensively purified HZ from P. falciparum cultures is not contaminated with LPS. These data clearly demonstrate that HZ activates a proinflammatory response in mice via MyD88, indicating that one of the MyD88-dependent TLRs might be involved in the recognition of HZ. HZ activation of innate immune responses is TLR9-dependent Further experiments were performed using spleen cells and DCs obtained from TLR2–/–, TLR4–/–, TLR7–/–, or TLR9–/– mice to examine whether HZ-induced innate immune activations are impaired or altered. HZ stimulated FL-DCs in wild-type, TLR2–/–, TLR4–/–, and TLR7–/– mice to up-regulate CD40 and CD86 in both CD11c+ B220+ plasmacytoid and CD11c+ B220– myeloid DC subsets (Fig. 3 a). In contrast, both subsets of FL-DCs derived from TLR9–/– mice failed to up-regulate CD40 and CD86 in response to HZ (Fig. 3 a). Similarly, FL-DCs derived from wild-type, TLR2–/–, TLR4–/–, and TRL7–/– mice, but not from TLR9–/– mice, produced TNF, IL-12p40, MCP-1, and IL-6 in response to HZ (Fig. 3 b and not depicted). It is of note that HZ did not induce IFN by FL-DCs, suggesting that the HZ-induced cytokine profile is similar to that of the K-type CpG ODN (also known as B type), but distinct from that of the D-type ODN (also know as A-type) or known natural DNA ligands for TLR9 such as bacterial or viral DNA (Fig. 3 c; reference 12). Nevertheless, these data clearly demonstrate that TLR9 and MyD88 are critical for HZ-induced activation of murine spleen cells and DCs. HZ activation of proinflammatory cytokines is MyD88/TLR9 dependent in vivo To further confirm that HZ activation is mediated by TLR9 and dependent on MyD88 in vivo, purified P. falciparum HZ was injected i.p. into wild-type and MyD88–/– or TLR9–/– mice, and serum cytokine productions were monitored. HZ injection significantly increased serum levels of MCP-1 and IL-6 in wild-type mice, which peaked between 1 and 4 h and declined within 6 h (Fig. 4 a). In contrast, such increases were completely abrogated in MyD88–/– and TLR9–/– mice. After 6 h, the cytokine levels declined in wild-type mice. These data clearly demonstrate that HZ-induced proinflammatory responses were mediated by TLR9 and MyD88 both in vitro and in vivo. Synthetic HZ (?-hematin, synthesized from monomeric heme in laboratory conditions) is structurally similar to HZ formed naturally by parasites (13, 14) and free of contaminant derived from parasites or culture. To examine whether synthetic HZ also activates the innate immune system in a TLR9-dependent manner, wild-type or TLR9–/– mice were injected with synthetic HZ, and then IL-6 and MCP-1 production in sera was monitored. Synthetic HZ injection into wild-type mice resulted in the production of MCP-1 and IL-6 in serum peaked at 4 h (Fig. 4 b). In contrast, such responses were abrogated in TLR9–/– mice (Fig. 4 b). These data suggest that synthetic HZ as well as natural HZ purified from P. falciparum stimulates the innate immune system in mice, excluding the contribution of the other possible contaminant(s) to HZ-induced, TLR9-mediated innate immune activation. To further exclude any possible contaminants in or during HZ preparation from the P. falciparum culture, a series of analyses was also performed to determine the purity of HZ in addition to the extensive purification method described above (11). DNA or RNA was not detectable in HZ or DNase-treated HZ solution (1 mM) by either ethidium bromide–stained agarose gel (Fig. 5 a) or with a spectrophotometer. DNase treatment or heat inactivation had no effect on HZ-induced DC production of TNF and IL-12p40 (Fig. 5 b). Furthermore, genomic DNA and RNA isolated from P. falciparum did not activate innate immune responses significantly, including the production of TNF and IL-12 (unpublished data and reference 15). No protein or lipid was detected in the HZ solution (1 mM) by all of the methods we tested. Taken together, these data strongly suggest that HZ-induced, TLR9-mediated innate immune activation is not due to the other contaminants. HZ-induced innate immune activation is CQ sensitive The antimalarial drug CQ has been shown to inhibit proinflammatory responses during malaria infection in addition to endo/lysosomal maturations and malaria HZ crystal formations, whereas the exact mechanism of the antimalarial effects of CQ is still under debate (8, 16). Recent evidence suggests that CQ also inhibits TLR9-mediated innate immune activation (17). To examine the effect of CQ on the HZ-induced proinflammatory responses, FL-DCs were stimulated with HZ in the presence of CQ. HZ-induced TNF and IL-12p40 productions were diminished by CQ (Fig. 5 b), suggesting that in addition to previously known mechanisms of CQ inhibition of malaria pathogenesis, CQ may also inhibit HZ-induced, TLR9-mediated innate immune responses during malaria infection, possibly contributing to its therapeutic effects. Although further studies are needed, it is possible that in addition to its ability to inhibit HZ formation, CQ may also interfere with the interaction of HZ with TLR9 or inhibit the maturation of HZ-containing phagosome, thereby inhibiting the following innate immune activation. It has been shown that TLR9 recognizes CpG motifs in microbial DNA or self-DNA–chromatin complex with specific IgG, whereas a non-DNA molecule(s) as a TLR9 ligand has not been reported (18, 19). This work provides the first evidence of a non-DNA ligand as recognized by TLR9. HZ is a crystal form of polymerized heme (ferriprotoporphyrin IX) produced in the food vacuole of parasites during degradation of hemoglobin from red blood cells. Once captured by phagocytes such as macrophages and DCs, HZ accumulates in the phagosome where TLR9 can be recruited from the endoplasmic reticulum via PI3 kinases (20, 21), indicating that TLR9 may recognize HZ in phagosome. HZ is also extremely hydrophobic, which may contribute to its immunostimulatory effect according to the hypothesis proposed recently (22). It is of interest that HZ is originally derived from hemoglobin in the host red blood cells, but modified by parasites from free heme (toxic to parasite) into HZ (nontoxic to parasite), self-molecules (heme) become active "non-self" molecules in the innate immune system during malaria infection. The physiological role of HZ-induced, TLR9-mediated innate immune activation in malaria infection and the host defense against it is currently under investigation. Preliminary results suggest that innate immune responses to malaria parasite are dependent on multiple factors in the host, including TLRs as well as Plasmodium. In particular, it is important to study whether TLR-mediated innate immune activation during malaria infection contributes to host-protective immunity or to malarial immune escape mechanism. It is of our interest as well to study the molecular mechanisms by which TLR9 discriminates between CpG DNA and HZ. Nonetheless, these findings clearly demonstrate that HZ, heme metabolite during malaria infection, activates the innate immune system via a TLR9-mediated, MyD88-dependent, and CQ-sensitive pathway, which may open a key to further understanding of malaria parasite–host interactions in innate immunity. Materials and Methods Mice Mutant mice (MyD88-, TRIF-, TLR2-, TLR4-, TLR7-, and TLR9-deficient) either on a 129/Ola x C57/BL6 or C57/BL6 background were generated as described previously (23–27). Age-matched groups of wild-type and mutant mice were used for the experiments. For the in vivo studies, 1,500 μg HZ purified from P. falciparum culture and synthetic HZ (?-hematin) were injected i.p. (28) into wild-type, MyD88–/– or TLR9–/– mice. Serum was collected from tails at 1, 2, 4, and 6 h for the cytokine ELISA. Reagents Synthetic CpG ODNs D35 (29) were synthesized and purchased from Hokkaido System Science. LPS from Salmonella minnesota Re-595, hemin chloride, and CQ was purchased from Sigma-Aldrich. DNase (DNase-I) was purchased from Invitrogen. Preparation of HZ and synthetic HZ (?-hematin) HZ (nonsoluble crystalloid structure) was purified from P. falciparum (3D7 strain)–infected erythrocytes as described previously (11, 13, 14). In brief, after saponin lysis of erythrocytes, parasites were sonicated and washed seven to eight times in 2% SDS. The pellet was incubated with 2 mg/ml proteinase-K at 37°C overnight. The pellet was then washed three times in 2% SDS and incubated in 6 M urea for 3 h at room temperature on a shaker. After three to five washes in 2% SDS and then in distilled water, the HZ pellet was resuspended in distilled water and sonicated again before use. In some experiments, HZ was either heat inactivated at 95°C for 15 min or treated with 100 U/ml DNase-I for 1 h as described previously (30). DNase-I treatment was successful in removing genomic DNA completely from the P. falciparum crude extract (Fig. 5 a). Quantification of nucleic acid and protein was performed by using a spectrophotometer or by ethidium bromide staining in agarose gel, BCA (Bio-Rad Laboratories), or the pyrogallol red method (Wako Pure Chemical Industries, Ltd.). Total lipid was measured by TLC using the Bligh-Dyer method (Toray Research Center) or by an enzymatic method using the Iatron LQ (Mitsubishi Kagaku Iatron Inc.). Synthetic HZ (?-hematin) was purified from hemin chloride using the protocol of Jaramillo et al. (28) based on the acetic acid treatment and alkaline bicarbonate wash. To avoid endotoxin contaminations, all solutions were prepared using endotoxin-free PBS or distilled water. Endotoxin levels measured by LAL assays (Bio-Whittaker) were <0.001 EU for each nmole HZ used. Quantification of HZ or synthetic HZ (?-hematin) The concentration of HZ or synthetic HZ was determined by depolymerizing heme polymers in 20 mM sodium hydroxide/2% SDS solution for 2 h at room temperature, and then the OD was read at 400 nm (14). The molar extinction coefficient for heme is 105 at 400 nm and 25 μg P. falciparum HZ equals 29 nmole of heme content (14, 28). Cells Single cell suspensions of spleen cells (5 x 105 cells/well) were cultured in complete RPMI 1640 medium supplemented with 10% FCS for 48 h. FL-DCs (105 cells/well) were generated by culturing bone marrow cells with Flt3-ligand (100 ng/ml; PeproTech) for 8–9 d in DMEM medium containing 10% FCS. Cells were stimulated in the presence of the indicated stimuli and supernatants were collected for cytokine ELISA. Cytokine ELISA. Mouse TNF, IL-12p40, MCP-1, IL-6 (R&D Systems), and IFN (PBL Biomedical Laboratories) were measured either from the supernatants or the serum by ELISA according to the manufacturer's instructions. Flow cytometric analysis of costimulatory molecule expressions. Cell surface molecule expression of stimulated cells was measured as described previously (21). In brief, stimulated cells were washed with ice cold PBS, fixed, and stained with FITC-, PE-, CyChrome-, and APC-labeled antibodies in the presence of anti-CD16 antibody for 30 min at room temperature. Stained cells were washed, resuspended in PBS/0.1% BSA/0.1% NaN3, and analyzed by FACSCalibur followed by analysis using CELLQuest software (Becton Dickinson). All antibodies were obtained from Becton Dickinson. Statistical analysis Statistically significant differences were analyzed by using Student's t test. P < 0.05 was considered significant. Acknowledgments We thank T. Mitamura, N. Arisue, N. Palacpac, and other members of the Horii lab for helpful discussions and support. We also thank all members of our laboratory, especially Y. Torii and Y. Fujita for generous support. The authors have no conflicting financial interests. Submitted: 7 September 2004 Accepted: 19 November 2004 References Gursel, M., D. Verthelyi, I. Gursel, K.J. Ishii, and D.M. Klinman. 2002. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J. Leukoc. Biol. 71:813–820Ishii, K.J., K. Suzuki, C. Coban, F. Takeshita, Y. Itoh, H. Matoba, L.D. Kohn, and D.M. Klinman. 2001. Genomic DNA released by dying cells induces the maturation of APCs. J. Immunol. 167:2602–2607...查看详细 (16559字节)
☉ 11120222:Gingival Carcinogenicity in Female Harlan Sprague-
ABSTRACT We evaluated gingival toxicities induced by chronic exposure of female Harlan Sprague-Dawley rats to dioxin and dioxin-like compounds (DLCs) and compared them to similarly induced oral lesions reported in the literature. This investigation represents part of an ongoing initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For two years, animals were administered by gavage 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. A full complement of tissues, including the palate with teeth, was examined microscopically. In the groups treated with TCDD and the mixtures of TCDD, PCB126, and PeCDF; PCB126 and 153; and PCB126 and 118, the incidences of gingival squamous hyperplasia increased significantly. Moreover, in the groups treated with TCDD, PCB126, and the mixture of PCB126 and 153, squamous cell carcinoma (SCC) in the oral cavity increased significantly. This investigation constitutes the first report documenting that chronic administration of dioxin-like PCBs can induce gingival SCC in rats. These results indicate that dioxin and DLCs target the gingiva of the oral cavity, in particular the junctional epithelium of molars. Key Words: gingival squamous hyperplasia; squamous cell carcinoma; rat; dioxin; dioxin-like compounds. INTRODUCTION Polyhalogenated aromatic hydrocarbons (PHAHs) comprise a large class of compounds including polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), and polybrominated diphenyl ethers (PBDEs). Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly termed dioxin, receives the most attention. Certain PCDDs, PCDFs, and coplanar PCBs have the ability to bind to the aryl hydrocarbon receptor (AhR) and exhibit biologic actions similar to those of TCDD; they have been commonly designated dioxin-like compounds (DLCs). Dioxin and DLCs enter the environment primarily through pyrolysis activities, at sites of municipal, hospital, and hazardous-waste incineration and metal smelting and refining, and as unintentional trace contamination formed during the manufacture, use, and disposal of chlorinated organics (Bosetti et al., 2003; Brambilla et al., 2004; Brouwer et al., 1998). They may induce developmental, endocrine, and immunological toxicity and multi-organ carcinogenicity in animals and/or humans (ATSDR, 1998, 2000; Brouwer et al., 1998; Huff et al., 1994; McGregor et al., 1998; Steenland et al., 2001; United States Environmental Protection Agency, 1996). The incidences of cancer have been evaluated in several human populations that received elevated exposures to TCDD and DLCs (ATSDR, 1998, 2000). A study in Seveso, Italy, indicated that exposure apparently induced an increase in all cancers combined and several specific cancers, such as lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and myeloid leukemia (Bertazzi et al., 2001). More than 2000 people each in Japan (1968) and Taiwan (1979) were reported to ingest accidentally PCBs and PCDFs in rice bran oil (Asahi, 1993; Guo et al., 1999; Miller, 1985; Wang et al., 2003); follow-up studies indicated increased mortality chiefly from liver cancer and other liver disease (McGregor et al., 1998). Several reports discuss whether exposure to PCBs causes an increased incidence of human cancer (Kimbrough, 1985; McGregor et al., 1998), and experimental studies provide meaningful evidence that they exert probable carcinogenic effects in humans (ATSDR, 2000; United States Environmental Protection Agency, 1996). Although much evidence exists of induction of cancer in humans by DLCs, a conclusive link between these compounds and increased incidences of oral tumors has not been established. Recently, the NTP conducted two-year bioassays in female rats to evaluate the chronic pathology and carcinogenicity induced by dioxin and DLCs, structurally-related PCBs, and mixtures of these compounds, such as TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachloro-dibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); the Toxic Equivalency Factor (TEF) tertiary mixture of TCDD, PCB126, and PeCDF; and the binary mixtures of PCB126 and 153 and PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118) (National Toxicology Program, 2004a,b,c,d,e,f,g). In these studies, in several tissues, a significant increase occurred in the incidence of neoplastic and nonneoplastic effects, such as cholangiocarcinoma and/or hepatocellular adenoma of the liver and cystic keratinizing epithelioma of the lung (Brix et al., 2004; Jokinen et al., 2003; National Toxicology Program, 2004a,b,c,d,e,f,g; Nyska et al., 2004; Tani et al., 2004; Walker et al., submitted manuscript). Additionally, the incidences of gingival squamous hyperplasia (GSH) and/or squamous cell carcinoma (SCC) were increased in all studies except PCB 153. Ten chemicals that induce oral tumors in rats have been reported by the National Toxicology Program (NTP) (Table 1, NTP Web Site: http://ntp-server.niehs.nih.gov/htdocs/pub.html). This article, one of a series of works highlighting specific findings from the NTP dioxin-TEF-evaluation studies, focuses on the incidences and morphologic aspects of oral lesions across these studies. In addition, we discuss the literature concerning DLC-related oral pathology and potential mechanisms of gingival tumor induction and compare oral lesions induced by these compounds used in the NTP studies with those lesions reported previously in animals and humans. MATERIALS AND METHODS Study design. The original studies comprised part of a series of analyses undertaken by the NTP to determine the suitability of the TEF methodology for predicting chronic toxicity and carcinogenicity of TCDD and DLCs. Female Harlan Sprague-Dawley rats were used, since the Sprague-Dawley rat has proven sensitive to the effects of TCDD (Kociba et al., 1978). A range of 50 to 66 animals per group was treated for two years with several doses of TCDD; PCB126; PeCDF; PCB153; a TEF tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 118 (Tables 2 and 3). Groups that received the highest doses of TCDD, PCB126, PeCDF, PCB153, and the mixture of PCB126 and 118 for 30 weeks, followed by vehicle treatment through the termination of the two-year study, were designated stop-exposure groups. Animals were dosed once daily for five days per week by oral gavage. The doses of all compounds were based on the TEF values selected by the World Health Organization (Van den Berg et al., 1998) (Table 2). TCDD has been considered the most potent DLC and the reference compound to which all DLCs are compared in the TEF methodology. TCDD has a TEF value of 1.0, and PCB126, PeCDF, and PCB118 have values of 0.1, 0.5, and 0.0001, respectively. PCB 126, a non-ortho-substituted PCB, has been deemed the most potent dioxin-like PCB congener present in the environment, accounting for 40–90% of the total toxic potency of PCBs exhibiting "dioxin-like" activity. PeCDF, a dioxin-like PHAH, represents the most potent PCDF present in human tissues. PCB118 is a mono-ortho-substituted PCB with partial dioxin-like activity; controversy exists, however, over whether mono-ortho-substituted PCBs should be included in the TEF methodology. In contrast, PCB153, which is a di-ortho-substituted nonplanar PCB, exhibits no dioxin-like activity and, therefore, merits no inclusion in the TEF methodology (Van den Berg et al., 1998). Chemicals. Dose formulations of TCDD (The IIT Research Institute, Chicago, IL), PCB 126 (AccuStandard, Inc., New Haven, CT), PeCDF (Cambridge Isotope Laboratories, Cambridge, MA), PCB153 (Radian International LLC, Austin, TX), and PCB118 (Radian International LLC, Austin, TX) were prepared for administration by gavage by mixing the test chemical in a corn-oil vehicle containing 1% USP-grade acetone. Animals. All experiments, for the duration of these studies, were conducted in the AAALAC-accredited facility of Battelle-Columbus Laboratories (Columbus, OH). Animal handling and husbandry met all NIH guidelines (Grossblatt, 1996). Female Harlan Sprague-Dawley rats were approximately eight weeks of age at the start of the study. Animals were randomly assigned to control or treated groups and housed five to a cage in solid-bottom polycarbonate cages (Lab Products, Inc., Maywood, NJ). The animal rooms were maintained at 69–75°F with 35–65% relative humidity and 12 h each of light and darkness. Irradiated NTP-2000 pelleted feed (Zeigler Bros., Inc., Gardner, PA) and water were available ad libitum. Pathology. Moribund and all scheduled-to-be-sacrificed animals were euthanized by carbon dioxide. Complete necropsies were performed on all animals using standardized methodology. At necropsy, all tissues, including masses and macroscopic abnormalities, were removed and fixed in 10% neutral buffered formalin. The maxillae, including the nose, were decalcified in a 5% Nitric Acid Decal Solution (Poly Scientific, Inc., Bay Shore, NY) for three days. Three nasal sections that included oral tissues were examined. The maxilla was trimmed just posterior to the upper incisors (Section I), midway between the incisors and first molar at the anterior surface of the incisive papilla (Section II), and at the middle of the first molar (Section III). After fixation and/or decalcification, all of the tissues were trimmed, dehydrated, cleared, embedded in paraffin, sectioned into 5-μm-thick sections, stained with hematoxylin and eosin (H&E), and examined microscopically. The severity of lesions was graded on a four-point scale of 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. The pathology results underwent comprehensive NTP peer review by Pathology-Working-Group pathologists (Boorman et al., 2002). The tongue and mandible were not routinely examined histopathologically, and no gross abnormality was observed in these organs. Statistical analysis. The probability of survival was estimated by the product-limit procedure of Kaplan-Meier (Kaplan and Meier, 1958). The incidences of lesions were evaluated statistically by the poly-3 test (Bailer and Portier, 1988; Portier and Bailer, 1989), which makes adjustments for survival differences among groups. For animals in the two-year studies, the total-lesion incidences, including findings from animals that survived until study termination and early-death animals, were included in the analysis. RESULTS Incidences of Oral GSH and SCC The incidences of GSH were quite variable in the control groups of our TEF studies, showing the range of 2–40%. In the groups treated with TCDD; the TEF mixture of TCDD, PCB126, and PeCDF; and the mixture of PCB126 and 118, the incidences of GSH increased significantly (Table 3). Doses of 3 ng/kg or greater of TCDD induced GSH, and the average severities increased at higher dosing levels. In the groups receiving 46 ng/kg or greater of TCDD, the incidence of oral SCC increased, and a statistically significant difference occurred in the highest dosed group (incidence rate: 19%), compared to the control group (2%). In the 100 ng/kg stop group, the incidence of oral GSH was still increased significantly, and the tendency toward an increased occurrence rate of SCC could be seen (incidence rate: 10%), compared to the control group (2%). Although an increased incidence of GSH in PCB126-treated rats in the two-year study could not be detected, 1000 ng/kg of PCB126 induced oral SCC significantly (incidence rate: 13%), compared to controls (0%). In the 550 ng/kg and 1000 ng/kg stop groups, the incidences of oral GSH and SCC were slightly higher than those of the control group, but without a statistically significant difference. In the TEF study of TCDD, PCB126, and PeCDF, all doses in the study induced GSH significantly with no differences in severities. The incidence of oral SCC, however, did not increase with any statistical significance. In the mixture study of PCB126 and 153, the levels greater than 100 ng/100 μg/kg induced GSH significantly; however, the average severities did not increase at higher dosing levels. In groups administered 300 ng/300 μg/kg or greater, the incidences of oral SCC increased significantly compared to those of control groups. Dosages of 22 ngTEQ/kg or higher of the mixture of PCB126 and 118 induced GSH significantly. The incidence of oral SCC did not, however, increase with any statistical significance. In the 360 ngTEQ/kg stop group, the incidence of oral GSH was increased significantly, similar to that of the 360 ngTEQ/kg continuous treatment group. In contrast to the above-mentioned studies, no significant differences occurred in the incidence and severity of oral GSH and SCC between controls and any dosed groups in the PeCDF and PCB153 two-year studies. Histopathological Characteristics of Oral GSH and SCC Cases showing the presence of hair shafts with or without inflammation in the periodontal tissue adjacent the molar tooth in nasal Section III were noted sporadically in control and dosed animals in all studies (Figs. 1a and 1b). The inflammation manifested as gingivitis may have contributed to the grade of hyperplasia. No significant statistical differences in the incidences of gingivitis occurred between control and dosed groups in all studies (data not shown). Gingival squamous hyperplasia induced by TCDD and some DLCs constituted a focal lesion that manifested itself in the stratified squamous epithelium (SE) of oral mucosa adjacent the molar tooth in Section III (Figs. 1c and 1d). It consisted of varying degrees of thickening of the epithelium, often with the formation of epithelial rete pegs that extended a short distance into the underlying connective tissue. Endophytic proliferation of tissue of GSH below the stratified SE occurred most commonly, while exophytic projections above the stratified SE were rarely present. Proliferative squamous epithelial cells became larger than normal gingival cells, and lesions exhibited hyperkeratosis and parakeratosis (Fig. 1d). The proliferative cells producing keratin displayed prominent intercellular bridges and resembled normal stratified SE with keratohyaline granules and distinct cellular boundaries. Severe hyperkeratosis and parakeratosis sometimes characterized GSH, thus resembling the wall of an epidermal inclusion cyst or keratoacantoma in the skin (Fig. 1e). In addition, dysplastic changes of proliferative epithelial cells appeared. The dysplastic cells contained large nuclei with clear nucleoli, eosinophilic cytoplasm, mitotic figures, and coexistent dyskeratotic and apoptotic changes (Fig. 1f), suggesting cellular atypia. Squamous cell carcinoma induced by treatment with TCDD and DLCs occurred within the oral mucosa of the palate, located mainly lateral to the molar tooth (Fig. 2a), and was characterized by irregular cords and clusters of stratified SE that invaded deeply into the underlying connective tissue, as well as cellular atypia (Fig. 2b). Both cauliflower-like exophytic projections into the oral cavity and endophytic invasion into the maxillary bone occurred, with the formation of keratin pearls composed of concentric layers of squamous cells around central layers of keratin. Cells with SCC exhibited frequent mitotic figures, formed buds and nets, and often infiltrated into the nasal cavities and destroyed nasal structure (Fig. 2c). No evidence for metastasis of SCC was noted in any other organ. DISCUSSION Our investigation constitutes the first report showing that chronic administration of DLCs and dioxin-like PCBs can induce gingival SCC. The incidences of GSH increased significantly in the groups treated with TCDD; the TEF mixture of TCDD, PCB126, and PeCDF; the mixture of PCB126 and 153; and the mixture of PCB126 and 118. The incidences of spontaneous GSH in the control groups of the seven studies ranged from 1/53 (2%) to 21/53 (40%). While the highest spontaneous incidence was noted in the PCB 153 control group, the control groups from the other DLC experiments exhibited much lower incidences that are considered most representative of the overall background occurrences; thus, the PCB 153 rate was considered to be related to biological variation that is sometimes seen in studies of this type. In addition, spontaneous gingival reactive lesions may be induced by impingement of hair shafts or coarse food particles upon the gingival mucosa, causing inflammation (Garant and Cho, 1979). In assessing pathological data, the most relevant evidence for potential treatment-related effect is comparison with the concurrent control. In all studies in which GSH was considered related to treatment, the incidence in the exposed groups was significantly higher than that in the concurrent controls. Moreover, the incidences of oral SCC increased significantly in the groups administered higher doses of TCDD, PCB126, and the mixture of PCB126 and 153. PeCDF and PCB153 did not induce oral lesions. The mixture of PCB126 and 118 did not induce SCC with any statistical significance; however, the incidence rate of SCC in the highest-dose group of PeCDF exceeded that seen in the historical control data (0–2%) and therefore may be considered related to treatment. In the 72 ngTEQ/kg group of the mixture study of PCB126 and 118, the incidence rates of SCC exceeded the rate of our historical data (0–2%), even though not being statistically significant. The groups receiving higher dosages than 216 ngTEQ/kg, extremely low survival rates were noted at the end of the study (data not shown, Table 3), thus likely preventing an accurate estimate of induction of SCC. Given the significant increase in SCC at higher TEQ doses in the study of PCB 126, it suggests that the incidence rate of SCC in the 72 ngTEQ/kg group in the mixture of PCB126 and 118 may be related to treatment. It is not clear what the association is between GSH and gingival SCC. In this examination of the H&E slides from the NTP studies, dystrophic changes manifested themselves as large nuclei with clear nucleoli and eosinophilic cytoplasm. Cells contained bizarre mitotic figures and cellular atypia that added complexity to the GSH. Nauta et al. (1996) described oral epithelial dysplasia with distinctive histological features in the Wistar rats: drop-shaped rete ridges, irregular epithelial stratification, basal cell hyperplasia, loss of intercellular adherence, loss of polarity, anisocytosis and anisonucleosis, pleomorphic cells and nuclei, mitotic activity, and/or bizarre mitoses. Squamous epithelium displaying cellular atypia has been diagnosed as epithelial dysplasia characterized by abnormally differentiated squamous layers usually accompanied by thickening of the epithelium. This lesion possesses the potential to progress to squamous cell tumors in oral cavities (Nauta et al., 1996; Okazaki et al., 2002; Umeda et al., 2004). Our findings of a histopathological similarity between SCC and dysplastic change accompanied by cellular atypia could indicate that GSH associated with dysplasia may develop into SCC in TCDD- and DLC-treated animals. Responses to TCDD and DLCs are mediated by the AhR, a ligand-activated transcription factor, which acts in concert with the AhR nuclear translocator protein (Denison and Nagy, 2003; Peters et al., 1999; Schmidt and Bradfield, 1996). Planar PCBs (e.g., PCB126) interact predominantly with the AhR (Hestermann et al., 2000; Poland and Knutson, 1982); however, unlike planar compounds, nonplanar PCBs (PCB153) do not have dioxin-like activity. For the PCB 126 and 118 mixture, the predominant dioxin-like activity is mostly due to the PCB126 component. In oral tissues, AhR can be detected in molar teeth buds and palatal epithelial cells, in particular from the late embryonic stage in rodents and humans (Abbott et al., 1994a,b; Gao et al., 2004; Sahlberg et al., 2002). In in vivo and/or in vitro studies of rats, mice, and/or humans exposed to TCDD during morphogenesis of the palate (Abbott and Birnbaum, 1989, 1990, 1991), TCDD becomes distributed rapidly to the secondary palate (Abbott et al., 1996), downregulates the AhR throughout the palate (Abbott et al., 1994b), and alters the differentiation and proliferation of palatal epithelial cells, followed by abnormal production of a stratified SE (Abbott et al., 1999). Ligand-dependent activation of AhR enhances terminal differentiation in skin cells and the palatal epithelium (Greenlee et al., 2001), and TCDD accelerates differentiation and proliferation in human keratinocytic and/or oral SCC cell lines (H?bert et al., 1990; Ray and Swanson, 2003). Table 4 presents a comparison of oral lesions in animals and humans exposed to TCDD and DLCs. Previous researchers have shown several effects on oral cavities induced by TCDD in animals, and developmental dental aberrations, such as enamel defect and hypodentia, were reported recently in humans (Alaluusua et al., 2004). TCDD induced developmental dental defects, particularly prevention of molar development, in young rats by in utero and/or lactational exposure (Kattainen et al., 2001; Lukinmaa et al., 2001). In contrast, exposure to a large amount of TCDD caused abnormal incisor development in adult rats (Alaluusua et al., 1993; Gao et al., 2004). Many studies of TCDD administered for longer times of exposure utilized several kinds of animals (ATSDR, 1998), such as rats (Kociba et al., 1978; National Toxicology Program, 1982a), mice (Della Porta et al., 1987; National Toxicology Program, 1982a,b; Toth et al., 1979), monkeys (McNulty, 1985), hamster (Rao et al., 1988), and mink (Render et al., 2000b, 2001). Only three of these reports describe TCDD-induced oral proliferative lesions. In a TCDD feed study by Kociba and colleagues (1978), increases occurred in the incidences of SCC of the hard palate/nasal turbinate in Spartan Sprague-Dawley rats. Gingival hyperplasia characterized by cystic nests and infiltrative SE in periodontal ligament appeared in mink during TCDD exposure (Render et al., 2000b, 2001). These gingival lesions occurred in locations adjacent the molars. Oral lesions from our studies appear similar to those reported in the studies of Kociba et al. (1978) and Render et al. (2000b, 2001). Several reports have dealt with oral-cavity lesions related to PCB exposure in animals or humans (Guo et al., 1999; Hashiguchi et al., 1987, 1995, 1997; Miller, 1985; Shimizu et al., 1992; Wang et al., 2003; Yamashita and Hayashi, 1985). In the adult human exposed to PCBs and PeCDFs, elongation of the depth of periodontal pockets, gingival pigmentation (Hashiguchi et al., 1995), and the sensation of "elevated" teeth (Yoshimura and Hayabuchi, 1985) occurred. Recently, the concentration of PCBs in saliva appeared to be one of the important risk factors for periodontal disease (Ogawa et al., 2003). PCB126 induced oral lesions in animals includes GSH in mink (Render et al., 2000a, 2001) and GSH, hyperkeratosis, dyskeratosis, keratocystic formation, squamous metaplasia of the ameloblast surrounding unerupted teeth, and/or hyperpigmentation in the monkey (Hashiguchi et al., 1983, 1987; McNulty, 1985; Tryphonas et al., 1986; Yoshihara et al., 1979). These lesions, however, were not observed in longer-exposure studies of PCBs in rats and monkeys (Arnold et al., 1999; Chu et al., 1994, 1995; Hori et al., 1982; Kimbrough, 1985; Schaeffer et al., 1984). Although several reports have indicated that exposure to PCBs causes an increased incidence of cancer in animals and possibly humans, no reports have documented the increased incidence of oral tumors as a result of such exposure (ATSDR, 2000; Kimbrough, 1985; United States Environmental Protection Agency, 1996). The location of the appearances of these oral lesions merits discussion. In our studies, we detected them lesions within the area of the molar-tooth ligament (tooth pocket, gingival sulcus). In the rat, the gingiva consists of a keratinized stratified squamous epithelium, connective tissue with fibroblasts, and the extracellular matrix composed chiefly of collagen fibers and ground substance containing sulfated glycosaminoglycans (Brunet et al., 1996; Haschek and Rousseaux, 1998; Tintari, 1983). The gingival epithelium in the molar-tooth area is classified as gingival oral epithelium, sulcular epithelium, and junctional epithelium, which appears nonkeratinized and forms the floor of the gingival sulcus. Gingival epithelium manifests a higher proliferative capacity and higher rate of absorption of drugs and chemicals than the skin (Haschek and Rousseaux, 1998; Shojaei, 1998). Mitotic activity appears greatest at the dento-gingival junction of molars, especially within the junctional epithelium (L?e et al., 1972; Shimono et al., 2003; Watanabe et al., 2004). Absorption of chemicals and the conversion by cytochrome P450 proteins to xenobiotic metabolites can occur in gingival epithelia (Vondracek et al., 2001). Our literature search revealed that, in animals, the molar teeth and their gingivae seem to be most sensitive to dioxin-induced toxicity. The junctional epithelium of molars, with high proliferative and metabolic activity, may change pathologically and constitute the earliest gingival change induced by TCDD and some kinds of DLCs. Reports implicate several underlying mechanisms of chemical induction of GH. Three classifications of drugs administered to humans–calcium channel blockers (e.g., nifedipine), immunosuppressants (e.g., cyclosporine), and anticonvulsants (e.g., phenytoin)—comprise the main causative agents of drug-induced gingival hyperplasia (GH) (Abdollahi and Radfar, 2003; Brunet et al., 1996; Butler et al., 1987; Guggenheimer, 2002). In association with lesions induced by phenytoin and cyclosporine, the occurrence of oral SCC has also been reported (McLoughlin et al., 1995; Varga and Tyldesley, 1991). Bacteria-associated inflammation manifested as gingivitis and the appearance of sulcular epithelium of the teeth have played essential roles in some cases of drug-induced GH (Brown et al., 1991). Although gingivitis appeared in all dosed groups in our rat studies, we observed no significant differences in its occurrence between control and dosed groups. Gingivitis with hair impaction has been occasionally observed in rat toxicity studies (Brown and Hardisty, 1990). A direct action upon the gingival epithelium, rather than the development of gingivitis, appears therefore to have played a key role in the induction of GSH in our rat studies. Indirect alterations in retinoid homeostasis in the liver may constitute another possible mechanism for the action of DLCs in the oral cavity. In rodents, mobilization of retinoid stores by TCDD and DLCs leads to a disruption in retinoid homeostasis, as well as vitamin A deficiency (Fattore et al., 2000; Fiorella et al., 1995; Schmidt et al., 2003; Van Birgelen et al., 1994, 1995). Abnormal epithelial differentiation creating a keratinized squamous phenotype characterizes retinoid deficiency (Lancillotti et al., 1992; Lotan, 1994). The action of DLCs may therefore involve a disruption of retinoid action leading to altered growth and differentiation of the oral gingival epithelium that result in development of GSH and, ultimately, neoplasia. Since the mechanisms of dioxin- and DLC-induced GSH and SCC remain to be elucidated, concentration on gene- and protein-related functions could enhance understanding of the pathogenesis of oral lesions. Additional research is needed to analyze the mechanism(s) of this induction and provide understanding of the potential extrapolations to humans of dioxin-induced oral lesions. ACKNOWLEDGMENTS The authors would like to thank all those involved in the design and conduct of these NTP studies, with special thanks to John Bucher, Rick Hailey, Angelique Braen (nee Van Birgelen), and Milton Hejtmancik. We gratefully acknowledge Dr. Rodney Miller, Dr. Adriana Doi, and Ms. JoAnne Johnson for critical review of the manuscript and Norris Flagler for expert preparation of the illustrations. The authors declare that they have no competing financial interests. REFERENCES Abbott, B. D., and Birnbaum, L. S. (1989). TCDD alters medial epithelial cell differentiation during palatogenesis. Toxicol. Appl. Pharmacol. 99, 276–286. Abbott, B. D., and Birnbaum, L. S. (1990). Rat embryonic palatal shelves respond to TCDD in organ culture. Toxicol. Appl. Pharmacol. 103, 441–451. Abbott, B. D., and Birnbaum, L. S. (1991). TCDD exposure of human embryonic palatal shelves in organ culture alters the differentiation of medial epithelial cells. Teratology 43, 119–132. Butler, R. T., Kalkwarf, K. L., and Kaldahl, W. B. (1987). Drug-induced gingival hyperplasia: Phenytoin, cyclosporine, and nifedipine. J. Am. Dent. Assoc. 114, 56–60. Chu, I., Villeneuve, D. C., Yagminas, A., LeCavalier, P., Hakansson, H., Ahlborg, U. G., Valli, V. E., Kennedy, S. W., Bergman, A., Seegal, R. F., and Feeley, M. (1995). Toxicity of PCB77 (3,3',4,4'-tetrachlorobiphenyl) and PCB118 (2,3',4,4',5-pentachlorobiphenyl) in the rat following subchronic dietary exposure. Fundam. Appl. Toxicol. 26, 282–292. Shimono, M., Ishikawa, T., Enokiya, Y. M., Muramatsu, T., Matsuzaka, K., Inoue, T., Abiko, Y., Yamaza, T., Kido, M. A., Tanaka, T., and Hashimoto, S. (2003). Biological characteristics of the junctional epithelium. J. Electron Microsc. (Tokyo) 52, 627–639. Shojaei, A. H. (1998). Buccal mucosa as a route for systemic drug delivery: A review. J. Pharm. Pharmaceut. Sci. 1, 15–30....查看详细 (30302字节)
☉ 11120223:Differential Expression of Mouse Hepatic Transport
ABSTRACT Drug-metabolizing enzymes and membrane transporters are responsible for the detoxication and elimination of xenobiotics from the body. The goal of this study was to identify alterations in mRNA expression of various transport and detoxication proteins in mouse liver after administration of the hepatotoxicants, acetaminophen or carbon tetrachloride. Therefore, male C57BL/6 J mice received acetaminophen (APAP, 200, 300, or 400 mg/kg, ip) or carbon tetrachloride (CCl4, 10 or 25 μl/kg, ip). Plasma and liver samples were collected at 6, 24, and 48 h for assessment of alanine aminotransferase (ALT) activity, total RNA isolation, and histopathological analysis of injury. Heme oxygenase-1 (Ho-1), NAD(P)H quinone oxidoreductase-1 (Nqo1), organic anion-transporting polypeptides (Oatp1a1, 1a4 and 1b2), sodium/taurocholate-cotransporting polypeptide (Ntcp), and multidrug resistance-associated protein (Mrp 1–6) mRNA levels in liver were determined using the branched DNA signal amplification assay. Hepatotoxic doses of APAP and CCl4 increased Ho-1 and Nqo1 mRNA levels by 22- and 2.5-fold, respectively, and reduced Oatp1a1, 1a4, and Ntcp mRNA levels in liver. By contrast, expression of Mrps 1–4 was increased after treatment with APAP and CCl4. Notably, a marked elevation of Mrp4 mRNA expression was observed 24 h after APAP 400 mg/kg (5-fold) and CCl4 25 μl/kg (37-fold). Collectively, these expression patterns suggest a coordinated regulation of both transport and detoxification genes during liver injury. This reduction in expression of uptake transporters, as well as enhanced transcription of detoxication enzymes and export transporters may limit the accumulation of potentially toxic products in hepatocytes. Key Words: acetaminophen; carbon tetrachloride; hepatotoxicity; transporters; Mrp4. INTRODUCTION Drug-induced liver injury accounted for more than 50% of all cases of acute liver failure in the United States from 1997 to 2002, with 40% of these attributed to acetaminophen (APAP) ingestion (Lee, 2003). Numerous chemicals, including APAP and carbon tetrachloride (CCl4), have been classically used in rodent models to investigate mechanisms of hepatotoxicity relevant to human exposure. Both APAP and CCl4 are bioactivated by Phase I cytochrome P450 enzymes to N-acetyl-p-benzoquinone imine (NAPQI) and trichloromethyl free radical, respectively. Toxicity resulting from these reactive metabolites is multifactorial and includes generation of oxidative stress and altered cellular redox status. In turn, expression of hepatic stress and detoxication genes, including microsomal heme oxygenase-1 (Ho-1) also known as heat shock protein 32, is induced in rat liver in response to chemical injury (Chiu et al., 2002; Nakahira et al., 2003). This up-regulation appears to be an adaptive mechanism to compensate for the dysregulated redox status. Importantly, chemical-induced liver injury impairs hepatobiliary function and results in altered disposition of xenobiotics. In turn, clinicians are required to adjust dosages and dosing intervals of pharmaceuticals to compensate for reduced hepatic function in patients. Altered drug disposition in patients with liver damage has been attributed to reduced hepatic albumin production, altered protein plasma binding, poor hepatic blood flow, and altered expression and activity of Phase I and II drug-metabolizing enzymes (Verbeeck and Horsmans, 1998). However, little is known about changes in transport processes during hepatic injury, which may contribute to altered disposition and the necessity for adjustments in drug therapy. Extraction of compounds from portal blood and subsequent excretion of the parent compound and its metabolites occurs via basolateral and canalicular transporters in hepatocyte plasma membranes (Arrese and Accatino, 2002). Constitutively expressed uptake carriers, such as organic anion-transporting polypeptides (Oatps) and the sodium/taurocholate-cotransporting polypeptide (Ntcp), transport xenobiotics and bile acids across the basolateral membrane into the hepatocyte. Subsequent excretion of these chemicals is mediated by numerous export transporters, including multidrug resistance proteins (Mdrs) also known as p-glycoprotein (Pgp), multidrug resistance-associated proteins (Mrps), bile salt export pump (Bsep), and breast cancer resistance protein (Bcrp). Canalicular transporters, such as Mrp2, Mdrs, Bcrp, and Bsep, are responsible for excretion of compounds and their metabolites from hepatocytes into bile, whereas basolateral transporters, such as Mrp 1, 3–6, are thought to mediate efflux of chemicals from hepatocytes into blood. Despite the high incidence of drug-induced liver injury in the United States, little is known about the expression of hepatic membrane transporters and their influence on xenobiotic disposition in individuals with acute liver damage. Limited data demonstrate altered expression of xenobiotic transporters during chemical-induced hepatotoxicity. Administration of CCl4 results in reduced expression of rat Ntcp, Oatp1a1 [previously called Oatp1 (Slc21a1)], and Oatp1a4 [previously called Oatp2 (Slc21a5)] mRNA, with no change in levels of Mrp2, Bsep, and Oatp1b2 [previously called Oatp4 (Slc21a10)] (Geier et al., 2002). Altered canalicular clearance of substrates for Pgp, Bsep, and Mrp2 was observed in hepatic membrane preparations from rats given CCl4 (Song et al., 2003). Treatment with APAP results in the up-regulation of canalicular Mrp2 and Pgp protein in rat liver (Ghanem et al., 2004). Additionally, administration of the hepatotoxicant bromobenzene increases the hepatic expression of Mrp1-3 mRNA in rat liver (Heijne et al., 2004). In order to investigate the regulation of transporter expression following chemical-induced liver injury, mice were injected with doses of APAP (200–400 mg/kg) or CCl4 (10–25 μl/kg) that resulted in varying degrees of hepatic damage over a 48-h time period. The analysis of transporter expression was extended to include numerous transporters not previously investigated during chemical-induced liver injury in rats. The inclusion of three time points (6, 24, and 48 h) for analysis of transporter expression enabled comprehensive characterization of temporal changes in relation to injury and recovery. The two hepatotoxicants were studied due to similarities and differences in their mechanisms of toxicity. Notably, APAP-induced hepatotoxicity has been associated with covalent adduct formation, depletion of cellular antioxidants such as glutathione, as well as generation of reactive oxygen and nitrogen species. Conversely, CCl4-mediated liver injury is generally characterized by formation of lipid peroxides and altered redox status. In this study, hepatotoxic doses of APAP and CCl4 resulted in the coordinated up-regulation of hepatic oxidative stress and efflux transport genes, as well as the concomitant reduction of uptake transporters. MATERIALS AND METHODS Chemicals. APAP, CCl4, propylene glycol, and corn oil were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of reagent grade or better. RNAzol B was purchased from Tel-Test Inc. (Friendswood, TX). Treatment of animals. Male C57BL/6 J mice, aged 10–12 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME). Mice acclimated 1 week upon arrival. Animals were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment. The mice were fed laboratory rodent diet (No. 5001, PMI Feeds, St. Louis, MO) ad libitum. APAP was dissolved in 50% propylene glycol:water. CCl4 was diluted in corn oil. Groups of mice (n = 3–7) were administered APAP (200, 300, or 400 mg/kg, 10 ml/kg, ip), CCl4 (10 or 25 μl/kg, 5 ml/kg, ip) or the respective vehicle control. The doses of APAP and CCl4 were selected in order to achieve mild to moderate, but not overt toxicity. Livers and plasma were collected 6, 24, or 48 h after APAP or CCl4 administration. Portions of each liver were removed for fixation in formalin. The remaining liver tissue was removed and snap-frozen in liquid nitrogen. Frozen tissues were stored at –80°C until assayed. All animal studies were conducted in accordance with National Institutes of Health standards and the Guide for the Care and Use of Laboratory Animals. Alanine aminotransferase (ALT) activity. Plasma ALT activity was determined as a biochemical indicator of hepatocellular necrosis using Infinity ALT Liquid Stable Reagent (Thermotrace, Melbourne, Australia) according to the manufacturer's protocol. Histopathology. Liver samples were fixed in 10% neutral-buffered formalin prior to routine processing and paraffin embedding. Liver sections (5 μm in thickness) were stained with hematoxylin and eosin. Sections were examined by light microscopy for the presence and severity of hepatocellular degeneration and necrosis. Centrilobular liver injury was scored using a grading system described previously (Manautou et al., 1994). Histopathology scoring was as follows: no injury = grade 0; minimal injury involving single to few hepatocytes = grade 1; mild injury affecting 10–25% of hepatocytes = grade 2; moderate injury affecting 25–40% of hepatocytes = grade 3; marked injury affecting 40–50% of hepatocytes = grade 4; or severe injury affecting more than 50% of hepatocytes = grade 5. RNA extraction. Total tissue RNA was extracted using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) according to the manufacturer's protocol. RNA pellets were resuspended in diethyl pyrocarbonate-treated deionized water. RNA samples were analyzed by agarose gel electrophoresis, and integrity was confirmed by visualization of intact 18S and 28S rRNA under ultraviolet light. Branched DNA signal amplification (bDNA) assay. Mouse Mrp1, 2, 3, 4, 5, 6, Bcrp, Oatp1a1, 1a4, 1b2, Ntcp, Nqo1, and Ho-1 mRNA were measured using the branched DNA signal amplification assay (Quantigene? High Volume bDNA Signal Amplification Kit, Genospectra, Fremont, CA) according to the method of Hartley and Klaassen (Hartley and Klaassen, 2000). Mouse gene sequences of interest were acquired from GenBank. Multiple oligonucleotide probe sets [capture extender (CE), label extender (LE), and blocker (BL) probes] were designed using Probe Designer software version 1.0 (Bayer Corp. Emeryville, CA), to be highly specific to a single mRNA transcript. Probesets for mouse Mrp1, 2, 4, 5, 6, Bcrp, Oatp1a1, 1a4, 1b2, Ntcp, Nqo1, and Ho-1 are listed in Supplementary Table 1. Probes to detect mouse Mrp3 have been previously described (Cherrington et al. 2003). All oligonucleotide probes were designed with a melting temperature of approximately 63°C. This enabled stringent hybridization conditions to be held constant (i.e., 53°C) during each hybridization step for each oligonucleotide probe set. Each probe designed in ProbeDesigner was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic local alignment search tool (BLASTn) to ensure minimal cross-reactivity with other mouse sequences. Oligonucleotides with a high degree of similarity to other mouse gene transcripts were eliminated from the design. Probes were synthesized by QIAGEN Operon (Alameda, CA). Briefly, 10 μl of sample RNA (1 μg/μl) were added to each well of a 96-well plate containing 50 μl of capture hybridization buffer and 100 μl of diluted probe set. Total RNA was allowed to hybridize to probe sets overnight at 53°C. Subsequent hybridization steps were carried out according to the manufacturer's protocol, and luminescence was measured with a Quantiplex? 320 bDNA Luminometer interfaced with Quantiplex? Data Management Software version 5.02. The luminescence for each well was reported as relative light units (RLU) per 10 μg total RNA. Statistical analysis. Data from control animals at 6, 24, and 48 h were pooled and designated 0 h for gene expression analysis. No changes in basal transporter expression from control livers were seen over the 48 h time period. Quantitative results were expressed as means ± standard error of the mean (n > 3). Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (p < 0.05). RESULTS Plasma ALT Activity after Treatment with APAP and CCl4 Administration of APAP and CCl4 to male C57BL/6 J mice resulted in hepatic injury as measured by plasma ALT levels (Fig. 1). The lower APAP doses (200 or 300 mg/kg) did not increase plasma ALT activity, whereas the highest APAP dose (400 mg/kg) increased mean plasma ALT activity at 6 h to 230 U/l. Plasma ALT activity remained elevated through the 48-h time period, although a statistical increase was only observed at 24 h. The low CCl4 dose (10 μl/kg) increased ALT values at 24 and 48 h to 160 and 290 U/l, respectively. The greatest increase in plasma ALT activity was observed after administration of 25 μl CCl4/kg. ALT increased at 6 h (mean ALT 145 U/l) and continued to increase through 48 h after CCl4 (mean ALT 4558 U/l). From these data, the highest dose of CCl4 induced a much higher plasma ALT activity compared to the highest dose APAP Liver Histopathology after Treatment with APAP and CCl4 Adverse histologic changes were absent in livers from APAP- and CCl4-treated mice at 6 h. Mild centrilobular hepatocellular injury (grade 2) was observed in livers from 50% of APAP (400 mg/kg)- and 100% of CCl4 (25 μl/kg)-treated mice at 24 h (Table 1). Resolution of APAP-induced liver injury occurred by 48 h. Conversely, centrilobular injury progressed in CCl4-treated mice, with all animals exhibiting grade 3 or 4 histological changes at 48 h (Table 2). In addition to the greater centrilobular injury, an increased number of mitotic figures indicated hepatocellular proliferation adjacent to areas of injury. Stress and Detoxication Gene Expression after Treatment with APAP and CCl4 Prototypical oxidative stress (Ho-1) and detoxification (NAD(P)H quinone oxidoreductase-1, Nqo1) genes were selected for analysis in both models. Up-regulation of microsomal Ho-1 has been previously documented in rats treated with hepatotoxic doses of APAP and CCl4 (Chiu et al., 2002; Nakahira et al., 2003). This study demonstrates similar up-regulation of mouse Ho-1 mRNA levels during chemical-induced liver injury (Fig. 2). Maximal Ho-1 expression in liver was seen after APAP (400 mg/kg) and CCl4 (25 μl/kg) treatment, respectively. The up-regulation of Ho-1 in liver occurred at 6 h and returned to baseline by 48 h. Induction, but of a lesser magnitude, of Ho-1 expression in liver was also observed 6 h after administration of the lower doses of APAP (300 mg/kg) and CCl4 (10 μl/kg). Nqo1 is a cytosolic enzyme responsible for detoxication of reactive quinone species through a two-electron reduction. Expression of Nqo1 mRNA in liver was increased 2.5- and 2.8-fold at 24 h after APAP (400 mg/kg) and CCl4 (25 μl/kg), respectively (Fig. 2). Nqo1 mRNA levels in liver returned to baseline by 48 h after CCl4 treatment, but remained elevated 48 h after APAP administration. Uptake Transporter Gene Expression after Treatment with APAP and CCl4 In general, APAP and CCl4 treatment resulted in decreased expression of basolateral uptake transporters that are responsible for influx of chemicals from blood into hepatocytes. Specifically, transcripts for Oatp1a1, Oatp1b2, and Ntcp were reduced 24 and 48 h after treatment with the highest dose of APAP (400 mg/kg) or CCl4 (25 μl/kg) (Fig. 3). APAP treatment (400 mg/kg) decreased Oatp1a1 mRNA levels in liver to 10% of control levels at 48 h. The reduction in Oatp1a1 mRNA levels after APAP treatment was not observed in livers from mice treated with CCl4. However, the higher dose of CCl4 reduced Oatp1b2 expression by 60% at 48 h. Similarly, CCl4 decreased Ntcp expression 45 and 70% at 24 and 48 h after exposure, respectively. Interestingly, there was a 1.7-fold increase of Oatp1a4 mRNA at 6 h in both hepatotoxicity models. Efflux Transporter Gene Expression after Treatment with APAP and CCl4 Similar to the patterns observed with uptake transporter expression, efflux transporters responsible for basolateral (Mrp1, 3, 4) and canalicular (Mrp2) export were differentially expressed following APAP and CCl4-induced hepatic injury (Fig. 4). Increased liver Mrp1 mRNA levels were seen with CCl4 (25 μl/kg) at 24 (2.7-fold) and 48 h (4-fold). These changes were not observed with APAP treatment. Conversely, APAP, but not CCl4, increased Mrp3 expression 2-fold at 6 and 48 h. Whereas selective up-regulation of Mrp1 and Mrp3 was observed following either APAP or CCl4 administration, similar changes in Mrp2 and Mrp4 expression were observed with both hepatotoxicants. APAP and CCl4 treatment increased Mrp2 mRNA levels in liver 2-fold. Mrp4 mRNA levels were increased by APAP (400 mg/kg) and CCl4 (25 μl/kg) at time points for which hepatotoxicity was observed. APAP administration increased Mrp4 mRNA levels in liver 5-fold at 24 h and 3-fold at 48 h. A more marked elevation of Mrp4 mRNA expression (37-fold) was associated with CCl4-induced injury at 24 h and remained elevated (6.4-fold) at 48 h. APAP and CCl4 treatment did not alter the expression of Bcrp, Mrp5, or Mrp6 (data not shown). Notably, minimal changes in Mrp expression were seen with nonhepatotoxic doses of APAP and CCl4, suggesting a dependency on hepatic injury for altered mRNA levels. DISCUSSION Other models of hepatic injury are accompanied by similar changes in transporter expression. Obstructive cholestasis and lipopolysaccharide-induced cholestasis in humans, mice, and rats results in the differential up-regulation of Mrp isoforms and down-regulation of Oatp/Ntcp isoforms (Cherrington et al., 2004; Donner and Keppler, 2001; Gartung et al., 1996; Wagner et al., 2003). Reduced levels of rat and human Ntcp/NTCP, Mrp2/MRP2, and Oatp/OATP isoforms are observed following intrahepatic and obstructive cholestasis (Donner and Keppler, 2001; Gartung et al., 1996; Kullak-Ublick et al., 2004; Zollner et al., 2003). Additionally, up-regulation of MDR1, MRP1, and MRP3 is documented in patients with hepatitis or chronic cholestasis (Ros et al., 2003). Similar patterns in the expression of mouse transporters have been observed during cholestasis. Common bile duct ligation in mice results in elevated levels of Mrp3 and Mrp4 with reduced expression of Ntcp (Wagner et al., 2003). Furthermore, feeding cholic acid to mice also increases expression of Mrp2 and Bsep (Fickert et al., 2001). The induction of Bsep and Mrp isoforms in cholestatic mice appears to be an adaptive mechanism to enhance excretion of toxic bile acids and conjugates into the bile and/or portal blood. Similarly, CCl4 treatment reduces the expression of Oatp1a1, Oatp1a4, and Ntcp in rat liver. More recent work shows the up-regulation of Mrp2 and Pgp expression and function following APAP treatment of rats (Ghanem et al., 2004). The work presented in this manuscript more comprehensively documents the down-regulation of mouse uptake carriers (Oatp and Ntcp) and up-regulation of Mrp efflux and stress (Ho-1 and Nqo1) genes. Collectively, these data support the hypothesis that the liver alters gene expression following injury to limit the accumulation of chemicals within the hepatocyte. To date, this is the first study that documents a marked induction of hepatic Mrp4 transcript in mouse liver. Limited data exists regarding the potential role of Mrp4 in liver injury. Mrp4 mRNA is up-regulated in bile duct-ligated mice (Wagner et al., 2003), although to a much lesser extent (three-fold). Mrp4 substrates include nucleotide chemicals, including cyclic AMP and GMP, prostaglandin E1 and E2, as well as some HIV antiviral drugs (Borst et al., 2000; Reid et al., 2003; Sampath et al., 2002). Additionally, Mrp4 transports sulfated compounds, including the steroid, dehydroepiandrosterone 3-sulphate (Assem et al., 2004). The coordinated transcriptional regulation of Mrp4 and sulfotransferase 2a1 has been reported to occur through constitutive androstane receptor (CAR)-mediated signaling pathways (Assem et al., 2004). Interestingly, modulation of CAR alters susceptibility of mice to APAP-induced hepatotoxicity (Zhang et al., 2002). Therefore, activation of CAR during liver injury may represent one mechanism contributing to the up-regulation of Mrp4 in these studies. Although the up-regulation of efflux and down-regulation of uptake transporters during hepatotoxicity appears to represent a general pattern in response to injury, some of the changes were specific to either APAP or CCl4. In some instances, different isoforms of Mrp and Oatp were altered by APAP (Mrp3, Oatp1b2) and CCl4 (Mrp1, Oatp1a1, Oatp1b2), while similar changes in Mrp2, Mrp4, Oatp1a4 were seen by both. This differential regulation may reflect the different pathogenic and transcriptional pathways elicited by APAP and CCl4. For example, down-regulation of Oatp1b2 and Ntcp during CCl4-induced hepatotoxicity may represent an attempt to limit influx of potentially harmful bile acids. On the contrary, the increase in Mrp1 and Mrp2 expression in response to CCl4 may enable efficient removal of the lipid peroxide 4-hydroxynonenal generated during injury (Reichard et al., 2003; Renes et al., 2000). It should be noted that APAP and CCl4 are not thought to be substrates for uptake transporters and instead appear to enter the hepatocyte by diffusion (McPhail et al., 1993). The induction of Mrps may be an attempt by the hepatocyte to remove residual APAP metabolites, such as APAP-glucuronide, which is a substrate of Mrp2 and Mrp3 (Chen et al., 2003; Slitt et al., 2003; Xiong et al., 2002). It is presently unknown if Mrp4 transports APAP-glucuronide. Based upon the temporal up-regulation of Mrp genes at 24 and 48 h in this study, these changes would be futile, because these conjugates are efficiently cleared in mice during the first 24 h (Wong et al., 1981). Instead, these changes in transport may represent an attempt to better dispose of these metabolites upon a second challenge with APAP. In addition to the transporter isoform-specific changes, the magnitude of altered expression differs between APAP and CCl4-treated mice. Presently, it is difficult to determine whether the marked increase (37-fold) in Mrp4 expression following CCl4 treatment compared to the moderate increase (5-fold) with APAP is related to differences in the mechanisms of injury, intrinsic xenobiotic properties, or the extent of hepatic injury achieved in these studies. Further analysis of additional hepatotoxicants, such as bromobenzene and chloroform, as well as higher doses of APAP may address this discrepancy in the magnitude of change in Mrp4 expression between the two hepatotoxicity models. Inclusion of Ho-1 and Nqo1 in our analysis may offer additional insight into the regulatory mechanisms underlying the observed changes in transporter expression. Inducible expression of Ho-1 in mouse liver is in part regulated by cytokine signaling, including interleukin-6 (IL-6) (Masubuchi et al., 2003). Studies with IL-6 null mice demonstrate a role for IL-6 in the regulation of Mrp2, Mrp3, and Ntcp expression following lipopolysaccharide treatment (Siewert et al., 2004). Similarly, exogenous administration of IL-6 reduces murine expression of Mrp2, Oatp1a1, and Oatp1a4 mRNA (Hartmann et al., 2002). The critical role of IL-6 extends to modulation of hepatotoxicity, proliferation, and repair pathways in response to APAP and CCl4 (James et al., 2003; Masubuchi et al., 2003). Coordinated regulation of both detoxication and transport genes through IL-6 signaling during liver toxicity may represent a recovery mechanism by the injured hepatocyte. Nuclear transcription factor-E2 p45-related factor 2 (Nrf2) is a transcription factor that regulates expression of multiple hepatic detoxification and stress genes including Nqo1, Ho-1, and glutathione-S-transferase during oxidative stress. Mice deficient in Nrf2 are more sensitive to the toxic effects of APAP (Chan et al., 2001; Enomoto et al., 2001). The enhanced sensitivity of Nrf2-deficient mice results, in part, from impaired compensatory up-regulation of these detoxication genes. Interestingly, Nrf2 is required for the constitutive and inducible expression of Mrp1 in mouse embryo fibroblasts (Hayashi et al., 2003). Further investigation is necessary to determine the potential role of Nrf2 in regulating hepatic membrane transporters in mice. This study comprehensively characterizes the temporal and dose-related changes in transporter expression during chemical-induced hepatotoxicity. A better understanding of this altered expression is necessary to address the contribution of transport mechanisms to the impaired hepatic clearance of xenobiotics during liver injury. Clinical management of patients with drug-induced liver disease should consider the role of altered transporter expression when selecting doses and dosing regimens for administration of pharmaceuticals. SUPPLEMENTARY DATA Supplementary data is available online. ACKNOWLEDGMENTS This work was supported by National Institute of Health Grant ES10093 and the University of Connecticut Research Foundation. Lauren Aleksunes is a Howard Hughes Medical Institute Predoctoral Fellow. REFERENCES Arrese, M., and Accatino, L. (2002). From blood to bile: Recent advances in hepatobiliary transport. Ann. Hepatol. 1, 64–71. Assem, M., Schuetz, E. G., Leggas, M., Sun, D., Yasuda, K., Reid, G., Zelcer, N., Adachi, M., Strom, S., Evans, R. M., et al. (2004). Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. J. Biol. Chem. 279, 22250–22257. Borst, P., Evers, R., Kool, M., and Wijnholds, J. (2000). A family of drug transporters: The multidrug resistance-associated proteins. J. Natl. Cancer Inst. 92, 1295–1302.Abstract/Free Chan, K., Han, X. D., and Kan, Y. W. (2001). An important function of Nrf2 in combating oxidative stress: Detoxification of acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 98, 4611–4616. Chen, C., Hennig, G. E., and Manautou, J. E. (2003). Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats. Drug Metab. Dispos. 31, 798–804. Enomoto, A., Itoh, K., Nagayoshi, E., Haruta, J., Kimura, T., O'Connor, T., Harada, T., and Yamamoto, M. (2001). High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59, 169–177. Fickert, P., Zollner, G., Fuchsbichler, A., Stumptner, C., Pojer, C., Zenz, R., Lammert, F., Stieger, B., Meier, P. J., Zatloukal, K., et al. (2001). Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 121, 170– James, L. P., Lamps, L. W., McCullough, S., and Hinson, J. A. (2003). Interleukin 6 and hepatocyte regeneration in acetaminophen toxicity in the mouse. Biochem. Biophys. Res. Commun. 309, 857–863. Kullak-Ublick, G. A., Stieger, B., and Meier, P. J. (2004). Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126, 322–342. Lee, W. M. (2003). Acute liver failure in the United States. Semin. Liver Dis. 23, 217–226. Reichard, J. F., Doorn, J. A., Simon, F., Taylor, M. S., and Petersen, D. R. (2003). Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal. Arch. Biochem. Biophys. 411, 243–250. Xiong, H., Suzuki, H., Sugiyama, Y., Meier, P. J., Pollack, G. M., and Brouwer, K. L. (2002). Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment. Drug Metab. Dispos. 30, 962–969. Zhang, J., Huang, W., Chua, S. S., Wei, P., and Moore, D. D. (2002). Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science 298, 422–424. Zollner, G., Fickert, P., Silbert, D., Fuchsbichler, A., Marschall, H. U., Zatloukal, K., Denk, H., and Trauner, M. (2003). Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis. J. Hepatol. 38, 717–727....查看详细 (28610字节)
☉ 11120224:Compartmentalization of Redox Regulation of Cell R
In the November issue of Toxicological Sciences, Hansen et al. (2004) report results that they conclude show that a transcriptional regulator, NE-F2-related factor 2 (Nrf-2), exhibits compartmentally differential redox responses. The working model put forward by Hansen is conceptually straightforward. In the model, Nrf-2 is retained in the cytosol by Keap-1, but when critical thiols on Keap-1 are oxidized, Nrf-2 is released and translocated to the nucleus, where Nrf-2 participates in the transcriptional activation of numerous genes. For Nrf-2 to be fully functional in transcriptional activation, a key cysteine residue must be in the thiol (reduced) form. The model proposed by Hansen et al. overlooks the contributions of phosphorylation in activation of Nrf-2 (Huang et al., 2002; Nguyen et al., 2004) and other evidence that activation of Nrf-2 by metabolism of polyamines appears to be attributable to the actions of acrolein, which is a thiol alkylating agent, rather than through the production of H2O2 and presumed oxidation of cysteine residues (Kwak et al., 2003). Nevertheless, the model proposed by Hansen et al. is useful in expanding the discussion of oxidant stress responses and what advances will be needed to elucidate the critical elements of redox regulation of cell function and viability. Separate from the ultimate requirement of oxidation to provide energy for cell functions, the basic idea that properties of proteins can be changed by oxidation is a valid working hypothesis. A similar redox mechanism was proposed for the transduction of NK-B, in that its translocation to the nucleus was suggested to be dependent on oxidation, whereas its binding to DNA depended on reduction (Droge et al., 1994). With regard to cytotoxicity, the concept that toxicants kill cells by substantial (>50%) depletion of cellular thiols (Di Monte et al., 1984) has not been substantiated in subsequent studies of oxidant cell killing, particularly in vivo (Smith et al., 1985), and contributions of thiol oxidation or alkylation to cell killing appear to be far more specific than some expected initially. More recently, the increased appreciation of specificity in modulation of cell function by modifications of protein thiols has been interpolated into redox regulation of thiol/disulfide status. In general, thiols are more readily oxidized than are other cellular components. The facile reversibility of S-thiolation reactions, whether formation of intra- or interchain disulfides (ProtS-SProt) or glutathione (GSH)-protein mixed disulfides (ProtS-SG) makes the participation of such modifications attractive in working hypotheses for mechanisms of regulation of a wide variety of cellular processes, from conception to death. However, critical tests of hypotheses based upon thiol-disulfide-driven regulation of gene transcription, signal transduction, and related cell functions require that thiol redox status of specific thiols, meaning the specific modification of specific residues of specific proteins, be characterized and quantitated accurately, and with compartmental specificity. The challenges in meeting this goal are beyond present bioanalytical capabilities, but the necessity of developing and applying such methods and concepts should be recognized, so that the limitations of the implications of results not resolved at this level will not be so readily overlooked. Many of the proteins that are most important in regulation of cell functions are present in relatively low abundance, and present bioanalytical methods are challenged, at best, to distinguish thiol from S-thiolated or S-alkylated forms of such proteins. Methods are being developed and reported, but at the present time, many of these reports are limited to detecting changes effected by large doses of diamide, H2O2, or other oxidants in vitro. However, the critical questions of changes occurring under physiological and even under relevant pathophysiological conditions in vivo are much more difficult to address and are limited by robust analytical methods required for such analyses. In the absence of the ability to measure thiol status of specific residues in low abundance proteins, the more ready apparent availability of methods to measure GSH and GSSG contents are being used to link changes in cell functions with changes in GSH and GSSG contents (Kirlin et al., 1999; Schafer and Buettner, 2001). These studies are based principally on the assumption that the thiol/disulfide status of critical effector proteins are reflected by GSH and GSSG levels. In earlier work, Jones reported that cysteine/cystine and thoredoxin [Trx(SH)2/Trx(SS)] redox pairs are not in thermodynamic equilibria with GSH/GSSG, or with each other (Jones et al., 2004). Why, then, would equilibration of one or more of these thiol/disulfide pairs with an effector ProtSH/ProtSSX be expected? The present report by Hansen et al. suggests one possibility, namely compartmentalization. One simplifying interpretation of the authors' report is that a shift in the redox status of the cytoplasm, as reflected in the GSH/GSSG couple, results in dissociation of Nrf-2 from Keap-1, whereas the ability of Nrf-2 to form transcriptional activation complexes in the nucleus is determined by Nrf-2 redox status in the nucleus. This latter activity is determined or reflected in part by [Trx(SH)2/Trx(SS)] redox status. This working hypothesis, which hopefully is not an egregious misinterpretation of the authors' intent, requires restriction of the exchange of mass, in this case more specifically GSH, between the cytosolic and nuclear compartments. This requirement is not consistent with evidence that the movement of GSH across the nuclear membrane is facile (Smith et al., 1996); however, it has been reported that GSH is not equally distributed throughout the cell, even within the cytosoplasm (Soderdalh et al., 2002). Even more problematic with the use of principles of chemical equilibration to explain mechanisms of regulation of dynamic processes in living systems, even GSH/GSSG and [Trx(SH)2/Trx(SS)] redox couples are not equilibrated with their respective NADPH/NADP+ ratios (Kirlin et al., 1999; Schafer and Buettner, 2001), which should ultimately drive the redox ratios of other couples, at the corresponding thermodynamic equilibrium states. The application of the Nernst equation to assessments of thiol/disulfide status and defining a single redox poise, redox potential, or gas gauge level to a tissue, cell, or subcellular compartment is appealing, but the Nernst equation is derived from the Gibbs free energy change associated with a reaction. For the reaction the Gibbs free energy change is where the values represent the respective activities of the reactants and products. For reactants and products that do not dissociate, the activities are products of the respective concentrations and activity coefficients, and activity coefficients can approach unity at infinite dilution, meaning that concentrations can provide useful approximations of activities of non-dissociated species, for purposes of Nernst equation calculations. However, for species that dissociate in solution, the relationships between concentrations and activities are considerably more complicated (Daniels and Alberty, 1967). For example, the activity of something as simple as CaCl2 is proportional to the third power of the molal concentration ([Ca2+]3), and even that relationship assumes high dilution in homogeneous solution. The relationships between concentrations and activities of polyionic proteins or even simpler species like GSH and GSSG, would not be expected to be as simple as for CaCl2, especially in very heterogeneous mixtures that are characteristic of cells and tissues. Faced with the enormous difficulty in determining the thiol/disulfide status of a specific thiol in a specific low abundance protein in a biologically relevant human sample or experimental animal model, Hansen et al. confront us with yet another challenge, that of distinguishing the subcellular compartment in which the protein is represented. Protein distributions can be studied by a variety of methods, such as confocal microscopy, but the ability to determine directly and definitively whether the redox status of a specific residue differs on a low abundance protein, such as Nrf-2, between two subcellular compartments will require ingenuity, methodological advancements and considerable work. REFERENCES Daniels, F., and Alberty, R. A. (1967). Physical Chemistry. John Wiley & Sons, New York. Di Monte, D., Bellomo, G., Thor, H., Nicotera, P., and Orrenius, S. (1984). Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homostasis. Arch. Biochem. Biophys. 235, 343–350. Droge, W., Schulze-Osthoff, K., Mihm, S., Galter, D., Schenk, H., Ech, H. P., Roth, S., Gmunder, H. (1994). Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8, 1131–1138. Hansen, J. M., Watson, W. H., and Jones, D. P. (2004). Compartmentation of Nrf-2 redox control: Regulation of cytoplasmic activation by glutathione and DNA binding by thioredoxin-1. Toxicol. Sci. 82, 308–317. Schafer, F. Q., and Buettner, G. R. (2001). Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212. Smith, C. V., Hughes, H., Lauterburg, B. H., and Mitchell, J. R. (1985). Oxidant stress and hepatic necrosis in rats treated with diquat. J. Pharmacol. Exp. Ther. 235, 172–177....查看详细 (9700字节)
☉ 11120225:Aryl Hydrocarbon Receptor-Activating Polychlorinat
ABSTRACT Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The ‘dioxin-like’ PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the ‘non-dioxin-like’ compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 μM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs. Key Words: cell proliferation; tumor promotion; contact inhibition; PCBs; liver epithelial cells. INTRODUCTION Polychlorinated biphenyls (PCBs) are a group of structurally diverse and persistent environmental pollutants that became widely distributed throughout various environmental compartments as complex mixtures. Although their production has been banned, environmental PCB levels can still be very high, especially at the sites of their production and industrial use (Cogliano, 1998). The biological effects of individual PCB congeners strongly depend on the number and position of chlorine atoms. While the non-ortho-substituted coplanar PCBs are known to elicit a set of adverse ‘dioxin-like’ effects associated with the activation of the aryl hydrocarbon receptor (AhR) (van den Berg et al., 1998), di-ortho-substituted PCBs exhibit a different spectrum of toxic modes of action (Hansen, 1998; Robertson and Hansen, 2001). Both groups of compounds are carcinogenic to laboratory animals, and PCB mixtures have been classified as possible human carcinogens (IARC, 1987). Several studies have implicated low-molecular-weight PCBs as potential tumor-initiating compounds that may either induce oxidative DNA damage or formation of DNA adducts (Espandiari et al., 2003; Oakley et al., 1996). However, it has been extensively documented in various two-stage liver carcinogenesis models that PCB mixtures, as well as individual PCB congeners, are effective at promoting both gross tumors and putative preneoplastic lesions in rodent liver (reviewed in Glauert et al., [2001]). The tumor-promoting activity of PCBs has been suggested to be associated with their capacity to either directly or indirectly activate signal transduction pathways leading to increased cell proliferation, inhibition of negative growth control and programmed cell death, or inhibition of gap junctional intercellular communication (GJIC) (Glauert et al., 2001). The effects of dioxin-like PCBs are considered to be related predominantly to their capacity to activate genes regulated by AhR (Safe, 1994); however, the exact mechanisms of their action still remain elusive. Several studies have demonstrated the capability of AhR ligands to stimulate cell proliferation and to inhibit apoptosis in liver; however, opposite effects have also been reported (Puga et al., 2002; Schwarz et al., 2000; Tharappel et al., 2002; W?lfle et al., 1993). Activation of AhR might also play a role in inhibition of GJIC that is observed in hepatoma cells or in hepatocytes (De Haan et al., 1994; Hemming et al., 1991). However, neither dioxins, known as efficient liver tumor promoters, nor dioxin-like PCBs are able to suppress GJIC in rat liver epithelial "stem-like" WB-F344 cells (Hemming et al., 1991; Machala et al., 2003). This cell line, isolated from the liver of an adult male Fischer 344 rat (Tsao et al., 1984), is an in vitro model of oval cells, small oval-shaped epithelial cells that are considered to be liver progenitor cells. The oval cells can give rise to both hepatocytes and biliary epithelial cells, and they might play a significant role in hepatocarcinogenesis (Alison, 2003; Dumble et al., 2002; Roskams et al., 2003). In humans, both dedifferentiation of mature hepatocytes and maturation arrest of progenitor cells have been suggested to lead to hepatocellular carcinomas (Roskams et al., 2003). Therefore, the liver progenitor cells might represent a potential target for tumor-promoting chemicals. The rate of proliferation of most non-transformed adherent cells decreases with increased cell density as they become arrested in G1 phase of the cell cycle, which is a phenomenon known as contact inhibition (Dietrich et al., 1997; Levenberg et al., 1999). The loss of contact inhibition can lead to deregulated growth and is often associated with malignant transformation (Tsukita et al., 1993). A release from contact inhibition is a mechanism suggested to be an important part of effects of tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (Oesch et al., 1988). Interestingly, both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and AhR-activating polycyclic aromatic hydrocarbons have been found to interfere with mechanisms of contact inhibition in rat liver epithelial cells (Chramostová et al., 2004; Dietrich et al., 2002; K?hle et al., 1999). Contact inhibition usually leads to decreased expression of cyclins involved in regulation of G1-S phase transition and early S phase, which in turn decreases the activities of the cyclin-dependent kinases (cdks) in rat liver epithelial cells (Dietrich et al., 2002). Tetrachlorodibenzo-p-dioxin, a powerful liver tumor promoter, has been reported to upregulate cyclin A expression in confluent WB-F344 cells, and that upregulation has been associated with increased cdk2 activity. Therefore, the cyclin A/cdk2 complex activity could play a pivotal role in the TCDD-induced release of confluent cells from contact inhibition (Dietrich et al., 2002). The increased activity of cytochrome P450 1A1 (CYP1A1), one of the AhR-regulated genes, has been reported to correlate with induction of cell proliferation by dioxins in WB-F344 cells (K?hle et al., 1999). Taken together, AhR might be involved in the TCDD-induced release from contact inhibition. Because dioxin-like PCBs are potent AhR ligands, with PCB 126 being considered only 10 times less effective than TCDD itself (van den Berg et al., 1998), the AhR-activating PCB congeners might disrupt contact inhibition in rat liver stem-like cells, and thus contribute to tumor promotion. The present study aimed to verify the hypothesis that dioxin-like PCBs and their hydroxylated PCB derivatives could stimulate a release of rat liver stem-like cells from contact inhibition by a mechanism that would involve modulation of expression and/or activity of proteins involved in the control of G1-S phase transition and early S phase progression. MATERIALS AND METHODS Chemicals. 2,2',4,4'-Tetrachlorobiphenyl (PCB 47), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) were purchased from Promochem (Wesel, Germany). The hydroxylated PCBs 4'-OH-3,3',4,5'-tetrachlorobiphenyl (4'-OH-PCB 79) and 4-OH-2,2',3,4',5,5',6-heptachlorobiphenyl (4-OH-PCB 187) were kindly provided by Drs. L. W. Robertson and H.-J. Lehmler (University of Iowa, Iowa City, IA) and Dr. ?. Bergman (Stockholm University, Stockholm, Sweden), respectively. The structures of test compounds are shown in Figure 1. Tetrachlorodibenzo-p-dioxin was purchased from Cambridge Isotope Laboratories (Andover, MA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored in the dark. Mouse monoclonal antibody to mouse cyclin D1 (sc-450), rabbit polyclonal antibodies to cyclin A (sc-751), cyclin E (sc-481) and cdk2 (sc-163), and goat polyclonal antibody to cdk4 (sc-601-G) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to mouse p27 (K25020) was purchased from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibodies against cyclin A (Ab-1, E23) and cyclin D2 (Ab-4, DCS-3.1, and DCS-5.2) were obtained from Neomarkers (Fremont, CA). Mouse monoclonal antibody against a C-terminal part of human cyclin D3, which cross-reacts with the mouse homologue (DCS-22), was generously provided by Dr. J. Lukas (Danish Cancer Society, Copenhagen, Denmark). Mouse monoclonal antibody against ?-actin and horseradish peroxidase–conjugated anti-immunoglobulins were from Sigma-Aldrich (Prague, Czech Republic). Polyvinylidene difluoride (PVDF) membrane Hybond-P and chemiluminescence detection reagents (ECLPlus) were supplied by Amersham (Amersham, Aylesbury, UK). All other chemicals were provided by Sigma-Aldrich. Assessment of cell proliferation and cell cycle distribution. WB-F344 rat liver epithelial cells, originally isolated in the laboratory of Joe W. Grisham, National Cancer Institute, Bethesda, MD (Tsao et al., 1984), were kindly provided by James E. Trosko and Brad L. Upham (Michigan State University, East Lansing, MI). Cells were grown in modified Eagle's Minimum Essential Medium (Sigma-Aldrich, Prague, Czech Republic) with 50% increased concentrations of nonessential amino acids, and supplemented with 1 mM sodium pyruvate, 10 mM HEPES, and 5% heat-inactivated fetal bovine serum (Sigma-Aldrich). The cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. Only the cells at passage levels 15–22 were used throughout the study. The proliferative effects of PCBs on confluent WB-F344 cells were determined as described previously (Chramostová et al., 2004). Briefly, cells were seeded at an initial concentration of 30,000 cells per cm2 in 4-well cell-culture plates (Nunc, Roskilde, Denmark) and grown until they reached an approximate confluency. The cells were exposed to tested compounds dissolved in DMSO for 72 h. The final concentration of DMSO did not exceed 0.1% (v/v) in any of the samples. The medium with tested compounds was changed daily to ensure that the cells would receive an adequate amount of nutrients to enable proliferation at high cell densities. Following the exposure, the medium was removed, cells were harvested with trypsin and counted with a Coulter Counter (Model ZM, Coulter Electronics, Luton, UK). Cells were then washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1 M Tris-HCl – pH 8.0; 0.1 % Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 μg/ml; RNAse A 50 Kunitz units/ml) (Vindelov, 1977) and incubated at 37°C for 30 min. Cells were analyzed on FACSCalibur, using 488-nm (15 mW) air-cooled argon-ion laser for propidium iodide excitation, and CELLQuestTM software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME). Western blot analyses and detection of cdk2 activity. Confluent WB-F344 cells grown on 60-mm-diameter cell culture dishes were exposed for 48 h to tested compounds or 0.1% DMSO (vehicle). Proteins were extracted for 30 min in ice-cold lysis buffer (50 mM Tris/HCl [pH 7.4], 150 mM sodium chloride, 0.5% Nonidet P-40, 1 mM EDTA, 0.1 mM dithiothreitol, 50 mM sodium fluoride, 8 mM ?-glycerolphosphate, 100 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml tosylphenylalanine chloromethane). The extracts were cleared by centrifugation at 15,000 g for 15 min at 4°C and stored at –80°C until use. Concentrations of total protein were determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Prague, Czech Republic). For kinase assays, the extracts (150 μg of total protein per reaction) were first subjected to initial absorption with protein G agarose beads and then incubated with appropriate antibodies (sc-163 for cdk2, sc-751 for cyclin A, and sc-601-G for cdk4) for 1 h in an ice bath. Immunoprecipitates were collected on protein G agarose beads by overnight rotation, washed three times with lysis buffer, and twice with kinase assay buffer (50 mM HEPES, pH 7.5; 10 mM MgCl2; 10 mM MnCl2; 8 mM ?-glycerolphosphate; 1 mM dithiothreitol). The cdk2 kinase reactions were carried out for 30 min at 37°C in a total volume of 25 μl in kinase assay buffer supplemented with 100 μg/ml histone H1 (type III-S) and 40 μCi/ml [32P] ATP. Cdk4 kinase assay was performed with 80 μg/ml GST-pRb (a gift from Dr. J. Lukas) and 40 μCi/ml 32P-ATP. Reactions were terminated by mixing with 2x Laemmli sample buffer, and each total reaction mix was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Densitometry was performed using AIDA Image Analyzer software (raytest Isotopenme?ger?te, Starubenhardt, Germany). For Western blotting, equal amounts of total protein were subjected to 10% SDS PAGE, electrotransferred onto a PVDF membrane, immunodetected using appropriate primary and secondary antibodies, and visualized by ECLPlus reagent (Amersham, Little Chalfont, UK) according to the manufacturer's instructions. When required, membranes were stripped in 62.5 mM Tris/HCl pH 6.8, 2% SDS, and 100 mM ?-mercaptoethanol, washed, and reblotted with another antibody. After immunodetection, each membrane was stained by amidoblack to confirm equal protein loading. Real-time RT-PCR for quantification of CYP1A mRNA. Total RNA was isolated from cells using the RNeasy mini kit (Qiagen, Valencia, CA) including treatment with DNase I (Qiagen). The amplifications of the samples were carried out in a final volume of 20 μl in a reaction mixture containing 10 μL of QuantiTect Probe RT-PCR Master Mix, 0.2 μl of QuantiTect RT Mix (Qiagen), 2 μl of solution of primers and probe, 5.8 μl of water, and 2 μl of sample. The final concentrations of primers and probe were 1.0 μM and 0.2 μM, respectively. The probes were labeled with a 5' FAM reporter and a 3' BHQ 1 quencher. The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the following program: reverse transcription at 50°C for 20 min and initial activation step at 95°C for 15 min, followed by 45 cycles at 95°C for 0 s and 60°C for 60 s. The primers and probe for rat CYP1A1 (Lake et al., 2003) were: forward 5'-TGAGTTTGGGGAGGTTACTGGTT-3', reverse 5'-TGAAGGCATC CAGGGAAGAGT-3', probe 5'-ATACCCAGCTGACTTCATTCCTATCCTCCGTT-3'. The primers and probe for the reference gene porphobilinogen deaminase (EC 4.3.1.8) were: forward 5'-CCCAACCTGGAATTCAAGAGTATTCG-3, reverse 5'-TTCCTCTGGG TGCAAAATCT-GGCC-3', probe 5'-CCTCAACACCCGCCTTCGGAAGCT-3'. Statistical analysis. Data were expressed as means ± S.D. and analyzed by Student's t-test, or by analysis of variance (ANOVA), followed by Dunnett's test. A p value of less than 0.05 was considered to be significant. RESULTS The Proliferative Activity of PCBs and OH-PCBs Corresponds with Their Ability to Activate AhR To investigate effects of PCBs on contact inhibition in confluent WB-F344 cells, the compounds that are representative of di-ortho-substituted (PCB47 and 153), mono-ortho-substituted (PCB 105) and non-ortho-substituted (PCB 126) congeners were selected. The study also included determination of effects of 4-OH-PCB 187, a highly abundant and persistent hydroxylated noncoplanar PCB derivative found in mammalian blood (Fangstr?m et al., 2002), and 4'-OH-PCB 79, a planar metabolite of the non-ortho-substituted PCB 77 congener. As reported previously, WB-F344 cells seeded at 30,000 cells per cm2 show a significant drop in proliferation after 72 h of incubation, which corresponds with a marked decrease in percentage of S phase cells (Chramostová et al., 2004). However, when cultivated in the presence of PCB 126, PCB 105, and 4'-OH-PCB 79 for an additional 72 h, a significant concentration-dependent increase in percentage of cells in S phase was observed in confluent WB-F344 cells, with PCB 126 being effective at a concentration as low as 100 pM (Fig. 2). These results corresponded with significantly increased cell numbers that were found for all three compounds (Fig. 3). PCB 126 did not exert a general proliferative stimulus because it had no effect on proliferation of WB-F344 cells cultivated at subconfluent densities (data not shown), and this suggested that its effects on confluent cells are due to the release from contact inhibition. Contrary to the above results, none of the other three compounds, carrying two or more chlorines at ortho positions, had any effect either on cell numbers or on cell cycle (Figs. 2 and 3). To determine whether the proliferative activity was associated with the capacity of PCBs to act as AhR agonists, we compared their effects on cell proliferation and cell cycle with induction of CYP1A1 mRNA expression. There is only limited information on inducibility of AhR-regulated transcripts, such as CYP1A1 in rat oval cells. It has been suggested that WB-F344 cells may not contain inducible CYP1A1 activity (Herrmann et al., 2002). However, both we and others have found that AhR ligands, such as polycyclic aromatic hydrocarbons or polychlorinated dibenzo-p-dioxins, are capable of inducing AhR-dependent 7-ethoxyresorufin O-deethylase activity in WB-F344 cells (Chramostová et al., 2004; K?hle et al., 1999). As outlined in Figure 4, the concentration-dependent induction of CYP1A1 by PCB 126 in WB-F344 cells corresponded with induction of cell proliferation (Figs. 2 and 3). PCB126, PCB 105, and 4'-OH-PCB 79 induced both a significant increase in percentage of cells in S phase and an increased CYP1A1 mRNA expression when applied at a concentration of 1 μM. In contrast, the non-dioxin-like PCB 47, PCB 153, or 4-OH-PCB 187 had no effect on CYP1A1 mRNA levels (Fig. 4). Induction of Cell Proliferation by PCB 126 Is Associated with Increased Cyclin A Expression and Increased Cyclin A/Cdk2 Activity It has been reported that contact inhibition of WB-F344 cells leads to downregulation of cyclin D1 and D2, decreased cyclin D2/cdk 4 activity, downregulation of cyclin A, decreased cyclin A/cdk2 activity, and accumulation of p27Kip1 (Dietrich et al., 2002). The TCDD-induced release from contact inhibition has been shown to be associated with increased expression of D-type cyclins and cyclin A, and with a significant increase of cyclin A/cdk2 activity (Dietrich et al., 2002). Therefore, we compared the effects of PCB 126, which was found to be an efficient inducer of cell proliferation, with those of PCB 153, which had no effect on proliferation of confluent WB-F344 cells or on expression of proteins associated with control of G1-S phase transition and S phase progression. The confluent cells were exposed to the test compounds for 48 h. This time interval has been reported to be optimal for detection of expression and activities of cell cycle regulatory proteins in this cell model (Dietrich et al., 2002). As shown in Figure 5, PCB 126 was found to upregulate cyclin A expression, which was similar to the effect of TCDD. Both TCDD and PCB 126 also induced a significant increase in cyclin D2 protein levels. In contrast, the levels of p27Kip1, a major cdk2 inhibitor known to be involved in contact inhibition of cell growth and S phase entry, remained unaffected by either TCDD or PCB 126. PCB 126 had no effect or only marginal on the expression of cyclins D1, D3, and E, or on protein levels of cdk2 and 4. Because the levels of cyclin A and D were significantly increased by PCB 126 treatment, we next examined the activities of cdks, which depend on the association with the above cyclins. As outlined in Figure 6A, PCB 126 induced approximately a twofold increase in total cdk2 activity. The level of cdk2 activity in untreated cells was very low, almost at background levels (data not shown), suggesting that cdk2 activity is strongly inhibited in confluent WB-F344 cells. The levels of cyclin A/cdk2 complex activity were significantly increased by both TCDD and PCB 126 treatment (Fig. 6B). In contrast, PCB 153 did not significantly affect cyclin A/cdk2 activity. Cdk4 activity in confluent cells was very low, and it was not altered by treatment with either PCB 126 or PCB 153 (Fig. 6A). DISCUSSION Polychlorinated biphenyls are known to exert tumor-promoting activity in liver, and it is conceivable that they might employ multiple tumor promoting mechanisms, because both dioxin-like and non-dioxin-like PCBs can act as hepatic tumor promoters (Glauert et al., 2001; Safe, 1994). The tumor-promoting activity of PCBs could be associated with their capacity to alter signal transduction pathways controlling cell proliferation and programmed cell death (reviewed in Glauert et al. [2001]). The effects of dioxin-like PCBs are considered to be predominantly related to activation of AhR. Several studies have demonstrated the capability of AhR ligands to modulate cell proliferation and/or to inhibit apoptosis in the liver; however, opposite effects have also been reported from various cellular models (Puga et al., 2002; Schwarz et al., 2000; Tharappel et al., 2002; W?lfle et al., 1993). Activation of AhR might also play a role in inhibition of GJIC (De Haan et al., 1994; Herrmann et al., 2002). The non-dioxin-like PCBs have been reported to inhibit apoptosis in rat hepatocytes or block GJIC in rat liver epithelial cells (Bohnenberger et al., 2001; Hemming et al., 1991; Machala et al., 2003). However, little is known about the role of PCBs in regulation of proliferation of hepatic progenitor cells, which may represent a potential target for tumor promoters. It has been shown that both TCDD and the AhR-activating polycyclic aromatic hydrocarbons induce a release from contact inhibition in rat liver epithelial stem-like WB-F344 cells (Dietrich et al., 2002; Chramostová et al., 2004). Therefore, we hypothesized that PCBs or their derivatives that act as AhR ligands might alter the normal control of cell proliferation via this mechanism. As shown in Figures 2 and 3, PCB 126, which is considered to be the most potent AhR agonist among coplanar PCBs (Glauert et al., 2001; van den Berg et al. 1998), was found to increase both cell numbers and percentage of S phase cells within the concentration range of 100 pM to 10 μM. These concentrations corresponded well with the levels of PCB 126 that were found to induce CYP1A1 mRNA expression in WB-F344 cells (Fig. 4). Similarly, the mono-ortho-substituted congener PCB 105 was found to increase CYP1A1 mRNA levels, as well as to increase the percentage of S phase cells and/or cell numbers in this cell model. Both PCB 126 and PCB 105 have been reported to induce promotion of altered hepatic foci (Dean et al., 2002; Haag-Gr?nlund et al., 1998; Hemming et al., 1995). In contrast, PCB 47 and PCB 153 had no effect on cell proliferation at concentrations up to 10 μM, nor did either compound induce CYP1A1 mRNA expression. To our knowledge, this is the first evidence that AhR-activating PCB congeners can increase cell proliferation of contact-inhibited liver epithelial cells. Such a mode of action could participate in the tumor-promoting effects of these PCBs. The concentrations of dioxin-like PCBs that induced cell proliferation in the present study correspond with the levels of PCBs used for various in vivo tumor-promotion studies in rats (Glauert et al., 2001; van den Berg et al., 1998). Polychlorinated biphenyls are known to be present at nanomolar levels in human blood (Kimbrough 1995); however, their concentrations can be significantly higher in other tissues. Significantly higher concentrations have also been found in individuals living in PCB-contaminated regions (Pavúk et al., 2004). The effects of PCBs within an organism can be further modified by formation of active hydroxylated metabolites. Hydroxylated PCBs (OH-PCBs) have been reported to disrupt estrogen and thyroid hormone signaling, or vitamin A transport (Connor et al., 1997; Kramer et al., 1997; Moore et al., 1997). Several highly chlorinated OH-PCBs have been reported to persist and accumulate in animal tissues at high concentrations (Fangstr?m et al., 2002). However, besides the fact that OH-PCBs can inhibit GJIC in vitro, little is known about their role in tumor promotion (Machala et al., 2004; Satoh et al., 2003). In the present study we found that the hydroxylated derivative of non-ortho-substituted PCB 77 and 4'-OH-PCB 79 can induce both the AhR-mediated induction of CYP1A1 mRNA expression and a release from contact inhibition in WB-F344 cells. In contrast, the noncoplanar hydroxylated PCB metabolite 4-OH-PCB 187 did not affect either CYP1A1 mRNA expression or cell proliferation. Disruption of normal control of the cell cycle is one of the important steps in carcinogenesis (Malumbres and Barbacid, 2001). Progression through the cell cycle is controlled by a sequential activation of cyclin/cdk complexes (Sherr, 2000). The D-type cyclins associate with cdk4 or cdk6, and they play an important role during both early and late G1 phase of the cell cycle. Cdk2 associates with cyclins E and A, which act at the G1-S phase transition and during S phase entry and progression, respectively (Ekholm and Reed, 2000; Sherr, 2000). Inhibition of cdk2 activity is known to be involved in maintenance of contact inhibition, and upregulation of its activity has been linked to the onset of proliferation (Chen et al., 2000). TCDD has been shown both to upregulate cyclin A levels and to increase cdk2 activity in confluent rat liver epithelial cells (Dietrich et al., 2002). This suggested that this mechanism might be activated by dioxin-like PCBs as well. We found that PCB 126, a model ‘dioxin-like’ PCB congener, can increase the expression of cyclin A and the activity of the cyclin A/cdk2 complex. In contrast, PCB 153 had no effect either on cyclin A levels or on cdk2 activity. Neither compound affected cdk4 activity, although PCB 126 increased cyclin D2 expression (Figs. 5 and 6). Although cdk4 activity was not increased, an increase in cyclin A expression and cyclin A/cdk2 activity alone could be sufficient to induce G1-S phase transition. It has been shown that the downregulation of cdk2 activity can be causative for the cell cycle inhibition, while the ectopic expression of cyclin A can restore cell cycle progression into S phase (Strobeck et al., 2000). The exact mechanism of PCB action in WB-F344 cells, leading to an increase in cdk2 activity, remains unclear. The dioxin-like compounds could activate several early-response genes involved in the regulation of cell proliferation, e.g., c-fos or c-jun, both of which have been shown to be upregulated by AhR ligands, or they could alter cell-signaling pathways stimulating cell proliferation, such as mitogen-activated protein kinases (Puga et al., 2002; Schwarz et al., 2000; Tharappel et al., 2002; W?lfle et al., 1993). It has been speculated that an increase in c-Src activity is responsible for the proliferative effects of TCDD on contact-inhibited WB-F344 cells (K?hle et al., 1999). AhR ligands have also been suggested to increase the activity of extracellular signal-regulated kinases (Tan et al., 2002). However, recent data suggest that neither c-Src nor ERK1/2 is involved in the effects of TCDD on cell proliferation in this in vitro model (H?lper et al., in press). Cyclin-dependent kinase 2 activity is controlled not only by its association with cyclin E and A, but also by cdk inhibitors, namely p27Kip1, which is an important regulator of contact inhibition in various cell types (Polyak et al., 1994). This inhibitor has been shown to be upregulated in confluent WB-F344; yet, it is also known to be upregulated by TCDD in rat hepatoma cells in an AhR-dependent manner (Dietrich et al., 2002; Kolluri et al., 1999). We found in the present study that release from contact inhibition induced either by TCDD or by PCB 126 is not associated with a decrease in p27Kip1 expression, suggesting that it is not involved in their effects on proliferation of confluent cells. Nevertheless, it cannot be excluded that the high levels of p27Kip1protein in cells released from contact inhibition by TCDD or by PCB 126 are also maintained through AhR-induced expression of this cdk inhibitor. Taken together, our data show that dioxin-like PCBs can release rat liver epithelial cells from contact inhibition by increasing cyclin A protein levels, which leads to upregulation of cyclin A/cdk2 activity. This effect correlates with induction of CYP1A1 expression, suggesting that AhR activation is involved. Disruption of cell cycle control in liver progenitor cells might play a role in the tumor-promoting effects of individual PCBs or their mixtures. Future studies should aim to describe in more detail the mechanisms responsible for this effect, as well as its in vivo significance. ACKNOWLEDGMENTS The authors thank Drs. L. Robertson and H.-J. Lehmler (University of Iowa) for providing 4'-OH-PCB 79, and Dr. ?. Bergman for providing 4-OH-PCB 187. This study was supported by grant 525/03/1527 from the Czech Science Foundation and by the Research Plan of the Academy of Sciences of the Czech Republic under grant. Z 5004920. REFERENCES Alison, M. R. (2003). Characterization of the differentiation capacity of rat-derived hepatic stem cells. Semin. Liver Dis. 23, 325–336. Bohnenberger, S., Wagner, B., Schmitz, H. J., and Schrenk, D. (2001). Inhibition of apoptosis in rat hepatocytes treated with "non-dioxin-like" polychlorinated biphenyls. Carcinogenesis 22, 1601–1606. Levenberg, S., Yarden, A., Kam, Z., and Geiger, B. (1999). p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry. Oncogene 18, 869–876. Pavúk, M., Cerhan, J. R., Lynch, C. F., Schecter, A., Petrík, J., Chovancová, J., and Kocan, A. (2004). Environmental exposure to PCBs and cancer incidence in eastern Slovakia. Chemosphere 54, 1509–1520. Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994). p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9–22. Puga, A., Xia, Y., and Elferink, C. (2002). Role of the aryl hydrocarbon receptor in cell cycle regulation. Chem. Biol. Interact. 141, 117–1130. Sherr, C. J. (2000). The Pezcoller Lecture: Cancer cell cycles revisited. Cancer Res. 60, 3689–3695. Schwarz, M., Buchmann, A., Stinchcombe, S., Kalkuhl, A., and Bock, K. (2000). Ah receptor ligands and tumor promotion: Survival of neoplastic cells. Toxicol Lett 112–113, 69–77. Strobeck, M. W., Fribourg, A. F., Puga, A., and Knudsen, E. S. (2000). Restoration of retinoblastoma mediated signaling to Cdk2 results in cell cycle arrest. Oncogene 19, 1857–1867. W?lfle, D., Becker, E., and Schmutte, C. (1993). Growth stimulation of primary rat hepatocytes by 2,3,7,8- tetrachlorodibenzo-p-dioxin. Cell Biol. Toxicol. 9, 15–31....查看详细 (32258字节)
☉ 11120226:A Proposed Testing Framework for Developmental Imm
ABSTRACT A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, industry, and academia met in May 2003, in Washington, D.C., to reach consensus regarding the most appropriate methods to assess developmental immunotoxicology (DIT) for hazard identification, including under what conditions such testing might be required. The following points represent the major conclusions from this roundtable discussion: (1) the rat is the preferred model; (2) any DIT protocol should be based on immune assays already validated; (3) DIT methods should be incorporated into standard developmental and reproductive toxicity protocols to the extent possible rather than a "stand-alone" protocol; (4) the approach to address DIT potential should be similar for chemicals and drugs, but the experimental design should be flexible and should reflect the specific questions to be answered; (5) it is possible to utilize a study design that assesses all critical windows in one protocol, with the results leading to further study of specific effects, as warranted; (6) animals should be exposed throughout the treatment protocol; (7) the triggers for DIT may include structure-activity-relationships, results from other toxicity studies, the intended use of a drug/chemical and/or its anticipated exposure of neonates and/or juveniles. Key Words: developmental immunotoxicology; DIT; immune system; developmental and reproductive toxicology; risk assessment; roundtable; study design; testing methods. While immunotoxicology has evolved to the point where guidelines exist within many regulatory frameworks, there is still a lack of consensus on the most appropriate experimental approaches and assays available to assess developmental immunotoxicology (DIT). From a scientific perspective, the interest in DIT has been predicated around the hypothesis that the developing immune system demonstrates greater susceptibility to chemical perturbation than the adult system. In light of some experimental data, DIT may be operationally defined by greater susceptibility, manifested as a qualitative difference (i.e., a chemical affects the developing immune system differently than the adult immune system; for example, demonstrates different profiles of immunotoxicity, or affects different immune parameters), a quantitative difference (i.e., a chemical affects the developing immune system at lower doses than it would affect the adult immune system; for example, demonstrates a similar profile of immunotoxicity or affects similar immune parameters, but shows a shift in the dose-response), or a more persistent effect (i.e., demonstrates a different pattern of recovery after exposure in younger animals as opposed to adult animals). From a regulatory perspective, there is concern that existing guideline immunotoxicity studies conducted exclusively in young adult animals would not detect this greater susceptibility. Moreover, although there is no question that there has been increasing interest in designing appropriate developmental toxicity protocols over the last five or more years, the integration of parameters reflecting the immune system has been quite minimal to date. This is best illustrated by the fact that immune organs are still not routinely included as potential target organs in most developmental toxicity protocols. It is with this background that ILSI HESI convened a panel of international experts from academia, government, and industry in May 2003 for a roundtable discussion on DIT. It is generally accepted that development of the immune system extends well beyond the early neonatal period in rodents (Holsapple et al., 2003; Landreth, 2002; Luster et al., 2003). Furthermore, the U.S. Environmental Protection Agency, in its Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), defines developmental toxicology as "the study of adverse effects on the developing organism that may result from exposure prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation." It is with this definition of developmental toxicology in mind that roundtable participants worked to reach agreement regarding the most appropriate methods to assess DIT, including under what conditions such testing might be required. A series of pre-determined discussion questions were identified (Table 1) and key observations and points of consensus were captured during the discussion. A summary of these points was prepared and distributed to all roundtable participants to ensure accuracy and completeness. The consensus conclusions of the roundtable discussion are addressed in this article. It is anticipated that this summary will contribute significantly to efforts to develop an appropriate DIT guideline protocol. Triggers A key issue in determining the need to conduct DIT studies is the identification of appropriate "triggers," that is, cause(s) for concern. Several general triggers were identified during the roundtable discussion. 1. Structural alerts. There are two general considerations with respect to chemical structure: structure-activity relationships (SAR) identified using computational methods and chemical class effects. There appears to be no public database which could be used for computational determination of immunotoxic potential. However, if a compound belongs to a class known to be associated with immunotoxicity (Dietert et al., 2001), this could be taken to indicate the need for DIT studies. 2. Findings in toxicology studies. A wealth of toxicology data is generated to support the safety assessment of pharmaceuticals and industrial or agricultural chemicals. These studies are generally conducted in adult rodent and non-rodent species, and include screening level assessments of a range of endpoints across multiple organ systems, following various durations of exposure via the route that is expected to be most relevant to humans. A typical toxicology database will also include standard studies that assess effects of prenatal and/or postnatal administration of the material to animal models. Treatment-related perturbations to immune system structure or function may be identified. These could be observed as adverse clinical findings (e.g., susceptibility to disease) that indicate inadequacy in immune system function; alterations in immune system functional endpoints (e.g., T-cell dependent antibody response; TDAR) in a guideline immunotoxicity study in adult animals; alterations in lymphoid organ weights, macroscopic findings, or histopathology of spleen, thymus, or lymph nodes; alterations in differential white cell counts or serum globulin/immunoglobulin levels; or cellular alterations that are nonspecific indicators of an effect on the immune response (e.g., increased numbers of macrophages in lung tissue or an increased incidence of inflammatory dermal lesions). Thus, compounds that are immunotoxic in adults should be considered likely to be immunotoxic to the developing immune system in general, although qualitative and quantitative differences may exist, as discussed above. 3. Findings in humans. Occasionally, evidence of immunotoxicity may be observed in humans, either due to intentional (e.g., drugs) or unintentional (e.g., foods or environmental chemicals) exposure. These findings, such as increased infections, are probably rare and would likely only be definitively detected in controlled clinical trials. However, if such effects are observed, and follow-up toxicology studies confirm immunotoxic potential, consideration should be given to conducting DIT studies. 4. Intended use. If a compound has been identified as causing immunosuppression, under most circumstances, inclusion of immunotoxicity determinations in developmental toxicology studies should be considered. The labeled indication often represents only a subset of real world use (which can include off-label use as well), and this fact should be taken into consideration in determining the need for DIT studies. Another issue to consider is the ability of the test compound to cross the placenta and/or be secreted in milk. If fetal/neonatal exposure appears to be so low as to be of no real consequence, this could be taken to indicate that DIT studies would be of little use in assessing risk. Characteristics of the intended patient population should be considered as well. For example, if a drug is intended to be used to treat HIV infection, immunotoxicology studies are usually needed because of the apparent increased vulnerability of these patients to drug-induced immunosuppression. In this circumstance, DIT studies would be needed for a comprehensive evaluation of risk, especially if the drug is intended to be used to prevent perinatal transmission of HIV infection. With respect to use of a drug in pediatric patients, there is currently no general expectation that juvenile animal toxicology studies need to be conducted if there is sufficient confidence in the established hazard assessment database (U.S. FDA, 2004). However, this issue is typically addressed on a case-by-case basis, and if a compound is a demonstrated immunotoxicant in mature animals, consideration should be given to conducting an appropriate immunotoxicology study in a juvenile animal model in order to estimate relative risk. A General Protocol for Assessment of Developmental Immunotoxicology Historically, evaluation of developmental immunotoxic effects has most often involved in utero exposure of pups, with dams being exposed at a variety of times during gestation and/or lactation and immune evaluation occurring when pups had attained adulthood (Fig. 1A). The period of non-exposure prior to immune evaluation may make this protocol more applicable for the evaluation of persistent immune effects, but leaves open the possibility that biologically significant effects may occur at earlier times and could be missed due to recovery during the period of non-exposure. Roundtable participants were asked to consider the extent to which DIT endpoints could be included in existing reproductive and/or other developmental protocols. Though not specifically discussed at the roundtable, incorporation of DIT endpoints into a standard prenatal developmental toxicology (teratology) study is not feasible because fetuses are evaluated prior to birth and exposure often occurs only during a specific stage of development (e.g., gestation days 6–17). Thus, other stages of immune system development would be missed. Consensus was achieved among roundtable participants that it may not be technically possible to add more endpoints to a developmental neurotoxicology (DNT) protocol (U.S. EPA, 1998c) because, logistically, this protocol is already quite complex. Participants did agree, however, that addition of DIT endpoints to standard reproductive/developmental toxicology protocols is generally possible. The feasibility of the latter approach has been previously demonstrated in studies described by Smialowicz and colleagues (2001). Such a protocol would assess the potential immunotoxic impact of a chemical during all critical windows of immunological development (Fig. 1B). In this proposed design, exposure of dams would begin prior to or at the time of conception, and would continue either through lactation or until such time as the pups could be directly dosed. Direct dosing would continue post-weaning until the pups were evaluated either on postnatal day (PND) 42, or when pups were 8–10 weeks old (young adults) if dosing into adulthood was desired. It should be noted that the purpose of the suggested protocol is not risk assessment, but rather, hazard identification. If one desires, modifications of this design could be made to assess the exact window(s) of sensitivity; for example, whether observed developmental immunotoxic effects are induced during in utero exposure (Fig. 1C) or during neonatal/juvenile exposure (Fig. 1D). When considering the proposed DIT protocol, assurance of offspring exposure to the test compound must be addressed. Roundtable participants agreed that pups in DIT studies should be exposed throughout the entire developmental period for the purpose of identifying potential adverse effects. While exposure cannot be specifically controlled in utero (placental transfer is a function of the chemical being tested), offspring can be exposed to compounds either via lactational exposure or through direct oral dosing. Participants also agreed that the decision to direct-dose pre-weaning pups may depend on the compound. If data on the test chemical indicate that lactational exposure does not occur, then direct-dosing may be necessary to assure exposure throughout the treatment protocol. Participants also agreed that the criteria for pre- and post-natal exposure should be consistent across developmental toxicity guidelines/protocols (e.g., developmental neurotoxicity) and should be predicated on the need to ensure exposure of the young during the critical window of organ system development under assessment. Several important considerations were discussed; specifically, limitations on our ability to quantify internal dosimetry under different dosing regimens and across different windows of susceptibility. Roundtable participants agreed that an understanding of the maximum tolerated dose (MTD) in pups was important because, depending on the chemical, the MTD may be higher or lower than that observed in the adult since the developmental status of the gastrointestinal tract and general metabolic processes, and/or potential target organ systems may have significant impact on the absorption, distribution, metabolism, and elimination characteristics of a compound in these young animals. It was questioned whether there was a need for a dose range-finding study in young animals. While consensus was reached on the ability to direct-dose rat pups beginning at PND 7, some investigators reported experience in oral dosing as early as PND 4. Participants acknowledged that determination of plasma levels in rat pups was technically challenging and consensus was not specifically reached on whether acquisition of toxicokinetic endpoints in pups was critical. For risk assessment purposes, it was felt that an understanding of these parameters was important, but this specific information may not be required for hazard identification (although it was considered beneficial). Roundtable participants also agreed that, if a study was conducted assessing all critical windows of exposure at once as suggested in the proposed DIT protocol (Fig. 1B) and that study were negative, then there would be no need to conduct further immunotoxicity testing solely in adult animals. In this instance, carrying exposure out to PND 42 in the proposed DIT protocol prior to functional evaluation would likely identify a similar response as would be seen if a standard immunotoxicology study were conducted in adult animals only. Methods to Assess Developmental Immunotoxicology (DIT) Although researchers have exposed animals in utero to chemicals or drugs, the majority of studies evaluating DIT to date are based on the measurement of immune status in the adult (Barnett, 1996; Dietert et al., 2000 (Fig. 1A)). This is not surprising, as most procedures developed for assessing the effects of chemicals and drugs on the immune system have undergone extensive evaluation in adult rodents only (Luster et al., 1988, 1992). As demonstrated by Luster et al. (1988, 1992, 1993), the most effective and sensitive methods of detecting immunotoxic alterations in adult B6C3F1 mice are those that assess immune function (e.g., the primary humoral immune response). Moreover, the EPA's immunotoxicity testing guidelines (U.S. EPA, 1998d) requires assessment of a functional parameter (i.e., the primary antibody response to a T-dependent antigen), together with measurement of immune organ weights as the first tier of testing. EPA also recommends that histopathology of immune system organs be assessed in subchronic and chronic studies. FDA, in their immunotoxicicty testing guidelines (U.S. FDA, 2002), also recommends the use of functional assays to assess the immune system of adult animals. Roundtable participants suggested that, when possible, methods to assess DIT should be added onto existing reproductive/developmental toxicology protocols. It was agreed that histopathologic evaluation of immune organs could easily be incorporated into existing reproductive/developmental protocols. Currently, neither EPA (U.S. EPA, 1998a,b) nor FDA (International Conference on Harmonisation, 1998) recommends histopathology of immune organs in guideline reproduction and developmental studies. It was agreed that histopathologic evaluation of immune organs could easily be incorporated into existing protocols. Despite this general agreement, there was much discussion around exactly what role pathology should play in assessing DIT. Some participants suggested routine histopathology was not sensitive enough to detect all potential immunotoxic effects, as noted in the recent article by Germolec et al. (2004). These participants argued that this may be particularly true for the developing immune system, and that more sensitive methods that assess immune function may need to be utilized. For example, thymus weight and limited histopathology of the thymus failed to detect the developmental immunotoxicity of lead, whereas immune functional assays indicated lead-induced alterations (Bunn et al., 2001b). However, other participants felt that histopathology could detect immunotoxic effects, although more defined methods (specifically immune markers, grading, and image analysis) may need to be identified. Roundtable participants emphasized the importance of capitalizing on already evaluated/validated assays and endpoints for assessing DIT. To this end, roundtable members agreed that an assay assessing the T-cell dependent antibody response (TDAR) was ready to be included in a DIT protocol. Either the plaque-forming cell (PFC) response (Holsapple, 1995) or an ELISA (Temple et al., 1993) may be used to measure the antibody response to a T-cell dependent antigen (e.g., sheep erythrocytes (SRBC)). Several other assays (i.e., phenotypic analysis of immune cells by flow cytometry, macrophage function, and natural killer cell activity) previously discussed at the NIEHS/NIOSH workshop (Luster et al., 2003) were also discussed. These assays were deemed to require further evaluation and validation before their utility in assessing DIT could be determined. Additionally, host resistance assays were not considered appropriate for a DIT screen (Holsapple, 2002), as these assays are considered to be a final tier of testing and are typically conducted only when data from a primary screen suggest alterations in immune parameters. The question of whether different assays were needed to assess different developmental windows of vulnerability was also discussed. Participants acknowledged that required tests would likely vary by chemical, based on the specific questions that each agency (EPA versus FDA) needs to have addressed. Data on whether test procedures optimized in adult animals are useful in assessing the functionally immature immune system of young animals, however, are limited. Results of a study by Ladics et al. (2000) suggest that it may not be possible to measure an antibody response in rat pups due to the immature status of their immune cells, whereas data in rat weanlings (i.e., PND 21 or older) indicate that an antibody response to SRBC of sufficient magnitude can be measured with the PFC assay. Roundtable members agreed that in rats, PND 30 SRBC responses were similar in magnitude to adult levels. Furthermore, although reports indicate that the delayed-type hypersensitivity (DTH) response can be assessed in weanling rats (Bunn et al., 2001a), participants agreed that data are lacking as to whether cell-mediated immune (CMI) assessments in younger animals are feasible. The degree of variability that occurs in the antibody response over different ages (i.e., stages of development) of a rat was also discussed; however, participants acknowledged there were too few data available to address this issue. Roundtable participants discussed whether inclusion of a TDAR, immune organ weights, and histopathology of immune system organs in the proposed DIT protocol was adequate for assessing DIT. Although data are limited, there are some examples of developmental immunotoxicants (i.e., lead and dioxin) that have been reported to affect only the CMI system (Bunn et al., 2001a,b; Gehrs and Smialowicz, 1999; Miller et al., 1998). It is important to emphasize, however, that the preferential alteration of CMI versus humoral immunity in the developing immune system was identified in a previous DIT workshop as an important data gap needing further investigation (Holsapple, 2002). Nevertheless, it was recommended at this roundtable and in previous DIT workshops that a CMI assay, such as the DTH, be considered in any proposed DIT protocol (Holsapple, 2002; Luster et al., 2003). For measurement of CMI, roundtable participants suggested that a ‘validated’ DTH or T-cell responses to anti-CD3 be evaluated. Previous results (Bunn et al., 2001a) have suggested that it is possible to avoid the use of a separate group of animals when assessing both the humoral and CMI responses. Data Interpretation It is important to emphasize that the roundtable discussion did not address the relevance of results from a DIT test to human clinical outcomes. As previously mentioned, the proposed DIT protocol is intended for hazard identification, not risk assessment. Furthermore, questions regarding the clinical relevance of results from developmental animal studies are not unique to DIT, but rather, should be addressed for all developmental and reproductive testing protocols (e.g., DNT). Nevertheless, some discussion regarding the applicability of results from animal studies to human health risk assessment were touched upon, although not resolved, at the roundtable. While immunotoxicology screening tests have undergone a series of validation exercises, and it is established that immunosuppression can lead to an increased incidence and/or severity to infectious and neoplastic diseases, interpreting results from immunological tests in DIT studies, or even from epidemiological studies conducted in children for quantitative risk assessment purposes, is problematic. This is particularly true when the immunological effects, as might be expected to occur from inadvertent exposures, are minimal-to-moderate in nature. Thus, it is important that a scientifically sound framework be established that allows for more accurate and quantitative interpretation of such data in the risk assessment process. Although experimental animal models provide an opportunity to establish more reliable exposure estimates and conduct more informative immune tests than human studies, extrapolating findings across species still requires the application of certain estimates or assumptions to account for differences in the integrity of the host's anatomical and functional barriers and the overall immunocompetence of an individual which can be affected by genetics, age, gender, use of certain medications, nutritional status, and environment (Morris and Potter, 1997). Conclusions and Future Needs The roundtable addressed a number of research needs previously identified in other forums. These include a recommendation that all critical windows be addressed in one protocol and that the results trigger subsequent studies, as warranted; the need for flexibility in any approach to address DIT potential and to understand the role of exposure in study design; and the recognition that immunotoxicity is most appropriately addressed by functional tests. Several assays were discussed for inclusion in a DIT study based upon their common application in clinical immunologic evaluations and immunotoxicity testing in adults. These include macrophage function, complement analysis, and surface marker analysis. In addition to the research needs identified for these specific assays, there was some emphasis placed on the need to more fully examine the relationship between adverse immune system responses in animals and estimations of human immunologic risk. Participants in the DIT roundtable emphasized a number of important research efforts currently underway in the area of DIT, which examine immune system responses following developmental exposures to known immunotoxicants. For example, an American Chemistry Council-funded research project involving two laboratories is evaluating whether the developing immune system shows differential susceptibility to chemical perturbation compared to the adult immune system (Dietert, 2003; Dietert and Lee, 2003; Dietert et al., 2003, 2004; Matulka et al., 2003). As well, studies are being conducted under the auspices of NIEHS to better understand the effects of endocrine disruptors on immune system development (Guo et al., 2002; Karrow et al., 2004). Inter-laboratory comparisons of such data will assist in further defining the standardization of methodologies used in DIT assessment. Methods for immune system hazard characterization currently focus on assessing immune suppression, and to some extent hypersensitivity, in adult animals. However, there is no established assessment model for the induction of autoimmunity or immune system over-stimulation—both important potential immunotoxic responses. Furthermore, the inclusion of an assessment of CMI response in a DIT test guideline was recognized at the roundtable as desirable, yet there remains some uncertainty regarding the status of validation for this methodology. Some methodological aspects of standardized DIT testing need to be further developed and supported through research. For example, while it is widely recognized that pharmacokinetic data could be useful in establishing dose levels for a study, there is no consensus regarding what those preliminary pharmacokinetic studies should entail. Also, concerns about ensuring exposure of the offspring via direct-dosing need to be further explored to determine the most relevant and scientifically valid manner in which to approach this issue. Additionally, the manner in which studies can be combined should be examined in order to reduce the number of animals used in testing. Further research is also needed to more effectively assess risks to developing humans. Foremost is a need to establish quantitative models to allow for accurate extrapolation of results from DIT screening studies to potential health effects (e.g., infectious disease, leukemia, etc.) in children. As well, more information is needed to determine whether an effect on an immune endpoint from an adult study can be applied to set safe levels in juveniles; specifically, information on expression of immune endpoints in juveniles is lacking. It is also not known whether juvenile humans are more sensitive to immune system perturbations than adults, although animal data have clearly identified age-related sensitivities for some chemicals (e.g., lead and dioxin). Additionally, the role of other factors in susceptibility to immune system perturbation is not known. These include polymorphisms in genes that are associated with immune system responses and the role of stress. Evaluations of the public health implications of immune system suppression will assist in further characterizing potential immunotoxic risks. NOTES views expressed in this document are those of the authors and do not necessarily reflect the views of policies of the U.S. Environmental Protection Agency or the U.S. Food and Drug Administration. As well, no official support or endorsement by these agencies is intended or should be inferred. ACKNOWLEDGMENTS The authors thank the Immunotoxicology Technical Committee (ITC) at the International Life Sciences Institute, Health and Environmental Sciences Institute (ILSI HESI) for hosting the May 2003 Developmental Immunotoxicology (DIT) Roundtable in Washington, DC, and supporting the preparation of this manuscript. The authors also acknowledge their fellow roundtable discussion participants: Peter Bugelski (Centocor, Inc.), Miklos Csato (F. Hoffmann-La Roche AG), Rodney Dietert (Cornell University), Ellen Evans (Schering-Plough Research Institute), Dori Germolec (NIEHS), Helen Haggerty (Bristol-Myers Squibb Company), Danuta Herzyk (GlaxoSmithKline), Dennis Hinton (U.S. FDA, CFSAN), Catherine Kaplanski (Merck Research Laboratories), Thomas Kawabata (Pfizer Global Research & Development), Hervé Lebrec (3 M Pharmaceuticals), Cynthia L. Mann (ExxonMobil Biomedical Sciences, Inc.), Barbara Mounho (Amgen, Inc.), Laurie G. O'Rourke (Novartis Pharmaceutical Corporation), Don O'Shaughnessy (D. O'Shaughnessy Consulting, Inc.), Marc Pallardy (Université de Paris), Richard W. Pfeifer (Wyeth Research), Lynnda Reid (U.S. FDA, CDER), Mary Jane Selgrade (U.S. EPA), Kimber L. White, Jr. (Medical College of Virginia), Daniel Wierda (Eli Lilly and Company), and Michael Woolhiser (Dow Chemical Company). The input of all involved was incorporated into the preparation of this manuscript. Finally, the authors thank Norbert Kaminski (Michigan State University) and Robert Luebke (U.S. EPA) for their careful review of the document as part of ILSI HESI's peer review process. REFERENCES Barnett, J. B. (1996). Developmental immunotoxicology. In Experimental Immunotoxicology (R. J. Smialowicz and M. P. Holsapple, Eds.), pp. 47–62. CRC Press, Boca Raton, FL. Dietert, R. R., Lee, J. E., and Bunn, T. L. (2001). Developmental immunotoxicology: Emerging issues. Human Exp. Toxicol. 21, 479–485. Dietert, R. R., Lee, J.-E., Olsen, J., Fitch, K., and Marsh, J. A. (2003). Developmental immunotoxicity of dexamethasone: Comparison of fetal vs. adult exposures. Toxicology 194, 163–176. Dietert, R. R., Lee, J.-E., Olsen, J., Fitch, K., and Marsh, J. A. (2004). Dexamethasone-induced immunotoxicity following fetal vs. adult exposure. Toxicologist 78(S-1), 96. Holsapple, M. P. (1995). The plaque-forming cell (PFC) response in immunotoxicology: An approach to monitoring the primary effect function of B lymphocytes. In Methods in Immuntoxicology, Vol. 1 (G. R. Burleson, J. H. Dean, and A. E. Munson, Eds.), pp. 150–76. Wiley-Liss, New York. Karrow, N. A., Guo, T. L., Delclos, K. B., Newbold, R. R., Weis, C., Germolec, D. R., White, Jr., K. L., and McKay, J. A. (2004). Nonylphenol alters the activity of splenic NK cells and the numbers of leukocytes subpopulations in Sprague-Dawley rats: A two generation feeding study. Toxicology 196, 237–245. Ladics, G. S., Smith, C., Bunn, T. L., Dietert, R. R., Anderson, P. K., Wiescinski, C. M., and Holsapple, M. P. (2000). Characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen. Toxicol. Methods 10, 283–311. Landreth, K. S. (2002). Critical windows in development of the rodent immune system. Human Exp. Toxicol. 21, 493–498. Luster, M. I., Munson, A. E., Thomas, P. T., Holsapple, M. P., Fenters, J. D., White, K. L., Jr., Lauer, L. D., Germolec, D. R., Rosenthal, G. J., and Dean, J. H. (1988). Development of a testing battery to assess chemical induced immunotoxicity: National Toxicology Program's guidelines for immunotoxicity evaluation in mice. Fundam. Appl. Toxicol. 1, 2–19....查看详细 (31806字节)
☉ 11120227:Signaling Modulation of Bile Salt-Induced Necrosis
ABSTRACT Hydrophobic bile salts induce either necrosis or apoptosis depending on the severity of the injury caused by them. Since bile salt-induced apoptosis is influenced by Ca2+- and protein kinase-signaling pathways, and both necrosis and apoptosis share common initiating mechanisms, we analyzed whether these signaling cascades also influence bile salt-induced necrosis in isolated rat hepatocytes. Taurochenodeoxycholate (TCDC, 0.25–1.50 mM, 2 h) reduced, in a dose-dependent manner, the percentage of viable hepatocytes, and increased the release of the cytosolic enzyme, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), and that of the plasma membrane enzyme, alkaline phosphatase (AP). The PKC inhibitors, H7 (100 μM) and chelerythrine (2.5 μM), both prevented significantly TCDC-induced necrosis. On the contrary, the PKA activator, dibutyryl-cAMP, exacerbated TCDC-induced cell damage in a dose-dependent manner; this effect was more likely due to cAMP-mediated PKA activation, as the PKA inhibitor, KT5720 (1 μM), counteracted this effect. Instead, the intracellular Ca2+ chelator, BAPTA/AM (20 μM), was without effect. TCDC (1 mM) increased lipid peroxidation from 0.7 ± 0.2 to 7.5 ± 0.9 nmol of malondialdehyde per mg of protein, p 90%), as assessed by the trypan blue exclusion test (Baur et al., 1975). Treatments. Hepatocytes were resuspended in Krebs-Ringer-HEPES buffer, pH = 7.4, supplemented with 0.5% D-glucose and 3% BSA, to reach a final density of 2.5 x 105 cells/ml (unless otherwise indicated). The suspension was kept on ice no longer than 30 min before use. Four ml of this suspension were incubated without or with the hydrophobic BS, TCDC (0.25, 0.50, 1.00, and 1.50 mM) for 2 h in 20 ml in plastic beakers, immersed in a Dubnoff water bath at 37°C, under an atmosphere of 95% O2/5% CO2; TCDC was used as a tool, since it was shown to induce a dose- and time-dependent necrotoxic effect to hepatocytes, as apparent from loss of cell viability and leakage of cytosolic enzymes (Ohiwa et al., 1993; Sokol et al., 1993). The selection of the concentrations and the time of exposure of TCDC was based upon a previous study by Ohiwa et al. (1993), which showed that necrotoxic changes occur in hepatocytes at TCDC concentrations higher than 0.1 mM, and at exposure periods longer than 1 h. The effect of pre-incubation of the hepatocytes with a number of signaling modulators was studied to ascertain the respective roles of PKA-, PKC-, and the Ca2+-dependent signal pathways in the necrotic effect of TCDC. The compounds tested, their biological effects, their final concentrations and the volume and kind of vehicle used for delivery are also indicated in Table 1. Hepatocytes were pre-incubated with the signaling modulators for 15 min, and then exposed to increasing TCDC concentrations for a further 2-h period. The signaling modulators were kept in the incubation medium throughout TCDC exposure. In a separate set of experiments, we sought to determine whether addition of an antioxidant before exposure of TCDC attenuates TCDC-induced necrosis. For this purpose, hepatocytes were pretreated for 15 min with the antioxidant agent, DPPD (50 μM), before adding TCDC. Analytical Methods Assessment of hepatocellular integrity. At the end of the incubation period with TCDC, aliquots of hepatocytes were removed to assess cell viability, leakage of the cytosolic enzymes, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), as well as the release of the plasma membrane-associated protein, alkaline phosphatase (AP). Hepatocyte viability was assessed by the trypan blue exclusion test (Baur et al., 1975). For this purpose, 5 μl of cell suspension were added to 150 μl of trypan blue (1.3 g/l), dissolved in HEPES-supplemented Hanks' solution. Viability was calculated as the percentage of hepatocytes able to exclude the dye from their cell bodies, referred to the values recorded in control cells not exposed to TCDC. Impairment of barrier properties of the hepatocellular plasma membrane is a chief event in cellular necrosis. To evaluate plasma membrane integrity, leakage of the cytosolic enzymes, LDH (EC 1.1.1.27) and ALAT (EC 2.6.1.2), into the incubation medium was assessed. These enzymes were determined spectrophotometrically in the incubation medium (Perkin Elmer UV/Vis Spectrometer Lambda2S, überlingen, Germany), by measuring the rate of NADH consumption at 340 nm using commercial, kinetic kits (Wiener Lab., Rosario, Argentina). The capability of BSs to impair hepatocellular integrity is associated with their ability to remove membrane lipids, thus releasing plasma membrane-associated proteins into the incubation medium. We evaluated this process by studying the release of the plasma membrane protein, AP (EC 3.1.3.1), assessed by measuring the rate of the AP-catalyzed conversion of p-nitrophenyl phosphate to p-nitrophenol, using a commercial, kinetic kit (Wiener Lab., Rosario, Argentina). Correction of the inhibition of these enzyme activities by the TCDC present in the reaction medium where enzyme activities had been assessed was carried out. For this purpose, a rat serum sample previously subjected to assessment of LDH, ALAT, and AP activity was used as an internal standard, by adding it into the reaction medium after the enzyme activity in the cell incubation medium had been measured. Serum sample addition increases abruptly the rate of NADH consumption (for LDH and ALAT) or p-nitrophenol apparition (for AP), as it becomes proportional to the sum of the enzyme activities of both extracellular medium and serum. The apparent enzyme activity of the serum sample in the reaction medium subjected to TCDC-induced inhibition () can be therefore calculated as the difference between the slope of NADH consumption (or p-nitrophenol apparition) before and after the serum sample is added into the reaction medium. Inhibition of the activity of the exogenously added enzymes (I) can be then calculated as where AS is the initially measured enzyme activity in the serum sample, before adding it to the reaction medium. Actual enzyme activity in the extracellular medium (AECM) was then calculated by correcting the initially recorded activity () by the inhibition of the activity of the exogenously added enzyme as follows: The activity of the enzymes released into the incubation medium was expressed as the percentage of total enzyme cell activity, to minimize influence of interindividual differences in enzyme cellular content. For this purpose, aliquots of the cellular suspension were treated with Triton X-100 (0.1% v/v), followed by centrifugation at 9000 x g for 2 min. Treatment with this tensioactive compound induces release of the total enzyme cellular content, both by dissolving membrane components and by releasing cytosolic enzymes due to loss of membrane barrier integrity. Assessment of lipid peroxidation. ROS production in the presence of TCDC was assessed by measuring generation of lipid peroxidation products, by a modification of the thiobarbituric acid-reactive substances (TBARS) method (Buege and Aust, 1978). Briefly, 0.2 ml of a cell suspension containing 106 cells/ml were added to 0.5 ml of trichloroacetic acid (10% w/v) and 50 μl of the antioxidant, DPPD (60 μM). The resulting supernatant was then added to 1 ml of thiobarbituric acid (0.7% w/v), and heated in a water bath to 100°C for 15 min. After cooling and centrifugation (1000 x g for 10 min), absorbance was measured at 532 nm. A standard curve using 1,1,3,3-tetramethoxypropane, which is converted mol for mol into malondialdehyde (MDA), was routinely run. Protein content in the aliquots of cell suspension used for the assay was measured by the method of Lowry et al. (1951). TBARS were then expressed as nmol of MDA equivalents per mg of proteins. Measurement of intracellular Ca2+ concentration ([Ca2+]i). The effect of the pre-treatment with the intracellular Ca2+ chelator, BAPTA/AM (20 μM, 15 min), on TCDC (1 mM)-induced increase in [Ca2+]i was assessed 15 min after the administration of the BS, using Fura-2/AM as a probe. For this purpose, 2 x 106 hepatocytes were resuspended at 37°C in 3 ml of a PBS buffer solution (pH = 7.4), containing 3 mM CaCl2, and then supplemented with 10 μM Fura-2/AM. Fluorescence intensities (F) were measured by using alternating excitation of 340 and 380 nm, and a fluorescence emission wavelength of 510 nm (3 nm bandwidth), using a spectrofluorometer Shimadzu RF-5301 PC. [Ca2+]i was calculated from the 340 nm/380 nm Fura-2/AM fluorescence intensity ratio (R), according to the following equation (Grynkiewicz et al., 1985): where Kd is the dissociation constant of the complex, Fura-2/Ca2+ (135 nM), Rmax and Rmin are R values measured sequentially by addition of 100 μg/ml digitonin to the Fura-2-loaded cells before and after chelating Ca2+ with 5 mM EGTA/Tris solution (pH 8.7), respectively. Statistical analysis. The results were expressed as mean ± SE. When requirements for parametric analysis were met, a Student's unpaired t-test was used for comparison between two groups; comparisons between groups that did not meet this criterion were made by using the Mann-Whitney's rank sum test. The Kruskal-Wallis' test (one-way ANOVA by ranks) was used when more than two groups were compared, followed by the Dunn's multiple-comparison, post hoc test for pairwise comparisons, if ANOVA reached any statistical significance among groups. P values lower than 0.05 were judged to be significant. RESULTS Effect of PKC Inhibitors on TCDC-Induced Hepatocellular Damage The incubation of freshly isolated hepatocytes for 2 h in the absence of TCDC administration led to a very small decrease in hepatocellular viability (–5 ± 1%) and to a slight increase in the activity in the incubation medium of LDH, ALAT, and AP (+3 ± 1%, +5 ± 2%, and +4 ± 1%, respectively). In contrast, TCDC exposure resulted in a far more severe, dose-dependent decline in cells viability (Fig. 1). This decline was associated with a gradual increase in the release of LDH, ALAT, and AP, suggesting a dose-dependent impairment in plasma membrane integrity. Activation of PKC by BSs has been well documented (Bouscarel et al., 1999). Therefore, we ascertained whether PKC activation plays a role in BS-induced toxicity. Figure 1 shows the effect of pretreatment of isolated hepatocytes with H7, a preferential PKC inhibitor which exhibits a slight PKA inhibitory effect as well. This inhibitor prevented partially the disruption of plasma membrane integrity induced by TCDC from a dose of the BS of 0.5 mM onwards, as visualized by the improvement in cell viability and a reduction in the release of the enzymes, LDH, ALAT, and AP. To confirm whether the toxic effect induced by TCDC actually involves activation of the PKC-dependent pathways, we pretreated hepatocytes with the more specific PKC inhibitor, Che. Like H7, this compound partially attenuated the hepatocellular damage, as revealed by the improvement in cell viability and a reduction in the TCDC-induced release of LDH, ALAT, and AP into the incubation medium (Fig. 2). The protective effect of Che reached a significant difference in all the parameters of cell integrity evaluated at the TCDC concentration of 1 mM, although cell viability showed an improvement in a wider range (0.25–1 mM), with a tendency towards protection with the remaining parameters evaluated. Lack of involvement of PKA inhibition as an artifact in the evaluation of the protective effect of H7 was further confirmed by using the specific PKA inhibitor, KT5720, which failed to affect per se TCDC-induced necrosis (data not shown). Effect of DB-cAMP on TCDC-Induced Hepatocellular Damage DB-cAMP, a freely diffusible cAMP analogue which renders the second messenger, cAMP, by non-selective, esterase-mediated hydrolysis, is a selective PKA activator. We used this tool to ascertain whether activation of PKA-dependent pathways modulates BS-induced necrosis. As shown in Figure 3, DB-cAMP, administered at the concentration range of 0.05–1 mM, exacerbated in a concentration-dependent manner the hepatocellular injury induced by TCDC, as revealed by both a more marked decrease in cell viability and an exacerbation of the release of LDH, ALAT, and AP. Since, apart from activating PKA, DB-cAMP evokes cytosolic Ca2+ elevations (Staddon and Hansford, 1986), we verified whether the exacerbation of the necrotoxic effect of TCDC depends on PKA activation, by using the specific PKA inhibitor, KT5720. As shown in Figure 4, the deleterious effect induced by DB-cAMP (0.05 mM) was dependent on PKA activation, since KT5720 attenuated significantly its harmful effect. Role of Cytosolic Ca2+ Elevations in TCDC-Induced Hepatocellular Damage BSs act as Ca2+ ionophores and induce both Ca2+ influx from the extracellular medium and mobilization of Ca2+ from intracellular stores (Bouscarel et al., 1999). To ascertain the role of Ca2+ elevations in BS-induced necrosis, we compared the capability of TCDC to induce hepatocellular damage in the presence or in the absence of the intracellular Ca2+-chelating agent, BAPTA/AM. As shown in Table 2, TCDC induced a 86% increase in [Ca2+]i. BAPTA/AM significantly reduced basal [Ca2+]i by 69%, and completely prevented the increase in this concentration induced by TCDC, maintaining Ca2+ levels at values even lower than controls, despite the presence of TCDC in the incubation medium. In spite of this, BAPTA/AM did not protect against TCDC necrotoxic effect at any of the BS concentrations tested Role of Oxidative Stress in TCDC-Induced Hepatocellular Damage Evidence has been provided for the involvement of oxidative stress in BS-induced hepatocellular death (Sokol et al., 1993, 2001). We tested in our experimental setting the contribution of this pathomechanism by assessing the protective effect of the antioxidant, DPPD, on TCDC toxicity. As depicted in Table 3, TCDC, at the dose of 1 mM, increased more than one order of magnitude lipid peroxidation levels, as assessed by MDA generation. Pretreatment with the ROS scavenger, DPPD (50 μM), completely blocked this increase. As shown in Figure 6, DPPD prevented significantly the drop of cell viability and the release induced by TCDC of the three hepatocellular enzymes studied only at the higher concentration studied (1.5 mM), although a tendency towards protection was apparent at the TCDC concentration of1 mM, which did not reach statistical significance for LDH and ALAT. Effect of PKC/PKA Modulators on TCDC-Induced Lipid Peroxidation Since both PKC and PKA activation affected BS-induced hepatocellular damage, and this effect was partially dependent on BS-induced ROS formation (see above), we evaluated whether PKC and PKA modulators affected BS-induced lipid peroxidation. As can be seen in Table 3, 1 mM TCDC increased more than one order of magnitude lipid peroxidation levels, as assessed by MDA generation. This increase was slightly, but significantly, counteracted by the PKC specific inhibitors, Che and staurosporine (SP); TCDC induced only a 8.1- and 8.6-fold increase in MDA generation in the presence of Che and SP treatment, respectively, as compared with the 10-fold increase for TCDC alone. On the contrary, the PKA activator, DB-cAMP, was without effect on this parameter. DISCUSSION The mechanisms by which BSs exert their hepatotoxic effect have not been completely elucidated. However, several lines of evidence suggest that ROS are generated and participate actively in its pathogenesis. Sokol et al. showed an association between BS-induced hepatotoxicity and the generation of ROS both in isolated rat hepatocytes (Sokol et al., 1993) and in the intact rat (Sokol et al., 1998), since BS-induced hepatotoxicity in both models was significantly attenuated by antioxidants; these previous findings were confirmed in this study by using the ROS scavenger, DPPD (see Fig. 6). These data, together with other works emphasizing the role of hydrophobic BSs as mitochondrial toxins due to their MPT-inducing properties (Botla et al., 1995), support a key role of oxidative stress in the pathogenesis of BS-induced necrosis. However, our observation that DPPD did not completely counteract the diminution in hepatocellular viability and the release of cytosolic and plasma membrane enzymes despite its full prevention of TCDC-induced lipid peroxidation, clearly suggests that pathomechanisms other than oxidative stress are involved. Although a full prevention of TCDC-induced necrotic damage had been previously reported to occur in the isolated rat hepatocytes treated with the antioxidant, D--tocopheryl succinate (Sokol et al., 1993), the TCDC concentration employed (0.2 mM) was lower than those used in this study (0.25–1.5 mM). Therefore, other mechanisms operating at higher concentrations may have been overlooked in that study. A likely mechanism for this additional damage is the detergent action of lipophilic BSs on plasma membranes, a contention supported by their well-recognized tensioactive properties, derived from their amphoteric structure. Our results showing a progressive release into the incubation medium by increasing concentrations of TCDC of the plasma membrane-constitutive protein, AP, support this possibility. This enzyme binds to the plasma membrane via a glycan-phosphatidylinositol anchor, which interacts strongly with plasma membrane fatty acids (Low, 1987). At TCDC concentrations higher than its critical micellar concentration (4 μM), like that employed in this study (250–1500 μM), AP incorporates into TCDC micelles, which favors its stability and solubility in the extracellular aqueous medium (Coleman, 1987). Although we cannot rule out a contribution of AP from cells other than hepatocytes present in the cell preparation (e.g., cholangiocytes), this is likely to be negligible, as our isolation procedure yields hepatocyte preparations with high (>95%) purity (Berry and Friend, 1969). A cross talk exists between both oxidative stress and signal-transduction pathways (Kamata and Hirata, 1999). Since BSs induce ROS generation (Sokol et al., 1993, 2001), it is not surprising that BS-induced hepatocellular damage is influenced by the cellular signaling status. In line with this view, previous studies carried out in primary hepatocyte cultures showed that apoptosis induced by glycochenodeoxycholate is counteracted by PKC inhibitors (Jones et al., 1997), suggesting that PKC-dependent signaling pathways play a key role in hydrophobic BS-induced apoptosis. Taking into account the existence of common mechanisms between BS-induced apoptosis and necrosis (e.g., MPT formation, oxidative stress), it is possible to infer a similar protective effect of PKC inhibitors on TCDC-induced necrosis. Our results agree with this view. H7, a preferential PKC inhibitor (although it can inhibit in certain extent PKA as well) prevented partially the necrotoxic damage induced by TCDC (see Fig. 1). Furthermore, the specific PKA inhibitor, KT5720, was without effect, suggesting that H7 protective effect depended exclusively on its capability to block PKC activity. This was supported further using the specific PKC inhibitor, Che, which mimicked H7 protective effect. The mechanisms by which PKC inhibition protects against BS-induced necrosis can be multifactorial in nature. Our results showing here that PKC inhibitors prevented partially TCDC-induced ROS formation suggest that mitochondrial ROS production is facilitated somewhat by PKC activation. In line with this observation, lipid peroxidation induced to isolated rat hepatocytes by the oxidizing compound, tert-butyl hydroperoxide (tBOOH) (von Ruecker et al., 1989), or by the heavy metal, cooper (Mudassar et al., 1992), was prevented by the PKC inhibitor, H7, and exacerbated by PKC activators. Furthermore, H7 attenuates tBOOH-induced LDH leakage (Mudassar et al., 1992). The preventive effect of PKC inhibitors on TCDC-induced lipid peroxidation is, however, rather marginal, suggesting that other mechanisms must be involved. For example, PKC inhibition may favor TCDC efflux into the extracellular medium by stimulating the exocytic discharge of vesicles containing BSs, as PKC inhibits hepatocellular vesicular trafficking (Zegers and Hoekstra, 1998); this mechanism is thought to play a key role in BS overcharging conditions, like that occurring in our experimental setting (Erlinger, 1990). Furthermore, we have shown that PKC inhibitors blocked, and PKC activators stimulated, vesicle-mediated trafficking of vesicle-containing BS transporters towards the apical hepatocellular pole (Roma et al., 2000). In concordance with this, a study in isolated rat perfused liver showed that H7-induced PKC inhibition increased biliary excretion of TCDC at a concentration in the perfusate within the range used in this study (1 mM) (Nakazawa et al., 1996). Hydrophobic BSs induce elevation of [Ca2+]i by an inositol (1,4,5)triphosphate-independent mechanism (Combettes et al., 1988). Conceptually, Ca2+ elevations can activate different Ca2+-dependent proteases, phospholipases and endonucleases, with the consequent hepatocellular damage. Furthermore, Ca2+-elevations lead to activation of Ca2+-dependent PKC isoforms, which may be involved in TCDC-induced damage as well (see above). Therefore, we analyzed here whether the Ca2+-chelating agent, BAPTA/AM, has any beneficial effect against TCDC-induced hepatocellular necrosis. Despite this compound completely prevented TCDC-induced elevations in [Ca2+]i, the capability of TCDC to induce hepatocellular damage was not attenuated (see Fig. 5). This result, however, should not be conclusively interpreted to indicate that intracellular Ca2+ plays no role in BS-induced cytotoxicity. The predominance of other deleterious mechanisms not influenced by Ca2+ levels, e.g., the detergent properties of TCDC on cellular membranes, may have masked its contribution, particularly shortly after TCDC injury. Indeed, TCDC induced, in the perfused rat liver model, an early (4 min), transient increase in LDH release, followed by a subsequent time- and dose-dependent elevation in this parameter; only the first peak was significantly suppressed by pretreatment with the Ca2+- channel blocker, Ni2+ (Hasegawa et al., 2003). It is therefore possible that the protective effects of intracellular Ca2+ chelation have been overlooked in our model, which evaluate events occurring later in the necrotic process. Nevertheless, Ca2+ elevations are not a prerequisite for some forms of hepatocellular necrosis to occur, like that following ATP depletion due to metabolic inhibition (Nieminen et al., 1988). The second messenger, cAMP, an endogenous activator of the PKA-dependent signaling pathway, was shown to have protective, dose-dependent effects in several models of hepatotoxicity (Kasai et al., 1996). Although its hepatoprotective mechanism/s have not been completely elucidated, its stabilizing effect on intracellular membrane is probably involved (Ignarro et al., 1973). Furthermore, cAMP inhibits BS-induced apoptosis by blocking caspase activation and cytochrome c release (Webster et al., 2002). Paradoxically, cAMP exacerbated rather than prevented TCDC-induced necrosis in a dose-dependent fashion (see Fig. 3). Although how this signaling molecule aggravates TCDC-induced damage remains elusive, changes in TCDC hepatocellular bioavailability may be involved. cAMP was shown to stimulate the Na+-dependent BS uptake by the basolateral transporter, Na+-taurocholate cotransporting polypeptide (ntcp), in the isolated rat hepatocyte model. This was attributed to the capability of cAMP to hyperpolarize the plasma membrane via PKA-mediated Na+/K+-ATPase phosphorilation (Edmondson et al., 1985), and by stimulation of the PKA-dependent translocation of ntcp from an endosomal compartment to the sinusoidal membrane (Webster and Anwer, 1999). Based upon these previous observations, and our own results showing the PKA dependency of DB-cAMP-induced exacerbation of TCDC-induced necrosis (see Fig. 4), we proposed that putative protective effects of cAMP could have been masked by the simultaneous increase in TCDC intracellular concentration due to enhanced uptake. Our results are in apparent contradiction to a previous study showing a protective effect of cAMP-permeant analogues on TCDC-induced necrotoxicity in hepatocytes cultured overnight (Ohiwa et al., 1993). The explanation for these varied results may depend on the different experimental conditions employed. Whereas freshly isolated hepatocytes like those used in our study maintain an intact capability to take up BSs, this function decreases significantly under culture conditions (Follmann et al., 1990). Indeed, uptake of the model bile salt, taurocholate, decreases to approximately half of the value recorded in freshly isolated hepatocytes during a culture period compatible to that used by Ohiwa et al. (1993). Therefore, our approach more clearly reflects the situation of the hepatocytes in situ, at least in terms of bile salt uptake. In summary, the present findings clearly show that modulation of PKC- and PKA-dependent signaling pathways can modify the capability of hydrophobic BSs to induce hepatocellular necrotoxicity; whereas PKC inhibition attenuates BS-induced necrosis, PKA activation exacerbates this harmful effect. This seems to occur either or both by modulating differentially the intracellular availability of these endogenous, harmful compounds or by affecting the pathomechanisms involved in their deleterious effects. Although it is not possible at this point to establish whether these data have relevance to the situation in vivo, our results encourage future application of signaling molecules in the prevention/cure of hepatopathies occurring with elevated hepatocellular levels of endogenous BSs. ACKNOWLEDGMENTS This work was funded by CONICET, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT 05-08669), Beca ‘Ramón Carrillo-Arturo O?ativia 2003,’ from Ministerio de Salud de la Nación, and Fundación Antorchas (Subsidio de Emergencia para ‘Ex-Beneficiarios’ 14264/40). REFERENCES Baumgartner, U., Scholmerich, J., Leible, P., and Farthmann, E. H. (1992). Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts. Biochim. Biophys. Acta 1125, 142–149. Drew, R., and Priestly, B. G. (1979). Choleretic and cholestatic effects of infused bile salts in the rat. Experientia 35, 809–811. Zegers, M. M., and Hoekstra, D. (1998). Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells. Biochem. J. 336(Pt. 2), 257–269....查看详细 (35516字节)
☉ 11120228:Peroxisome Proliferator-Activated Receptors: Media
ABSTRACT Many phthalate ester plasticizers are classified as peroxisome proliferators (PP), a large group of industrial and pharmaceutical chemicals. Like PP, exposure to some phthalates increases hepatocyte peroxisome and cellular proliferation, as well as the incidence of hepatocellular adenomas in mice and rats. Most effects of PP are mediated by three nuclear receptors called peroxisome proliferator-activated receptors (PPAR,?,). An obligate role for PPAR in PP-induced events leading to liver cancer is well-established. Exposure of rats in utero or in the neonate to a subset of phthalate esters causes profound, sometimes irreversible malformations in the male reproductive tract. We review here the data that supports or discounts roles for PPARs in phthalate-induced testis toxicity including (1) toxic effects of phthalates on the male reproductive tract, (2) expression of PPARs in the testis, (3) activation of PPARs by phthalates, (4) role of PPAR in testis toxicity, (5) gene targets of phthalates involved in steroid biosynthesis and catabolism, and (6) interactions between PPARs and other nuclear receptors that play roles in testis development and homeostasis. Critical research needs are identified that will help determine the significance of PPARs in phthalate-induced effects in the rat male reproductive tract and the relevance of toxicity to humans. Key Words: phthalate ester; PPAR; testis; peroxisome proliferator; male reproductive tract. INTRODUCTION Most animal cells contain peroxisomes, subcellular organelles that perform diverse metabolic functions including H2O2-derived respiration, ?-oxidation of fatty acids, and cholesterol metabolism (Lock et al., 1989). Peroxisome proliferators (PP) are a large group of structurally dissimilar industrial and pharmaceutical chemicals that were identified as inducers of both the size and number of peroxisomes after in vivo exposure. PP include a large number of phthalate ester plasticizers which are, through their widespread use in various industries, widely distributed in the environment (Staples et al., 1997). Unlike many PP, phthalates are metabolized to active species by esterases found in the gut and other tissues. These esterases cleave one of the two side chains from the parent diester phthalate producing an active monoester phthalate. Rodent exposure to PP, including many phthalate esters leads to predictable adaptations in the liver consisting of hepatocellular hypertrophy and hyperplasia and transcriptional induction of fatty acid metabolizing enzymes (Lock et al., 1989). Chronic exposure to some PP causes an increased incidence of liver tumors in male and female mice and rats (Klaunig et al., 2003). Many studies also show that exposure to some phthalates results in profound, irreversible changes in the development of the male reproductive tract (Foster et al., 2001; Sharpe, 2001). Many of the adaptive consequences of PP exposure are mediated by a subset of nuclear receptor superfamily members called peroxisome proliferator-activated receptors (PPARs). The PPAR family includes three distinct subtypes, PPAR, PPAR? (also known as NUC1 or PPAR), and PPAR encoded by separate genes (Lemberger et al., 1996). PP activate PPARs leading to altered expression of genes with roles in metabolism, cell growth, and stress responses (Corton et al., 2000). Most, if not all responses to PP including some phthalates in the liver are dependant on PPAR (Klaunig et al., 2003). Given recent findings describing novel aspects of phthalate-PPAR interactions, we review here evidence supporting or discounting roles for PPARs in mediating the toxic effects of phthalates on the male reproductive tract. Effects of Phthalates on the Developing Male Reproductive Tract in Utero Exposure of male rats to some diester and monoester phthalates in utero results in profound changes in the testis and other androgen-dependent structures. The extent and severity of the changes depend in large part on the timing and duration of the exposure and how the compound is administered. Studies in which reproductive tract defects have been observed involve exposure of pregnant dams during the time of androgen-dependent sexual differentiation, estimated to begin in the rat gestation day 12 (GD 12). Studies in which di-n-butyl phthalate (DBP) or butylbenzyl phthalate (BBP) were dosed before GD 12 had no effect on reproductive tract development, as expected, but did produce other malformations (Ema et al., 2000; Ema and Miyawaki, 2002). Exposure regimens that resulted in the most consistent and robust effects involved administration of the phthalate by daily gavage throughout development of the reproductive tract until birth or shortly thereafter (Gray et al., 2000; Mylchreest et al., 1999). A number of studies showed common defects in the male rat reproductive tract after in utero exposure to DBP (Barlow and Foster, 2003; Barlow et al., 2004; Fisher et al., 2003; Mylchreest et al., 1999, 2000, 2002; Wine et al., 1997), di-(2-ethylhexyl) phthalate (DEHP) (Moore et al., 2001; Parks et al., 2000), and BBP (Nagao et al., 2000; Piersma et al., 2000; Tyl et al., 2004). These effects included those in the testis as well as other organs that depend on testosterone for proper development (Table 1). Some effects caused by DBP were found to be permanent including gross lesions in the testis, vasa deferentia, seminal vesicles, prostate, and penis, as well as decreases in anogenital distance and nipple retention (Barlow et al., 2004). In addition, DBP at 1% in the diet (Wine et al., 1997) and BBP at 750 mg/kg/day (Tyl et al., 2004) but not 500 mg/kg/day (Nagao et al., 2000) decreased total number of sperm and indices for mating, fertility, and pregnancy in F1 generation rats in multi-generation exposure studies. In contrast, di-isononyl phthalate (DINP) was a weak reproductive toxicant as in utero exposure led only to increased nipple retention (Gray et al., 2000). Dimethyl phthalate and diethyl phthalate had no adverse effects on the development of the male reproductive tract (Gray et al., 2000). Monobutyl phthalate (MBP), the principle metabolite of DBP, disrupted the descent of the fetal testis (Imajima et al., 2001; Shono and Suita, 2003). Reproductive effects of DBP are not exclusively found in rats. Exposure of male tadpoles of the frog species Rana rugosa increased the incidence of undifferentiated gonads developing into those having complete or partial ovarian structure (Ohtani et al., 2000). Male rabbits exposed in utero exhibited decreased weights of testis and accessory sex glands, decreases in ejaculated sperm, and increases in abnormal sperm six weeks after exposure (Higuchi et al., 2003). The effects of the active phthalates on the male rat reproductive tract share similarities with a condition in humans termed testicular dysgenesis syndrome (TDS). As proposed by Skakkebaek et al. (2001), TDS includes testicular germ cell cancers, poor semen quality, crytorchidism and hypospadias, all thought to arise from disruption of embryonic gonadal development. That TDS is increasing in developing countries and that environmental exposure to man-made male reproductive toxicants are responsible for TDS is unresolved, partly because of the lack of high quality data allowing comparison of studies (Sharpe, 2001). Only increases in testicular cancer over the last 50–90 years have been established (Bergstrom et al., 1996; Toppari et al., 1996). TDS, like exposure to phthalates is associated with abnormal function in both Leydig and Sertoli cells, resulting in downstream consequences in the testis and sex organs (Fig. 1). Exposure of rats to DBP in utero increased the proportion of immature Sertoli cells unable to support spermatogenesis and the number of multinucleated gonocytes, possibly through alterations in Sertoli cell-gonocyte interactions (Fisher et al., 2003). How DBP treatment interferes with these interactions and in particular, gonocyte cytokinesis is not understood. Phthalates which produce changes in male sex organs may be operating through an anti-androgen-like mechanism. The effects of DBP and DEHP overlapped with, but were distinct from androgen receptor antagonists including linuron (Gray et al., 1999) and flutamide (Mylchreest et al., 1999). Unlike other anti-androgens, some active phthalates target the Leydig cell testosterone biosynthetic machinery (discussed in detail below). Decreases in fetal testicular testosterone levels or serum testosterone in young postnatal male rats (PND 35) were observed after in utero exposure to DBP (Fisher et al., 2003; Mylchreest et al., 2002; Shultz et al., 2001; Wilson et al., 2004), DEHP (Akingbemi et al., 2001; Parks et al., 2000; Wilson et al., 2004), and BBP (Nagao et al., 2000; Wilson et al., 2004). When exposed only in utero, testosterone levels returned to normal after PND 35 (Akingbemi et al., 2001; Fisher et al., 2003). Male rabbits exposed in utero to DBP also exhibited decreases in testosterone six weeks after exposure that were reversible at 12 weeks (Higuchi et al., 2003). In contrast, testicular descent is at least partially testosterone-independent and may be disrupted through an alternate mechanism involving decreases in insulin-like hormone 3 (INSL3) expression observed in fetal testis after exposure to DBP, DEHP, and BBP (Wilson et al., 2004). Overall, the data is consistent with some phthalates indirectly down-regulating the activity of the androgen receptor through decreases in testosterone levels, resulting in delayed or absent development of androgen-dependent male reproductive organs. Alteration of Testis Structure and Function by Phthalates in Postnatal and Adult Rats Exposure of postnatal, preadolescent, and adult rats to some phthalates produces a different set of sequelae in testis compared to that of male rats exposed in utero. The age of the rat at the time of exposure has a dramatic effect on the testis as younger animals are more sensitive to the effects of phthalates (Gray and Gangolli, 1986). Compared to rats, mice are relatively resistant at any age. Some phthalates induce testicular toxicity in guinea pigs (Gray et al., 1982) and ferrets (Lake et al., 1976). Resistance to phthalate toxicity in Syrian hamsters is likely due to inefficient metabolic activation of diesters to monoesters by gut esterases compared to that in rats (Gray et al., 1982). Exposure to phthalates in responsive species leads to decreases in seminiferous tubule diameter and testis weight. At least in rats, this occurs through increased germ cell apoptosis and necrosis preceding the sloughing of germ cells into the tubular lumen (Gangolli, 1982). The primary cellular target of toxic phthalates is the Sertoli cell which exhibits biochemical and morphological changes after exposure. FSH-stimulated cAMP accumulation in primary Sertoli cells was inhibited by Mon-(2-ethyl)-hexlphthalate (Heindel and Chapin, 1989; Lloyd and Foster, 1988), MBP and monopentyl phthalate, but not monomethyl or monoethyl phthalate (Heindel and Powell, 1992). Decreases in proliferation of Sertoli cells were observed after DEHP exposure in vivo (Li et al., 2000) or after exposure to MEHP in vitro (Li et al., 1998; Li and Kim, 2003), and may occur through a block in FSH-stimulated Sertoli cell proliferation (Li et al., 1998). Two genes which play roles in Sertoli cell proliferation and differentiation, Müllerian inhibiting substance and GATA-4, were decreased by MEHP in Sertoli cells within cultured testis from GD 18 and PND 3 rats (Li and Kim, 2003). Sertoli cell numbers recover somewhat during the course of exposure, as the decreases observed at one week were no longer evident after two to three weeks exposure to DEHP (Dostal et al., 1988). In rats exposed to MEHP, collapse in the Sertoli cell intermediate filament vimentin structure (indicative of changes in cell morphology) was observed as early as 3 h, followed by increases in perinuclear condensation of vimentin at 6–12 h. These changes preceded or were coincident with increased apoptosis in germ cells (Richburg and Boekelheide, 1996). Immunostaining for flamingo1/Celsr2, a G protein coupled receptor family member that may link cell-cell adhesion to G-protein dependent signaling was redistributed within 2 h of MEHP exposure and disappeared by 12 h, indicating that this protein is a proximate target of MEHP (Richburg et al., 2002). Germ cells exhibit changes that may be secondary to effects on Sertoli cells. In the intact testis DEHP increased the generation of reactive oxygen species with concomitant decreases in the concentration of glutathione and ascorbic acid, and in primary cultures, oxidative stress was selectively induced in germ cells but not in Sertoli cells treated with MEHP (Kasahara et al., 2002). MEHP may specifically target the stages IX–XI of the mouse seminiferous cycle as apoptosis was increased in cells of these timed tubule segments treated in vitro (Suominen et al., 2003). Sertoli cells from mice treated with DEHP express the Fas ligand (FasL) while associated spermatocytes express Fas, the receptor for FasL, indicating that activation of the Fas system originating in Sertoli cells could lead to subsequent apoptosis in spermatocytes (Ichimura et al., 2003). Experiments in gld mice which carry a defective FasL gene revealed that germ cell apoptosis after MEHP exposure had both Fas-dependent (Richburg et al., 2000) and Fas-independent components (Giammona et al., 2002). Additionally, MEHP alters the expression and activity of death receptors 4,5,6 in the testis which may act in concert with the Fas-signaling pathway to initiate germ cell apoptosis (Giammona et al., 2002). Leydig cells in postnatal and adult rats are also targeted by some active phthalates. DEHP (Jones et al., 1993; Kim et al., 2003) and di-n-octyl phthalate (Jones et al., 1993) decreased testosterone serum levels. Primary Leydig cells treated with MEHP or mono-octyl phthalate also exhibited decreases in testosterone secretion (Jones et al., 1993) although the concentrations used to achieve these effects were very high (10–3 M). Human chorionic gonadotropin-induced testosterone secretion was suppressed by prior exposure of Leydig cells in culture to PP (Biegel et al., 1995; Cook et al., 1992; Liu et al., 1996). No phthalates were tested in these studies, however. Decreases in testosterone levels may explain the decreased weights of accessory sex organs (seminal vesicle and prostate) in male rats exposed to DEHP that were partially reversed by co-administration of testosterone or gonadotropins (Gray and Gangolli, 1986). Decreased testosterone levels may also play a role in the genesis of Leydig cell tumors (Klaunig et al., 2003) through increases in compensatory Leydig cell proliferation observed with DEHP (Akingbemi et al., 2004) and the PP, WY-14,643 (Biegel et al., 1992). Two studies have shown increases in serum testosterone levels in male rats after exposure to DEHP at 200 mg/kg from PND 21–48 (Akingbemi et al., 2001) or relatively low doses of DEHP (10 or 100 mg/kg) for PND 21–90 (Akingbemi et al., 2004), which the authors attribute to an observed increase in Leydig cell numbers. Thus, short-term exposure to phthalates may lead to decreases in testosterone while longer exposures may stimulate Leydig cell proliferation resulting in consequent increases in testosterone. A more comprehensive time course would be useful to confirm these events for DEHP and other phthalates. Expression of PPARs in the Testes PPAR subtypes are expressed in the adult rat testes (Table 2). PPAR was expressed in Leydig and Sertoli cells of the adult rat (Braissant et al., 1996; Schultz et al., 1999). Conflicting reports exist as to the expression of PPAR in spermatocytes, with one report detecting expression (Schultz et al., 1999) and the other negative for expression (Braissant et al., 1996). PPAR? is expressed in Leydig and Sertoli cells but not in spermatocytes (Braissant et al., 1996). PPAR, in contrast, is either weakly expressed or not expressed at all in these cell types (Braissant et al., 1996). The spectrum of PPAR subtype expression in rat Leydig cells is similar to that in a Leydig mouse cell line (MA-10); PPAR and PPAR?, but not PPAR were expressed (Gazouli et al., 2002). Although there are no reports, that we know of, that examine PPAR target gene expression specifically in Sertoli cells or spermatocytes, there were increased levels of two PPAR gene targets involved in peroxisomal ?-oxidation (acyl-CoA oxidase, multifunctional protein-1) in Leydig cells after adult rats were fed the PP ciprofibrate in the diet for two weeks (Nemali et al., 1988) indicating that PPAR expressed in Leydig cells is responsive to PP. In contrast, neither WY or the PP ammonium perfluorooctanoate increased peroxisomal ?-oxidation in Leydig cells from chronically treated rats (Biegel et al., 2001). The PPAR gene exhibited stage-specific expression during the spermatocyte differentiation cycle, peaking during stages II–VI and having the lowest expression at stages VII–VIII and IX–XII as assessed in isolated rat seminiferous tubules. Strong PPAR expression was observed in Sertoli cell nuclei during stages XIII–I (Schultz et al., 1999), a time when cells are most sensitive to FSH (Parvinen, 1982). Consistent with this, PPAR expression was increased in cultured seminiferous tubules after FSH treatment during all stages of the cycle (Schultz et al., 1999). These results indicate that PPAR expression is controlled in part by FSH, and that PPAR carries out a specific functional role during different stages of the differentiation cycle. Only expression of PPAR has been examined in the early postnatal testis (Schultz et al., 1999). Expression of PPAR was high in rat seminiferous tubules at PND 1 followed by a steady decline until PND 30 and an increase at PND 60. Information about expression of PPAR subtypes in the rat testis in utero is lacking but PPAR-null (Lee et al., 1995) and PPAR?-null (Peters et al., 2000) mice remain viable and fertile indicating that these receptors are not essential for mouse testicular development and fertility. Compensatory mechanisms have not been ruled out (e.g., increased expression of another PPAR subtype) that would allow proper testicular development. PPAR was expressed in human Leydig cells and spermatocytes, but not in Sertoli cells (Schultz et al., 1999). PPAR and PPAR? were expressed in human testis homogenates (Hase et al., 2002). PPAR may also be expressed although conflicting data exists (Elbrecht et al., 1996; Hase et al., 2002). Overall, these results indicate that PPAR subtypes are expressed in some types of adult cells in both rat and human testis. Further work is needed to determine if PPAR subtypes are expressed at both the transcript and protein levels in the testis throughout development from humans as well as a range of species susceptible to phthalate-induced testicular toxicity. Activation of PPARs by Phthalate Esters Despite their structural diversity compared to ligands that activate other nuclear receptors, PP do have similar structural requirements for activating PPARs in vitro and for initiating biological effects in animals. Most PP are amphipathic molecules containing a hydrophobic backbone (aliphatic or aromatic) linked to an acidic group or a moiety that can be metabolized to an acidic group. The acidic group is essential for ligand activity and typically consists of a carboxylic acid present in the parent compound. In the case of phthalates, esterases cleave a side chain from the diester resulting in a monoester containing a carboxylic acid. Some PP resemble endogenous lipid activators of PPARs such as long-chain saturated and unsaturated fatty acids (Xu et al., 1999). Many phthalates activate PPARs in in vitro transactivation assays. In these assays either full length PPAR (Hurst and Waxman, 2003; Lampen et al., 2003; Lapinskas et al., 2004; Maloney and Waxman, 1999) or hybrid transcription factors consisting of the PPAR ligand binding domain cloned to the glucocorticoid receptor (Lampen et al., 2003) or GAL4 (Bility et al., 2004) DNA binding domains were co-transfected into a mammalian cell line with a reporter gene (e.g., luciferase) under control of either PPAR binding sites called peroxisome proliferator responsive elements (PPREs) (Hurst and Waxman, 2003; Lapinskas et al., 2004; Maloney and Waxman, 1999), glucocorticoid responsive elements (Lampen et al., 2003) or the binding site for GAL4, UASG (Bility et al., 2004). After time to express the receptor, the cells are treated with the phthalate esters. The reporter gene activity is then normalized to the activity of an additional reporter gene used as a transfection control and results are usually reported as a fold-change relative to a solvent control. Mouse PPAR and PPAR are activated by a large number of phthalate monoesters (Table 3). Monoester phthalates with longer aliphatic or aromatic side chains tended to be more potent activators. Monoesters with short straight chains either do not activate at all or activate weakly. Monooctyl phthalate possessing a longer side chain activated all three subtypes (Bility et al., 2004), consistent with the observation that the longer the length of the fatty acid (up to 21 carbons), the better the fatty acid is at activating PPARs in vitro (Xu et al., 1999). Monooctyl and monoheptyl phthalates are unique in that they can activate PPARs in vitro but do not act as PP because the aliphatic side chains are rapidly cleaved in vivo (Albro and Moore, 1974). Steric restrictions appear to play a part in the ability of some monoesters to activate. Monoesters with the bulkiest side chains were less potent activators than MEHP or were completely inactive (e.g., mono-(1-methyl)-2-norbornyl phthalate, mono-(2,2-dimethyl-1-phenylpropyl) phthalate). The DEHP metabolite 2-ethylhexanoic acid weakly activated PPAR and compared to MEHP. In two studies the diesters DEHP and BBP were able to activate PPAR and PPAR, albeit weakly (Lampen et al., 2003; Lapinskas et al., 2004) despite the fact that the monoesters are considered to be the active toxic species. However, it is possible that the activation reflects a low level of esterase activity in the cell lines used. Mouse PPAR? was activated by only a small number of phthalates and in general, higher concentrations of those phthalates were required to activate to the same level as PPAR and PPAR. PPAR? was activated by monoester phthalates with longer or bulkier side chains such as MEHP, mono-isohexyl phthalate, mono-(1-methyl)-heptyl phthalate, mono-isodecyl phthalate and monobenzyl phthalate but not monoesters with shorter side chains (Bility et al., 2004; Lampen et al., 2003). Human PPARs were also activated by phthalates. All human PPAR subtypes were activated by MEHP, mono-(1-methyl)-heptyl phthalate and monobenzyl phthalate. Mono-sec-butyl phthalate, DL-mono-(1-methyl)-hexyl phthalate, mono-isoheptyl phthalate, mono-isodecyl phthalate, mono-isohexyl phthalate and mono-2-(methacryloyloxy)ethyl phthalate were more selective, activating only one or two of the subtypes. Importantly, the human PPARs required higher concentrations of the monoesters to be activated to the same levels as the corresponding mouse receptor (Bility et al., 2004; Hurst and Waxman, 2003; Maloney and Waxman, 1999). Activation of PPARs comes about through two distinct mechanisms. Most PP are thought to bind directly to PPAR leading to activation. However, there is evidence that some PP may activate PPARs indirectly, through increasing the pool of endogenous activators, e.g., by displacing fatty acids from carrier proteins (Luebker et al., 2002). To determine whether phthalate esters interact directly with PPARs, the scintillation proximity assay (Nichols et al., 1998) was used to assess the ability of phthalate esters to bind to human PPAR and PPAR (Lapinskas et al., 2004). Some monoester phthalates interacted with both receptors, including, monohexyl phthalate, monooctyl phthalate, monobenzyl phthalate, but not shorter chain phthalates (monopropyl phthalate, monopentyl phthalate). Despite the short length of the side chain, monoethyl phthalate binds with low affinity to both PPAR and PPAR. BBP and DBP also bind weakly to both subtypes and this could explain why these diesters were able to weakly activate in transactivation assays. The data supports phthalate-induced changes in gene expression through binding to and activation of PPAR by monoester phthalates. Activation of Other Nuclear Receptors by Phthalates Phthalate esters have been tested for their ability to interact with sex hormone receptors. In transactivation assays, a wide range of diesters and monoesters did not act as androgen receptor agonists or antagonists in HepG2 cells (Gaido, personal communication). In addition, DEHP and its active metabolite, MEHP, do not bind to the human androgen receptor (Parks et al., 2000). Some phthalates behave like estrogen receptor ligands in that the phthalates inhibited binding of estrogen to isolated estrogen receptors and induced estrogen-responsive endpoints in vitro (summarized in Moore, 2000). The relevance of these findings is questioned as monoesters, considered to be the proximate metabolites, were inactive in these in vitro assays and the phthalate diesters that do alter estrogen receptor activity only do so at concentrations that approach solubility of the compound, which could lead to nonspecific effects. Importantly, the phthalates were uniformly negative in a number of in vivo tests for estrogenicity including the uterotrophic assay (Moore, 2000). Taken together, these studies indicate that phthalates do not mediate their effects through estrogen or androgen receptors, but rather, support PPAR subtypes as primary nuclear receptor targets for phthalate esters. Correlation of PPAR Activation to Male Reproductive Tract Malformations The strength of the monoester as a PPAR activator partially correlates with the ability of the parent phthalate to act as a male reproductive toxicant (Table 3). Monomethyl and monoethyl phthalate were either inactive or only weakly active as PPAR activators. The parent phthalates dimethyl and diethyl phthalate were inactive as in utero reproductive toxicants (Gray et al., 2000). In contrast, MEHP was an activator of all three mouse PPAR subtypes and DEHP exposure led to a spectrum of male reproductive tract changes in rats (Gray et al., 2000; Moore et al., 2001; Parks et al., 2000). BBP is hydrolyzed to either monobenzyl phthalate or monobutyl phthalate with the ratio of hydrolysis products in rats being approximately 3:5, respectively (Mikuriya et al., 1988). Monobenzyl phthalate activated all PPAR subtypes and BBP was positive as a reproductive toxicant (Gray et al., 2000; Nagao et al., 2000; Tyl et al., 2004). There are two outliers in this comparison. DINP was a very weak reproductive toxicant while MINP was a moderate activator of mouse PPAR and PPAR (Bility et al., 2004). DBP is a reproductive toxicant in rats despite the fact that MBP was only weakly active in PPAR transactivation assays (Bility et al., 2004; Hurst and Waxman, 2003; Lapinskas et al., 2004). The weak activity of MBP in PPAR transactivation assays was surprising given that DBP, like DEHP and BBP, acts like a typical PP in the rat liver (Corton et al., 1996; Marsman, 1995) and effects in the mouse liver induced by DBP as well as DEHP and DINP are dependent on PPAR (Anderson et al., 1999; Lapinskas et al., 2004; Valles et al., 2003; Ward et al., 1998). Although treatment with MBP induced the same effects as DBP in the juvenile rat testis (Foster et al., 1981; Oishi and Hiraga, 1980) indicating that MBP is the proximate metabolite, no studies have been carried out in which MBP metabolites were comprehensively examined for toxic effects in the testis as well as abilty to activate PPARs. Candidate metabolites include MBP-glucuronide, a dominant MBP metabolite in adult rats (Foster et al., 1983; Tanaka et al., 1978) and omega and omega-1 products of MBP (Tanaka et al., 1978). It is also possible that a metabolite of DBP or DBP itself activates PPARs indirectly, i.e., by releasing a lipid activator of PPAR as discussed above a process which would not necessarily be detectable using transactivation assays. The fact that two out of six phthalates are outliers in this comparison weakens the hypothesis that PPARs are generally involved in phthalate-induced male reproductive tract defects. Regulation of Sex Hormone Synthesis and Catabolism Genes by Phthalate Esters As discussed above, BBP, DBP, and DEHP decrease testosterone levels in the fetus and in the young male rat. Consistent with this, genes involved in testosterone biosynthesis were uniformly down-regulated by DBP exposure in the fetal testis (Barlow et al., 2003; Shultz et al., 2001; Thompson et al., 2004) (Fig. 2A). The genes include Scavenger Receptor Class B, type 1 (SR-B1) and steroidogenic acute regulatory protein (StAR) (Barlow et al., 2003; Shultz et al., 2001). SR-B1 mediates the selective uptake of cholesterol esters from high-density lipoproteins, and StAR carries cholesterol from the outer to the inner mitochondrial membrane. The peripheral benzodiazepine receptor (PBR), which also carries cholesterol into the mitochondria was down-regulated by WY-14,643 and DEHP in adult mouse testes in a PPAR-dependent manner (Gazouli et al., 2002). In the fetal rat testis, PBR mRNA was up-regulated by DBP but by immunohistochemistry the protein was decreased in Leydig cells (Lehmann et al., 2004). Leydig cell mitochondria isolated from fetuses exposed to DBP in utero, exhibited decreased uptake of cholesterol supporting altered cholesterol handling for the decreases in testosterone synthesis (Thompson et al., 2004). Other genes involved in steroid biosynthesis that were down-regulated by DBP included P450 side chain cleavage enzyme (P450scc) thought to be the limiting enzymatic step in testosterone biosynthesis, 3?-hydroxysteroid dehydrogenase (3?-HSD) and CYP17 (Barlow et al., 2003; Shultz et al., 2001). Uniform decreases in the expression of these steroidogenic genes especially in a dose-dependent manner (Lehmann et al., 2004) is consistent with decreased levels of testicular testosterone. The nuclear receptor steroidogenic factor 1 (SF-1) plays a prominant role in the development and differentiation of steroidogenic tissues and controls the expression of steroidogenic enzymes and cholesterol transporters required for steroidogenesis (Val et al., 2003). INSL3 (Emmen et al., 2000) and receptors for follicle-stimulating and leutinizing hormones (Val et al., 2003) are also regulated by SF-1. Genes altered by DBP exposure including SR-B1, StAR, P450scc, CYP17 and 3?-HSD, Type II (Barlow et al., 2003; Shultz et al., 2001) require SF-1 for basal promoter activity and for cyclic AMP induction. The hypothesis that phthalate ester exposure leads to suppression of SF-1 activity was tested in the Leydig cell line MA-10 in which expression of a reporter gene linked to the promoters of SR-B1, StAR, and CYP17 genes was measured after MBP exposure (Thompson et al., 2004). However, MBP had no effect on reporter gene expression. Although this finding indicates that phthalates inhibit steroidogenic gene expression independently of effects on SF-1 activity additional monoesters should be tested in this system before any definitive conclusion can be made of SF-1 involvement. The mechanism by which these genes are down-regulated by DBP and whether other phthalates target the same genes requires further study. Phthalate esters along with other PP, also alter the expression of testosterone and estrogen metabolism genes (Figs. 2A and 2B). Among these, 5-reductase which converts testosterone to the more potent androgen, dihydrotestosterone was up-regulated in the prepubertal rat testis by DEHP (Kim et al., 2003). Multiple cytochrome P450 family members which hydroxylate testosterone were altered by PP exposure but in ways that do not clearly indicate a role in decreasing testosterone levels. CYP2C7 and CYP2C11 were down-regulated while CYP3A2 was up-regulated in the male rat liver (Corton et al., 1998; Fan et al., 2004-a,b). CYP2C11 and CYP3A were both up-regulated in the testes (Kim et al., 2003). WY-14,643 increased expression of fatty acyl-CoA enzyme A:testosterone acyl transferase in the liver (Xu et al., 2001). The down-regulation of CYP2C11 by a PP required PPAR; the site within the CYP2C11 promoter required for down-regulation by PP was identified and likely interacts with transcription factors other than PPAR implying that PPAR down-regulates CYP2C11 through an indirect mechanism (Ripp et al., 2003). Serum estrogen levels were increased in male rats after DEHP exposure (Akingbemi et al., 2004; Eagon et al., 1994) and after exposure to a number of PP (Biegel et al., 2001; Liu et al., 1996). Phthalates alter the expression of many estrogen metabolizing enzymes (Fig. 2B). Aromatase exhibits both up-and down-regulation by PP depending on the tissue and compound (Biegel et al., 2001; Kim et al., 2003; Lovekamp and Davis, 2001; Lovekamp-Swan et al., 2003; Mu et al., 2001; Rubin et al., 2000; Toda et al., 2003). Genes that decrease estrogen levels were increased after PP exposure including 17?-hydroxysteroid dehydrogenase, type IV (Corton et al., 1996) and fatty acyl-CoA enzyme A:estradiol acyl transferase (Xu et al., 2001). However, these changes may be offset by decreases in the estrogen hydroxylases CYP1B1 after exposure to DBP, DEHP, and BBP in the testis (Seo et al., 2004) or CYP2C11 (Corton et al., 1998) and estrogen sulfotransferase (Fan et al., 2004-b) after WY, GEM, or DBP exposure in rat livers. A handful of these genes involved in testosterone or estrogen metabolism are directly regulated by PPAR either in the liver or testis as determined by studies in PPAR-null mice. These include 17?-HSD, type IV (Corton et al., 1996) and the CYP3A2 ortholog Cyp3a11 (Fan et al., 2004-b). These results point to a role for PPAR in regulating expression of some sex steroid metabolism genes upon phthalate exposure in the adult animal. Although these studies help us appreciate the gene targets of phthalates involved in regulation of steroid metabolism, more work is needed to determine the mechanism by which phthalates alter the expression of these genes, whether these genes are also regulated in parallel at the protein level and the contribution of these collective changes to phthalate toxicity in the postnatal testis. PPAR and Phthalate-Induced Testicular Toxicity The role of PPAR in phthalate-induced developmental and testicular toxicity has been determined directly in wild-type and PPAR-null mice in two studies. In the first study (Peters et al., 1997), pregnant dams were dosed with DEHP at GD 8 and GD 9 (before development of the male reproductive tract) and malformations of the fetuses were evaluated on GD 10 and GD 18. DEHP exposure decreased crown-rump length and increased the incidence of open neural tubes (failure of the hind- and mid-brain to close) in both DEHP-treated wild-type and PPAR-null mice compared to controls, demonstrating that DEHP-induced malformations are PPAR-independent. However, this study cannot be used to determine the role for PPAR in phthalate-induced male reproductive tract defects from in utero exposure, because DEHP treatment did not occur during the critical window of development of the male reproductive tract and to our knowledge, the rat model of in utero male reproductive tract defects has not been successfully recapitulated in mice. In the second study (Ward et al., 1998), male wild-type and PPAR-null mice were fed a diet containing 12,000 ppm DEHP and lesions in the liver and testis were examined. In the liver all of the expected effects of DEHP were dependent on PPAR. After four and eight weeks of exposure, testis from wild-type mice exhibited mild or moderate toxic effects including focal tubular degenerative lesions, decreased spermatogenesis, and giant cells within the epididymis. In sharp contrast, the testis from PPAR-null mice at these time points were predominantly normal except for a few tubules that lacked normal indicators of spermatogenesis. PPAR-null mice at 24 weeks exhibited moderate testicular effects. However, all of the wild-type mice were sacrificed at 12–16 weeks due to toxicity when there was severe testicular atrophy making it impossible to directly compare the effects in the two strains at the 24 week time point. The authors concluded that DEHP effects in the testis were the result of both PPAR-dependent and -independent mechanisms. This study indicates that PPAR determines the timing and severity of testicular toxicity by DEHP. To better characterize the role of PPAR in phthalate-induced testicular toxicity, additional studies using other phthalates should be carried out in wild-type and PPAR-null mice. Given that PPAR? is expressed in the testis, PPAR?-null mice could also be used to determine any role played by this receptor in phthalate-induced testicular toxicity. Determining a role for PPAR will require construction of testis-specific nullizygous mice as the PPAR-null mutation is embryonically lethal (Barak et al., 1999). Cross-Talk between PPAR and Nuclear Receptor Signaling: Potential Impact on the Male Reproductive Tract Several studies have demonstrated ‘cross-talk’ between PPAR and other nuclear receptors. Cross-talk occurs when signaling pathways other than the primary pathway are activated or repressed through interactions with components of the primary pathway. PPAR-induced interference of other nuclear receptor pathways occurs through competition between PPARs and other receptors for binding to (1) the same DNA response element or (2) a common heterodimerization partner (Fig. 3). In the Supplementary Materials we examine cross-talk between PPARs and nuclear receptors for estrogen, thyroid hormone, and retinoic acid that play roles in the development or homeostasis of the testis. Many of these studies supporting interactions between PPAR and other nuclear receptors were either performed with PP other than phthalates in in vitro cell models allowing only conjecture as to how PPAR-phthalate interactions may disrupt male reproductive tract function in vivo. Further work is needed before these proposed mechanisms should be considered to play a role in phthalate-induced effects in the male reproductive tract. Conclusions In this review we have assessed the involvement of PPAR subtypes in phthalate-induced effects on the male reproductive tract. Much of the evidence supporting a role for PPARs is correlative. A high level of PPAR activation and testicular toxicity requires metabolic conversion of the diester to the monoester. PPAR subtypes are activated by structurally diverse phthalate monoesters. Phthalate diesters activate weakly or not at all. PPAR and PPAR are usually more sensitive to monoester activation than PPAR?, and mouse PPARs are more sensitive than human PPARs. PPARs, principally the and ? subtypes are expressed in neonatal or adult Sertoli and Leydig cells, the target cells of phthalate ester effects. The testicular toxicity of DEHP occurred later and with diminished severity in PPAR-null mice, indicating PPAR acts as a modifier gene regulating the timing and severity of toxicity. There is a correlation between the ability of four of six diesters to induce reproductive tract changes and the corresponding monoesters to activate PPAR subtypes. Phthalates alter the expression of genes encoding sex steroid metabolizing enzymes and enzymes involved in testosterone biosynthesis, some in a PPAR-dependent manner. PPARs interact with and down-regulate the activities of other nuclear receptors that play roles in the developing testis including receptors for estrogen, thyroid hormone, and retinoic acid, leading to plausible mechanisms by which PPAR activation by phthalates could alter testicular function in vivo. Other evidence exists to argue for little, if any participation of PPARs in phthalate-induced testicular effects. Although phthalates and PP can activate PPARs, only the phthalates are generally considered testicular toxicants. However, it is not unprecedented that structurally distinct classes of nuclear receptor ligands (e.g., ER ligands) have unique properties as agonists, partial agonists, or antagonists depending on receptor-ligand conformations and subsequent interactions with tissue-specific co-activators/co-repressors (McDonnell et al., 2002). With this in mind, it is interesting to note that many genes normally up-regulated by PP in the adult rat liver were down-regulated by DBP in the fetal testis and include those that are known targets of PPAR including fatty acid ?–oxidation genes (Shultz et al., 2001). Phthalate effects in the adult testis can occur rapidly (within 2 h), likely before any phenotypic effects of PPAR-mediated gene regulation would be observed. Two phthalates, DBP and DINP, do not neatly fit into a relationship of inducing male reproductive tract malformations while the corresponding monoester induces PPAR activation. DBP is a strong male reproductive toxicant but MBP only weakly activates PPARs; DINP is a weak toxicant but MINP is a moderately strong PPAR activator. The fact that phthalate-induced testicular toxicity occurs in species that do not respond to PP-induced liver effects has been used to invoke a PPAR-independent mechanism, but comprehensive information on PPAR expression in the testis of responsive vs. nonresponsive species is lacking. One way to rationalize these seemingly disparate findings is to invoke a mechanism that at least in the neonatal testis, includes both PPAR-dependent and -independent events leading to testicular toxicity after phthalate exposure. Future investigations should be designed to directly determine the role of PPARs as potential mediators of phthalate effects. Resolving a role for PPARs will be expedited by a comprehensive determination of PPAR expression in the developing testis and testing the effects of multiple phthalates in established mouse models nullizygous for each PPAR gene during male reproductive tract development. SUPPLEMENTARY DATA Supplementary data are available online at www.toxsci.oupjournals.org. ACKNOWLEDGMENTS The authors thank Drs. Paul Foster and Kevin Gaido for a critical review of the manuscript and the Phthalate Esters Panel of the American Chemistry Council for funding to produce this review. We apologize to colleagues whose papers were not cited due to space limitations. REFERENCES Akingbemi, B. T., Youker, R. T., Sottas, C. M., Ge, R., Katz, E., Klinefelter, G. R., Zirkin, B. R., and Hardy, M. P. (2001). Modulation of rat Leydig cell steroidogenic funtion by di(2-ethylhexyl)phthalate. Biol. Reprod. 65, 1252–1259. Biegel, L. B., Liu, R. C., Hurtt, M. E., and Cook, J. C. (1995). Effects of ammonium perfluorooctanoate on Leydig cell function: in vivo, in vitro, and ex vivo studies. Toxicol. Appl. Pharmacol. 134, 18–25. Biegel, L. B., Hurtt, M. E., Frame, S. R., O'Connor, J. C., and Cook, J. C. (2001) Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats. Toxicol. Sci. 60, 44–55. Nichols, J. S., Parks, D. J., Consler, T. G., and Blanchard, S. G. (1998). Development of a scintillation proximity assay for peroxisome proliferator-activated receptor gamma ligand binding domain. Anal. Biochem. 257, 112–119. Parvinen, M. (1982). Regulation of the seminiferous epithelium. Endocr. Rev. 3, 404–417. Peters, J. M., Lee, S. S., Li, W., Ward, J. M., Gavrilova, O., Everett, C., Retiman, M. L., Hudson, L. D., and Gonzalez, F. J. (2000). Growth, adipose, brain, and skin alterations resulting from tageted disruption of the mouse peroxisome proliferator-activated reeceptor beta (delta). Mol. Cell Biol. 20, 5119–5128. Valles, E. G., Laughter, A. R., Dunn, C. S., Cannelle, S., Swanson, C. L., Cattley, R. C., and Corton, J. C. (2003). Role of the peroxisome proliferator-activated receptor alpha in responses to diisononyl phthalate. Toxicology 191, 211–225....查看详细 (44614字节)
☉ 11120229:PBPK Model for Radioactive Iodide and Perchlorate
ABSTRACT Detection of perchlorate () in several drinking water sources across the U.S. has lead to public concern over health effects from chronic low-level exposures. Perchlorate inhibits thyroid iodide (I–) uptake at the sodium (Na+)-iodide (I–) symporter (NIS), thereby disrupting the initial stage of thyroid hormone synthesis. A physiologically based pharmacokinetic (PBPK) model was developed to describe the kinetics and distribution of both radioactive I– and cold in healthy adult humans and simulates the subsequent inhibition of thyroid uptake of radioactive I– by . The model successfully predicts the measured levels of serum and urinary from drinking water exposures, ranging from 0.007 to 12 mg , as well as the subsequent inhibition of thyroid 131I– uptake. Thyroid iodine, as well as total, free, and protein-bound radioactive I– in serum from various tracer studies, are also successfully simulated. This model's parameters, in conjunction with corresponding model parameters established for the male, gestational, and lactating rat, can be used to estimate parameters in a pregnant or lactating human, that have not been or cannot be easily measured to extrapolate dose metrics and correlate observed effects in perchlorate toxicity studies to other human life stages. For example, by applying the adult male rat:adult human ratios of model parameters to those parameters established for the gestational and lactating rat, we can derive a reasonable estimate of corresponding parameters for a gestating or lactating human female. Although thyroid hormones and their regulatory feedback are not incorporated in the model structure, the model's successful prediction of free and bound radioactive I– and perchlorate's interaction with free radioactive I– provide a basis for extending the structure to address the complex hypothalamic-pituitary-thyroid feedback system. In this paper, bound radioactive I– refers to I– incorporated into thyroid hormones or iodinated proteins, which may or may not be bound to plasma proteins. Key Words: pharmacokinetics; human; perchlorate; radioactive iodide; inhibition; thyroid. INTRODUCTION Advances in detection sensitivity of ion chromatography have revealed widespread contamination of ground and drinking water with perchlorate () across the United States (Motzer, 2001; Urbansky and Schock, 1999; U.S. Environmental Protection Agency, 2003). The bulk of this contamination is associated with the use of ammonium perchlorate (NH4ClO4) as an oxidizing agent in missile and rocket fuel. Ammonium perchlorate is also used in pyrotechnics (fireworks) and air bag inflators. The salt is readily soluble in water, and its dissociation product, the perchlorate anion (), is very stable under environmental conditions and very mobile in most media (Motzer, 2001). Perchlorate is not metabolized in the body (Anbar et al., 1959; Yu et al., 2002). However, because has a similar hydrated ionic radius and carries the same charge as iodide (I–), it is able to affect biological systems by inhibiting I– uptake into the thyroid by the sodium-iodide symporter (NIS) (Anbar et al., 1959; Brown-Grant and Pethes, 1959). While it is known that competes with I– for NIS binding sites, whether is actually translocated into thyrocytes is the subject of debate (Eskandari et al., 1997; Riedel et al., 2001). The weight of evidence at this time, however, suggests is a competitive inhibitor of thyroid I– uptake, replacing I– as a substrate of NIS and crossing the basolateral membrane (Clewell et al., 2004, Van Sande et al., 2003). Reduced I– uptake may lead to a disturbance in the first stage of normal thyroid hormone genesis. Hence, there is reasonable concern that chronic exposure to low levels of in drinking water could induce thyroid hormone deficiencies and subsequent thyroid disorders. NIS resides in the basolateral membrane of thyroid epithelial cells and simultaneously transports two Na+ and one I– ion from extracellular fluid (plasma) into the thyroid epithelial cell (Spitzweg et al., 2000). NIS is expressed in the thyroid and other tissues including the GI tract, skin, mammary tissue, and placenta. However, only in the thyroid is I– organified to form thyroid hormones and iodinated proteins (Ajjan et al., 1998; Spitzweg et al., 1998). Thyroid hormone homeostasis is maintained through a complex feedback mechanism. A drop in circulating serum thyroid hormone levels signals the pituitary to produce more thyroid stimulating hormone (TSH), which in turn stimulates NIS expression. In rats, a decrease in free thyroxine (fT4) and subsequent increase in TSH occur quickly (within one day) after acute exposures (Wyngaarden et al., 1952; Yu et al., 2002). In humans, thyroid hormone conservation is more efficient, although thyroid hormone status is very dependant on the iodine status and life stage under consideration. Little or no significant change in T4, fT4, and TSH was seen in adults after 2 weeks of controlled exposure to via drinking water at 0.007 to 0.05 mg/kg/day (Greer et al., 2002) and 0.14 mg/kg/day (Lawrence et al., 2000), despite significant levels of thyroid I– uptake inhibition. However, significant drops in fT4, intrathyroidal iodine, as well as an increase in serum thyroglobulin (Tg) have been reported in humans after high levels of exposure (900 mg/day) for 4 weeks (Brabant et al., 1992). The dynamics of thyroid hormone homeostasis is very different for a late gestation fetus or neonate. Empirical measurement of intrathyroidal stores of thyroid hormone in human fetuses and neonates have shown that the amount of thyroid hormone stored in the colloid is less than that required for a single day (van den Hove et al., 1999). The extent of chronic low-level exposure required to cause significant hormone deficiencies in humans is not yet known. Thus, the question facing risk assessors and regulatory agencies is: what concentrations of perchlorate could be considered problematic? It is known that I– deficiency during the fetal and neonatal period affects physical and mental development (Laurberg et al., 2000; Porterfield, 1994). In the adult, effects of I– deficiency are less dramatic. Clinical and subclinical hypothyroidism is often overlooked due to the vague symptoms associated with the condition. Yet, hypothyroidism occurs in over 10% of older women and is associated with cognitive impairment (Volpato et al., 2002). The development of hyperthyroidism, especially in multinodular goiters with autonomous nodules, is also associated with long-term mild to moderate I– deficiency in adults. Hyperthyroidism is also often overlooked in the elderly and, if left untreated, may lead to cardiac arrhythmias, impaired cardiovascular reserves, osteoporosis, and other abnormalities (Laurberg et al., 2000). Therefore, perchlorate-induced I– deficiency may represent a public health concern not only during perinatal development, but also in the elderly and subpopulations with already compromised thyroid function. In order to better understand the effect of occupational and environmental exposure to perchlorate on the hypothalamus-pituitary-thyroid (HPT) axis, a few studies have been performed that directly correlate hormone changes to quantitative perchlorate doses. In two occupational health studies at U.S. production facilities, workers were exposed to ammonium perchlorate (NH4ClO4) dust in the air. Perchlorate exposure levels were estimated from monitoring breathing zone air over full work shifts (Gibbs et al., 1998; Lamm et al., 1999). Gibbs and coauthors categorized exposure groups based on job tasks and air monitoring results. Controls, selected from an associated plant, were not exposure free, but had exposures estimated to be several orders of magnitude below any of the ‘exposed’ groups. The researchers found no elevation in pre- and post-shift serum TSH, and no drop in serum free thyroxine (fT4) among any of the exposed workers. In the study by Lamm et al. (1999), control or ‘comparison group’ subjects worked at the same facility but at unrelated processes and were believed to have very low exposure to perchlorate-contaminated particulates. Daily perchlorate doses were estimated from breathing zone air monitoring of respirable particulates over full work shifts and by urinary measurements. No significant differences in triiodothyronine (T3) and T4 were reported between the exposure and comparison groups. However, the mean pre- and post-shift urine perchlorate measurements from the comparison group averaged, respectively, 64% and 22% of those from the lowest ‘exposed’ group. Ecological epidemiological studies on neonatal screening data from California, Nevada, and Arizona health departments have resulted in conflicting data. Studies by Lamm and Doemland (1999) and Li et al. (2000) showed no increase in incidence of congenital hypothyroidism or decrease in neonatal T4 associated with in drinking water up to 15 μg/l. In contrast, the studies of Schwartz (2001) and Brechner et al. (2000) both found effects on newborn thyroid hormones from exposures at similar environmental levels (1 to 15 μg/l). A retrospective study of school-age children and newborns in three Chilean cities with drinking water concentrations of 8 h). Golstein et al. (1992) reported that this apical channel also appears sensitive to inhibition, suggesting a lower Km for (KmTLp) than for I–. A KmTLp value of 1.0 x 108 ng/l was derived from Chow and Woodbury's (1970) data, as described in Merrill et al. (2003). Maximum velocities, Vmax(s), for anion uptake vary between tissues and species (Bagchi and Fawcett, 1973; Wolff, 1998). Humans tend to have lower Vmax values than other species (Gluzman and Niepomniszcze, 1983; Wolff and Maurey, 1961) when expressed per gram of tissue. The Vmax(s) (ng/h/kg) for I– uptake in the thyroid and plasma were estimated by visually optimizing the clearance portion of the curves to respective time-course data of Degroot et al. (1971). This was accomplished by keeping all other parameters fixed, while the Vmax value was adjusted so that the model prediction adequately approximated the observed mean. It may be noted that Vmax values for the thyroid follicle and lumen differ by up to an order of magnitude from preliminary values, reported in Clewell et al. (2001). This was attributed to the availability of new data sets, which allowed improved parameterization. For tissues lacking time-course data, the Vmax(s) were estimated to yield kinetics similar to those described by the male rat model (Merrill et al., 2003). For example, for I– kinetics in the stomach and skin, VmaxGi and VmaxSki respectively, were visually optimized to resemble tissue:serum concentration ratios seen in the rat, while maintaining the fits to human serum data. Because data was only available in serum and urine, Vmax(s) for NIS-containing tissues were scaled from the I– Vmax(s), using the ratios between corresponding I– and Vmax(s) established in the male rat model. Diffusion, concurrent with active uptake in the stomach, thyroid, and skin, was described using permeability area cross products (PA) (l/h-kg) and effective partition coefficients (P). In general, PA values were visually fit to the uptake portion of the curves, prior to setting Vmax(s), with partition coefficients, and all other parameters were set to the values in Table 2 and held fixed. The early time-course data of I– in gastric aspirations from Hays and Solomon (1965) were used to estimate PAGJci, representing 131I– transfer from the gastric juice into the gastrointestinal plasma (l/h-kg). To simulate the removal of gastric aspirations, the amount of 131I– reabsorbed by the stomach wall had to be mathematically eliminated or set to zero. Once parameters were established using the aspiration session data, stomach reabsorption was reintroduced, and the permeability cross product for 131I– transfer between gastric blood and tissue (PAGci) was fit to the corresponding increase in 131I– in plasma, thyroid, and urine seen in the control session (where gastric juices were not aspirated). The permeability area cross product between the thyroid stroma and follicle, PATFci, was visually optimized to the uptake portion of the thyroid I– data. The first-order clearance rates describing the organification of I– shortly after it enters the thyroid follicle (Clhormci) and the secretion of organic I– into systemic circulation (Clsecrci) were visually optimized to the clearance portion of thyroid 131I– data, as well as the later time points of the plasma PBI data from Degroot et al. (1971). The first-order rate, describing the body's overall deiodination rate (Cldeiodci) was also estimated through visual optimization of the later PBI timepoints, while maintaining the model fit to total plasma iodine and keeping all other parameters fixed. Later plasma time points of PBI reflect the contribution of hormone secretion and deiodination rates due to sufficient lapse of time for radioactive I– incorporation into thyroid hormones and precursors. Therefore, earlier PBI time points were visually fit to establish parameters for binding of inorganic I– to plasma proteins (e.g., KmBi, VmaxBi). Similarly, reversible plasma binding of perchlorate was described using a first-order rate constant (Clunbp), which was visually optimized to available serum data. Urinary excretion rates for both anions (ClUi and ClUp) were visually fit to available cumulative urine data (Degroot, 1971; Greer et al., 2002; Hays and Solomon, 1965). Because cumulative urinary perchlorate data was available in the Greer et al. study, ClUp was visually fit to each individual's data, and the average value was then used for the model parameter. Allometric scaling and rate equations. Differential equations used to simulate radioactive I– and transport were written and solved in ACSLTM (Advanced Continuous Simulation Language) (AEgis Technologies, Austin, TX). To account for body-weight-dependent variables and species extrapolations, allometric scaling was applied to Vmax(s), PA(s), Cl(s), tissue volumes (V), and blood flows (Q). The variety of dosing regiments and routes were simulated using various pulse function codes in ACSL. Rate equations describing I– transport in ng/h in the thyroid stroma, follicle and lumen (colloid) (RATSi, RATFi, and RATLi, respectively), as well as the rate of change in bound thyroid iodine (RAbndi) are provided below. These equations demonstrate diffusion-limited uptake, using P(s) and PA(s), and saturable uptake and competitive inhibition using M-M parameters. Equations used in the other compartments are expressed similarly. Subscripts i and p identify the anion as either I– or , QT is thyroid blood flow (l/h), CAi is the arterial blood concentration (ng/l), CVTSi,p is the thyroid stroma concentration (ng/l), CTFi,p is the follicular concentration (ng/l) of I– or , and CTbndi is the concentration of incorporated I– in the entire thyroid. PTFi, PTLi, PATFi, and PATLi are the partition coefficients and permeability cross products describing passive diffusion of I– across the basal (follicle:stroma) and apical (lumen:follicle) membranes. Michaelis-Menten equations are used to describe the rates of active uptake of I– into the follicle by NIS and into the lumen by the apical I– channel (RupTFi and RupTLi, respectively), including inhibition by . VmaxTFi, VmaxTLi, KmTFi,p and KmTLi,p are the maximum velocities (ng/h/kg) and affinity constants (ng/l) for transport of I– or into the follicle and lumen. Clhormi and Clsecri are first-order clearance values (h–1) for the organification of I– into thyroid hormones and the secretion of organified I– into systemic circulation. Transport of through the thyroid is calculated similarly, except there are no terms for organification of (Clhormi and Clsecri). In addition, inhibition of uptake by I– is not included. As described earlier, due to the lower affinity of I– (10-fold higher Km) than that of , I– does not significantly inhibit sequestration in NIS-containing tissues. Example equations of other compartments are shown elsewhere (Merrill et al., 2003). Sensitivity analysis of parameters. To assess the relative impact of each parameter on model predictions, a sensitivity analysis was performed. After finalizing all model parameters, the model was run at a drinking water dose below NIS saturation (0.1 mg/kg/day) for 240 h (to ensure equilibrium was reached) to determine the average serum concentration [area under the curve (AUC)]. The model was then repeatedly rerun, using a 1% increase in each parameter to determine the resulting change in predicted serum concentration AUC, and sensitivity coefficients for each parameter were then calculated using the equation below. Where A equals serum AUC with 1% increased parameter value, B equals serum AUC using original parameter value, C equals parameter value increased 1% from original value, and D equals the original parameter value. RESULTS TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES Model Parameterization Iodide kinetics. Effective partition coefficients (P) and affinity constants for the active transport mechanisms (Km), listed in Table 2, were kept consistent with those used in the male rat model. Parameterization of other I– parameters was obtained using available human time-course data as described in the Methods section. Figures 2A through 2D show the model simulations of the early distribution of 131I– in plasma, thyroid, gastric juice, and urine during both the control and gastric aspiration session by Hays and Solomon (1965). Degroot's plasma, urine, and thyroid time-course measurements extended nearly 11 days post-dosing, allowing greater calibration of parameters affecting both uptake and clearances in the thyroid and plasma. Model simulations of plasma inorganic I–, PBI, and total plasma iodine are presented in Figures 3A–3C. The model underpredicted urinary I– measured by Degroot et al. (1971) (Fig. 3D). However, considerable variations in I– excreted by humans exist, as it varies with thyroid function and dietary I– intake (NRC, 1996). Since increasing urinary clearance (ClUi) would result in underprediction of plasma iodine, the value for ClUi was not changed to fit this data set. View larger version (25K): [in this window] [in a new window] FIG. 2. 131I– in serum (A), thyroid (B), gastric juice (C), and urine (D) of nine healthy males after an iv dose of 10 μCi 131I– (3.44 ng/kg). Model simulations (lines) and actual values (stars) are presented for both the control and aspiration sessions (Hays and Solomon, 1965). View larger version (20K): [in this window] [in a new window] FIG. 3. Model simulations (lines) and measured 131I– in plasma (A), protein-bound iodine (B), thyroid (C), and urine (D) of euthyroid adult after a tracer dose of 131I– (Degroot et al., 1971). The subjects from the Hays and Solomon (1965) and Degroot (1971) studies had fasted at least 12 h prior to 131I– administration. The VmaxTFi values, visually optimized from these data, averaged 2.5 x 105 ng/h-kg. Using this value, however, individual 8 and 24 h radioactive I– thyroid uptake (RAIU's) from Greer and coworkers' (2002) study were over-predicted. Since no dietary restrictions were given to the subjects in the Greer study, differences in dietary I– among subjects in the three studies may account for the differences in uptake. Individually fit VmaxTFi values using the Greer (2002) data resulted in a lower average, 1.5 x 105 ng/h-kg, reflecting the high iodine intake (see Table 3 and Figure 4). View this table: [in this window] [in a new window] TABLE 3 Individually Fit ClUcp(s) and VmaxcTFi(s) View larger version (13K): [in this window] [in a new window] FIG. 4. Model simulations (lines) and observed mean ± SD (bars) 8 and 24 h RAIU measurements from 31 healthy subjects (Greer et al., 2002). Kinetic data for I– in human skin were not available. However, as mentioned earlier, NIS has been identified in human skin (Slominski et al., 2002). Assumptions that anion uptake and clearance in human skin were comparable to that in rat skin [e.g., slow uptake into and diffusion out of the tissue (Merrill et al., 2003)] lead to improved fits of serum 131I– concentrations. Perchlorate kinetics. Because I– parameterization, which was based on actual human time-course data, resulted in many values similar to those developed for the male rat, it is reasonable to expect that the proportional difference between the rat's I– and parameters would also apply to the human's parameters. Using these proportionally scaled parameters for the thyroid, stomach, and skin, cumulative in urine was visually fit for each individual in the 0.5, 0.1, and 0.02 mg/kg/day dose groups from Greer et al., (2002), resulting in an average urinary clearance constant for (ClUcp) of 0.13 ± 0.05 l/h-kg (Table 3) (Fig. 5). View larger version (17K): [in this window] [in a new window] FIG. 5. Model simulations (lines) and mean and standard deviations of the observed cumulative ClO4– in urine (cross bars) in male subjects dosed 0.02, 0.1, and 0.5 mg/kg-day (Greer et al., 2002). As mentioned earlier, reversible binding of to nonspecific human plasma proteins has been qualitatively demonstrated in other studies (Carr, 1952). Additionally, the model indicated the existence of plasma binding, as without it the model underestimated serum at 0.1 mg/kg/day, while simulations at 0.5 mg/kg/day produced adequate fits. Hence, at the lower level, plasma binding represents a larger proportion of the overall amount of serum . Serum levels from the 0.02 mg/kg/day dose group were below the detection limit and thus could not be compared to model predictions. The plasma protein affinity for (KmBp) was assumed to be similar to that used in the male rat model, given the same proteins (albumin and prealbumin) appear to be responsible (Carr, 1952; Merrill et al., 2003) (Fig. 6). View larger version (35K): [in this window] [in a new window] FIG. 6. Model simulations (lines) and mean ± SD (bars) of serum in volunteers who received 0.02, 0.1, and 0.5 mg/kg-day via drinking water, four times per day for 14 days. n = 8/dose group (Greer et al., 2002). Note: serum perchlorate levels at 0.02 mg/kg/day were below detection limits. Model Validation The ability of the model to predict human data from other experiments, analyzed at different labs, was tested using available data from other independent studies. Using the parameters in Table 2, the model slightly overpredicted plasma-bound 131I– fractions from several euthyroid patients; however, the prediction was remarkably close (within a factor of 0.75 of the mean values reported) (Fig. 7). Predictions of cumulative in urine after oral doses of approximately 9.07 mg/kg (Durand, 1938), 9.56 mg/kg (Kamm and Drescher, 1973), and 20 mg/kg (Eichler, 1929) were excellent (Figs. 8–10). View larger version (14K): [in this window] [in a new window] FIG. 7. Model simulations (lines) and mean ± SD (bars) of plasma bound 131I– fractions of 25 euthyroid patients after an oral dose of 100 μCi 131I– (Scott and Reilly, 1954). View larger version (11K): [in this window] [in a new window] FIG. 8. Model simulation (line) and observed (asterisks) cumulative in urine from a healthy male after an oral dose of 9.56 mg (Kamm and Drescher, 1973). View larger version (11K): [in this window] [in a new window] FIG. 9. Model simulation (line) and observed (asterisks) cumulative in urine from a healthy male after an oral dose of approximately 20 mg (Eichler, 1929). View larger version (11K): [in this window] [in a new window] FIG. 10. Model simulation (line) and observed (asterisks) cumulative amount of in urine from a healthy male after an oral dose of approximately 9.07 mg/kg of (Durand, 1938). The model was also able to predict serum concentrations at 12 mg/kg-day from the unpublished study performed by Dr. Georg Brabant at the Medizinische Hochschule, Hanover, Germany, described previously in the Methods section (Fig. 11). With the exception of the significantly higher dose, this study was very similar to Greer et al. (2002). Subjects received 12 mg/kg-day in drinking water at or near meal times. The wide range in the observed serum measurements was believed to reflect variability in the dosing regime, as the experimental protocol was less rigid than that used in Greer et al. (2002). Table 4 lists the predicted and measured average serum and percent inhibitions on exposure day 14 from each dose group in Greer et al. (2002). View larger version (34K): [in this window] [in a new window] FIG. 11. Model simulations (lines) and mean ± SD (bars) of serum concentrations in 5 males during exposure to 12 mg/kg-day of in drinking water. Subjects ingested the drinking water solution three times/day for 14 days (unpublished data, Brabant and Leitolf). View this table: [in this window] [in a new window] TABLE 4 Average Serum Perchlorate and Percent Inhibition Across Dose Groups The competition of I– and for NIS binding sites was also successfully predicted by the model. Predictions of thyroid radioactive I– uptake inhibition after 14 days of exposure to in drinking water at 0.5, 0.1, 0.02, and 0.007 mg/kg-day, also measured by Greer et al. (2002) in the same subjects, were well within the mean and standard deviations of the observed data (Figs. 12A–12D). Using this same set of I– and parameters, again the model accurately predicted discharge tests performed by Gray et al. (1972) on euthyroid subjects (Fig. 13). View larger version (23K): [in this window] [in a new window] FIG. 12. Model simulations and observed (mean ± SD) of 8 and 24 h RAIU measurements before exposure (upper lines and solid squares) and on day 14 of perchlorate exposure at (A) 0.5 mg/kg-day, (B) 0.1 mg/kg-day, (C) 0.02 mg/kg-day, and (D) 0.007 mg/kg-day (lower lines and open circles) (Greer et al., 2002). View larger version (11K): [in this window] [in a new window] FIG. 13. Model simulation (line) and observed perchlorate discharge tests performed on euthyroid subjects (Gray et al., 1972). Lastly, the model's utility for predicting I– and kinetics under altered thyroid function was evaluated. Hyperthyroidism, as manifest in Grave's disease, is marked by increased thyroid I– uptake, as well as elevated T4, T3, PBI, and TSH (Fenzi et al., 1985). Gluzman and Niepomniszcze (1983) reported elevated Vmax(s) at the NIS in thyroid specimens from subjects with Grave's disease. As expected, by increasing VmaxcTFi to 5.0 x 106 ng/l-kg, the model successfully simulates the thyroidal 131I– uptake in a male with Grave's disease (upper line in Fig. 14) (Stanbury and Wyngaarden, 1952). This value exceeds those estimated for normal subjects with varying levels of dietary I– by more than a factor of 10. Using this set of thyroid parameters, including the elevated VmaxcTFi, the model underpredicts the degree of inhibition after 100 mg K, but is within a factor of 2 from the data (bottom line Fig. 14). View larger version (14K): [in this window] [in a new window] FIG. 14. Model simulation (line) and observed (asterisks) amount of 131I– uptake in the thyroid of a male with Graves' disease after iv dose of 10 μCi 131I– before and after 100 mg K (Stanbury and Wyngaarden, 1952). Sensitivity Analysis Sensitivity analysis on serum AUC levels at 0.1 mg/kg/day indicated that the urinary clearance (ClUp) had the greatest influence. The sensitivity coefficient for ClUp was –0.84. Serum levels at this dose level are also sensitive to plasma binding parameters for maximum capacity (VmaxBp), the first-order dissociation rate (Clunbp), and plasma binding affinity constant (KmBp), with sensitivity coefficients of 0.26, –0.25, and –0.17, respectively. A comparison of model sensitivity to these parameters between the concurrently developed male rat model and this human model are shown in Figure 15. Sensitivity coefficients of all other parameters were below an absolute value of 0.1. View larger version (13K): [in this window] [in a new window] FIG. 15. Calculated sensitivity coefficients for model parameters with greatest impact on serum AUC at a drinking water dose of 0.1 mg/kg/day. Comparison shown for the male rat and human models. DISCUSSION TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES The validity of the model structure and its parameters are demonstrated by its ability to predict and I– in serum and urine data, as well thyroid I– inhibition data from various studies, which involve various dose levels and routes, while using a single set of parameters. The model adequately simulates serum and cumulative urine levels after drinking water exposure to spanning almost four orders of magnitude (0.02 to 12.0 mg/kg-day). Although serum levels were not available at 0.02 mg/kg-day, the model was able to simulate the cumulative urine from that dose group (Fig. 9). Comparison with parameters of the rat model indicates that humans have a considerably lower plasma binding capacity (VmaxcBp) for (approximately 20 times lower). Although binding of to plasma proteins has been directly measured in human serum, it is not surprising that it would occur to a lesser extent than in the rat. Carr (1952) tested the ability of to bind to various proteins in human blood, including albumin, pre-albumin and thyroxine binding globulin (TGB). They found that was able to bind to albumin and prealbumin, but not TBG. Thus, it would be expected that the binding capacity would be greater in the rat, whose primary binding protein is albumin, than the human, whose primary binding protein is TBG. Data available for calibrating and validating serum I– were limited to tracer dose levels. However, the kinetic behavior of I– is expected to be linear over a wide range of doses. Although the mechanism of transfer into the tissues with NIS is saturable, the value of Km (4.0 x 106 ng/l) indicates that very high doses would be required to saturate this mechanism. Additionally, similar parameter values and identical model structures in the corresponding rat models of various life stages have yielded validated serum predictions at dose levels ranging three or more orders of magnitude for both anions. Aspects of the model, which were supported in the literature or laboratory studies but could not be directly observed in humans, were incorporated if necessary to improve the fit of the model to available data. For example, active uptake of I– and into human skin and the relatively slow diffusion of both anions from skin back into systemic circulation were incorporated in spite of a lack of human time-course data. The literature supports this behavior, as NIS has been identified in human skin (Slominski et al., 2002), and slow diffusion has been noted with similar anions, such as pertechnetate. Hays and Green (1973) performed dialysis studies on intact human tissues with pertechnetate. They found skin had a relatively slow uptake of pertechnetate peaking at 18 h and, in fact, more retention after leaching dialysis than seen in brain, muscle, and serum. It is possible that the reason elevated I– in human skin has not been reported in clinical radioactive I– scans is the difficulty in differentiating skin radioactivity from background radioactivity. The large volume of the skin allows radioactive I– to be diffused over a large surface. However, this same property allows the skin to be an important pool for the storage and slow turnover of I–. Simulations with this model demonstrated that inclusion of active uptake in human skin was required to simulate serum data. Sensitivity analysis on the corresponding male rat model indicated that serum levels were highly sensitive to parameters of the skin and plasma binding and urinary excretion (Merrill et al., 2003). Kinetic data for establishing parameters for the gastric compartment were limited to the early I– data (3 h post-dosing) by Hays and Solomon (1965). Their gastric juice 131I– data indicated rapid transport of I– into the gastric mucosa (Fig. 2C). It is expected that uptake in the stomach would behave likewise, due to the similarity of the anions in size and charge. The time-course behavior of radioactive iodine in stomach contents of any species is complicated by the fact that it reflects more than sequestration of radioactive I– by NIS. Its appearance also reflects the accumulation of salivary radioactive I– that is swallowed involuntarily throughout the study. Several studies that examined sequestration of these anions in digestive juices have all shown high variability in the concentrations measured over time (Hays and Solomon, 1965; Honour et al., 1952). There is a tendency for the gastric juice:plasma ratio to be low when the rate of secretion of gastric juice and saliva is high (Honour et al., 1952). This is because the increase in secretions does not correspond with upregulation of NIS; therefore, the gastric juice concentration becomes diluted. Fluctuations in the secretion rate are probably the most important factor in determining the pattern of the concentration ratios in individuals. Therefore, variability in stomach or GI tract parameters between models is expected. However, the early rise in the gastric juice:plasma ratio mentioned earlier is a constant feature across these data sets, whether or not an attempt was made to eliminate contamination of gastric juices by dietary contents or saliva. Animal data that show both the anion uptake and clearance in the stomach (Yu et al., 2002), unlike the data in Figure 2C which only show uptake, indicate that the clearance portion is less rapid. This model, and the series of different life-stage rat models (Clewell et al., 2003a,b; Merrill et al., 2003;) consistently predict this same trend. Average urinary clearance values were found to be 0.11 l/h-kg for I– and 0.13 l/h-kg for . However, these values are not expected to be successfully applied to every euthyroid individual studied, though the use of these average values should provide a reasonable prediction of the euthyroid population. Individual differences in urinary I– are expected with variation in thyroid function and protein-bound I– in plasma. Iodide is ultimately removed or eliminated by two competing mechanisms, thyroidal uptake and urinary excretion. Thus, a higher amount of excreted I– in urine is indicative of reduced thyroid uptake. Historically, this relationship has been used to estimate thyroid function. A cumulative 24-h urinary clearance of less than 30% of a tracer dose is indicative of hyperthyroidism, whereas clearances exceeding 40% are often associated with normal or decreased thyroid function. However, a high degree of variability exists between human subjects. Such a significant degree of overlap exists in thyroid test results for normal, hyperthyroid, and hypothyroid patients, that it is often necessary to run several different additional screens in order to identify subclinical conditions (NRC, 1996). In addition to the expected variability in thyroid uptake parameters (VmaxcTFi values ranging from 5.0 x 104 to 5.0 x 105 ng/h-kg) between individuals, variability across data sets was also noted. However, the difference seen in the average VmaxcTFi obtained from the Greer et al. (2002) subjects (1.5 x 105 ng/h-kg) and those from Hays and Solomon (1965) (2.5 x 105 ng/h-kg) is easily explained by the difference in experimental conditions between the two studies. Hays and Solomon's subjects fasted 12 h prior to the administration of the 131I–, whereas Greer and coauthors' subjects had no dietary restrictions prior to 125I– administration. As a result, intrathyroidal I– levels would have been lower in the fasted individuals, and as anticipated, the average VmaxcTFi from Hays and Solomon (1965) would be increased. Dietary iodine and endogenous inorganic I– levels are clearly important in modeling I– and kinetics, because excessive I– levels cause the ion to inhibit its own uptake (Wolff and Chaikoff, 1948). The ability of the model to describe the bound and free I– fractions in the thyroid and serum provides the basis for subsequent modeling of hormone synthesis and regulation in humans. Measurements of tracer radioactive I– can be fitted to predict transfer rates. However, the use of these acute parameters is limited when attempting to describe long-term thyroid kinetics, unless the existing endogenous I– and the relationships between the regulating hormones are taken into consideration. Ultimately, such factors as preexisting thyroid conditions and regional dietary iodine might be addressed in a more comprehensive hormone feedback model. In its present state, our model is useful in predicting perchlorate's effect on thyroid I– uptake in what is considered the normal population: euthyroid individuals with adequate dietary I–. That the model is capable of predicting I– uptake in hyperthyroid subjects by increasing the VmaxTFi supports the usefulness of the current model structure. TSH increases the total amount of NIS in a membrane, thereby increasing VmaxTFi. Gluzman and Niepomniszcze (1983) reported elevated Vmax(s) in thyroid specimens from subjects with Graves' disease, toxic adenoma, and dishormonogenetic goiter. In future versions of the model, the increase of TSH and subsequent effect on this parameter can be described mathematically in order to predict the dose- and time-dependent response of the thyroid activity to various disease states. In specimens from nontoxic nodular goiter, Hashimoto's thyroid, or extranodular tissue from toxic adenoma, Vmax(s) were decreased. However, in all subjects there was little variation in the KmTi. Therefore, one would not expect the underprediction of thyroid inhibition in the subject with Graves' disease to be due to disease-induced lowering of Km, but rather the increased inhibition is mostly likely due to simple interindividual differences. Sensitivity analyses performed on the model for the rat indicates that model-predicted values of inhibition are highly sensitive to even small changes in Km for . Thus, it is quite possible that changing Km within the range of normal values would account for this apparent discrepancy in the model fit. The PBPK models developed for perchlorate-induced inhibition have been useful to the ongoing risk assessment of , and helped integrate the data from diverse data sets to evaluate the dose response of adverse effects from low levels of exposure (U.S. Environmental Protection Agency, 2003). The resulting parameters may be used in conjunction with those established for the male (Merrill et al., 2003) and perinatal rat models (Clewell et al., 2003a,b) to extrapolate to human gestational and lactational models (Clewell et al., 2001). In order to further assess model performance and to facilitate the use of these models in risk assessment, a more comprehensive statistical evaluation of model parameters may prove additionally useful. Sensitivity analysis provided insight into the relative importance of model parameters with respect to specific measures of dose. Comparing the highest sensitivity coefficients between the male rat and human models indicated that, at low doses, human serum levels are most sensitive to urinary clearance, whereas the rat's serum levels are more sensitive to plasma binding parameters (Fig. 15) (Merrill et al., 2003). The fact that data was available across multiple doses for establishing parameter values for urinary clearance and plasma binding adds confidence to the model's predictive ability. More useful to the application of the models, for human dosimetry predictions, is variability analysis that is performed with known distributions for model parameters. This tool can be applied to the model to allow the prediction of likely ranges of the dosimetrics within a human population. Modeled effects on hormone regulation are yet to be developed. Perturbations in hormones levels after exposure demonstrate complex differences in the hormone regulatory mechanisms between rats and humans, which are difficult to describe (Clewell et al., 2001; Merrill et al., 2001). However, the current model structures may provide a basis for evaluating thyroid effects from other environmental contaminants. For example, excessive exposure to other similarly behaving anions, such as sodium chlorate, thiocyanate, or nitrate, all found to also contaminate ground and surface waters, may contribute to environmental anti-thyroid effects in humans (Hooth et al., 2001; Wolff and Maurey, 1963). Further, the possibility of additive anti-thyroid effects to those of perchlorate from these cocontaminants may need to be considered (Kahn et al., 2004). NOTES The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. The U.S. Government has the right to retain a nonexclusive royalty-free copyright covering this article. 2 Current address: CIIT Centers for Health Research, Research Triangle Park, NC 12137. 3 Current address: Environmental Health Science, University of Georgia, Athens, GA 30602. ACKNOWLEDGMENTS The authors express special thanks to Drs. Monte Greer, Gay Goodman, Georg Brabant, and Holger Leitolf for supplying serum and urine samples from their studies for perchlorate analyses and Dr. Mel Andersen and Harvey Clewell for constructive comments. Also acknowledged are Lt. Col. Dan Rogers, Dr. Richard Stotts, and Dr. Dave Mattie for assistance in obtaining funding for this research from the U.S. Air Force, U.S. Navy, and NASA. Drs. Andrew Geller and Allan Marcus are thanked for their critical technical reviews of the draft manuscript. Lastly, this work would not have been possible without analytical support from Lt. Eric Eldridge, Latha Narayanan, Gerry Buttler, and SSgt. Paula Todd. Funding for this research was provided by the U.S. Air Force, U.S. Navy, and NASA. REFERENCES Motzer, W. E. (2001). Perchlorate: Problems, detection, and solutions. Environ. Forensics. 2(4), 301–311 Narayanan, L., Buttler, G. W., Yu, K. O., Mattie, D. R., and Fisher, J. W. (2003). Sensitive high-performance liquid chromatography method for the determination of low levels of perchlorate in biological samples. J. Chromatog. B. 788, 393–399 NRC (National Research Council) (1996). The Artic aeromedical laboratory's thyroid function study: A radiological risk and ethical analysis. Committee on Evaluation of 1950s Air Force Human Health Testing in Alaska Using Radioactive Iodine-131. National Academy Press, Washington, D.C. Pena, H. G., Kessler, W. V., Christian, J. E., Cline, T. R., and Plumlee, M. P. (1976). A comparative study of iodine and potassium perchlorate metabolism in the laying hen: Uptake, distribution, and excretion of potassium perchlorate. Poult. 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Acta 47, 467–474 Wolff, J., and Maurey, J. R. (1963). Thyroidal iodide transport: IV. The role of ion size. Biochim. Biophys. Acta. 69, 48–58 Wyngaarden, J. B., Wright, B. M., and Ways, P. (1952). The effect of certain anions upon the accumulation and retention of iodide by the thyroid gland. Endocrinology 50, 537–549 Yokoyama, N., Nagayama, Y., Kakezono, F., Kiriyama, T., Morita, S., Ohtakara, S., Okamoto, S., Morimoto, I., Izumi, M., Ishikawa, N., et al. (1986). Determination of the volume of the thyroid gland by a high resolutional ultrasonic scanner. J. Nucl. Med., 27, 1475–1479 Yu, K. O., Narayanan, L., Mattie, D. R., Godfrey, R. J., Todd, P. N., Sterner, T. R., Mahle, D. A., Lumpkin, M. H., and Fisher, J. W. (2002). The pharmacokinetics of perchlorate and its effect on the hypothalamus/pituitary-thyroid axis in the male rat. Toxicol. Appl. Pharmacol. 182(2), 148–159....查看详细 (69235字节)
☉ 11120230:Novel Progestogenic Activity of Environmental Endo
ABSTRACT Endocrine disruption is a major global health concern in the industrialized world. The induction of uterine calbindin-D9k (CaBP-9k), which belongs to a large family of intracellular calcium binding proteins, was used to assess the exposure of endocrine disruptors (EDs) in an immature mouse model. Sex steroid hormones have been demonstrated to regulate uterine CaBP-9k expression in the uterus of rats and mice. In particular, the mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly induced by 17-beta-estradiol (E2) in the uterus. In the present study, immature (14-day-old) female mice were injected with 4-tert-octylphenol (OP), nonylphenol (NP), bisphenol A (BPA), E2, or P4 to determine their effects on uterine CaBP-9k mRNA and protein expression. In addition, to specify estrogenic or progestogenic activity of EDs in the regulation of CaBP-9k, the mice were co-treated with ICI 182,780, an estrogen receptor (ER) antagonist, or RU486, a progesterone receptor (PR) antagonist,. Treatments with OP, NP, or BPA resulted in an increase in CaBP-9k mRNA and protein in the uterus of immature mice in a dose-dependent and time-dependent manner. The EDs-induced expression of CaBP-9k mRNA and protein was reversed or abolished by pretreatment with RU486 or ICI 182,780, suggesting that these synthetic chemicals may have both progestogenic and estrogenic properties by acting through PR or ER in the induction of uterine CaBP-9k mRNA and protein in this model. These results describe a novel in vivo model for detection of both estrogenic and progestogenic activities of EDs in the induction of CaBP-9k mRNA and protein in the uterus of immature mice. Key Words: calbindin-D9k; endocrine disruptor; estradiol; progesterone. INTRODUCTION Calbindin-D9k (CaBP-9k) is a member of a family of cytosolic calcium binding proteins that play an important role in the regulation and buffering of Ca2+ in the intestine, uterus, placenta, and kidney (Bruns et al., 1988; Delorme et al., 1983; Jeung et al., 1992; Krisinger et al., 1992). CaBP-9k is a major vitamin D target gene involved in calcium homeostasis and expression of the vitamin D–dependent calcium-binding protein (An et al., 2003b; Christakos et al., 1989). It has been shown that CaBP-9k in the mammalian intestine is induced transcriptionally by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the most active metabolite of vitamin D (Delorme et al., 1983; Li et al., 1998). It is of interest that CaBP-9k expression is not controlled by vitamin D in the uterus, despite the presence of vitamin D receptors in this tissue, but it is controlled by the sex steroid hormones (Delorme et al., 1983; L'Horset et al., 1990; Nie et al., 2000). During the estrous cycle in rats, a high level of expression of CaBP-9k mRNA was observed at estrus and proestrus (Krisinger et al., 1992). Transcription of CaBP-9k in the uteri of mature ovariectomized and immature rats is stimulated by E2 (L'Horset et al., 1993, 1990). However, in the mouse uterus, CaBP-9k expression is mainly regulated by progesterone (P4), not 17-beta-estradiol (E2). During the estrous cycle in mice, CaBP-9k was highest at diestrus and metestrus, whereas only basal levels of expression were detected at proestrus and estrus (Nie et al., 2000). This differential pattern of CaBP-9k expression during the estrous cycle suggests that distinct mechanisms exist in the regulation of CaBP-9k in rats and mice (Li et al., 1998; 2001). Interestingly, when the uterus of immature and ovariectomized mice was treated with E2 and P4 together, CaBP-9k expression was enhanced compared to treatment with P4 alone (Nie et al., 2000; Tatsumi et al., 1999). In addition, the action of P4 in target tissues was mediated through the progesterone receptor (PR), which can also be regulated by E2 in immature rats (An et al., 2003a). Recent research has demonstrated that environmental chemicals caused endocrine disruption through agonistic or antagonistic effects on steroid hormone receptors (Sumida et al., 2001). Endocrine disruptors (EDs) have been proposed to bind to one of the nuclear estrogen receptors (ERs), and xenoestrogens and phytoestrogens, except for genistein, showed an equal binding affinity to both ER subtypes; nevertheless it is unclear that this is their major or only mode of actions (Gutendorf and Westendorf, 2001). Examples of suspected environmental estrogenic chemicals include polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT), diethylstilbestrol (DES), 4-tert-octylphenol (OP), 4-nonylphenol (NP), and bisphenol A (BPA) (An et al., 2003a; Sumida et al., 2001). In addition, these chemicals have been demonstrated to exhibit similar affinities to steroid receptors, with the inhibition of receptor binding affinity concentrations of 0.7 μM for ER and 1.2–3.8 μM for PR, respectively (Laws et al., 2000). Alkylphenols, such as OP and NP, are derived from alkylphenol ethoxylates. Bisphenol is a monomer on polycarbonate plastics and a constituent of the epoxy and polystyrene resins used extensively in food packaging and dental sealants (Jorgensen et al., 2000; Witorsch, 2002). The mechanism of action of these compounds has not been established in all cases, but the resulting effect is consistent with ER-mediated action (Arnold et al., 1996; Nagel et al., 1997; White et al., 1994). The effects of NP and OP are due to their direct interaction with ER (Odum et al., 2001; Routledge and Sumpter, 1997). The differences in the biological potency and binding affinity of OP, NP, and BPA to ER have been demonstrated in a recent study (Jorgensen et al., 2000). The ER can bind to a variety of nonsteroidal compounds that are structurally similar to the alkyl-substituted phenol of E2 (Hu and Aizawa, 2003). Alkylphenolic compounds exhibit a weak ER binding affinity, 1/1,000 to 1/1,000,000–fold lower than endogenous E2, and they appear to be weak agonists for PR (Witorsch, 2002). Previous studies demonstrated that a significant increase in CaBP-9k mRNA and protein was observed in the uterus when immature rats were treated with OP, NP, or BPA (An et al., 2002; 2003a), and in the fetal uterus following maternal injection of rats (Hong et al., 2003). A pure antagonist for ER and ER?, ICI 182,780, blocked the effect of E2 and BPA (Papaconstantinou et al., 2001), and controlled ER and ER-regulated responses in the uterus (Cowley et al., 1997; Das et al., 1998). ICI 182,780 downregulated ER and antagonized E2-induced increases in uterine PR. In addition, ICI 182,780 antagonized E2-induced and BPA-induced effects on the localization of heat shock protein 90 in vitro (Papaconstantinou et al., 2001). RU486 has a high affinity to PR and glucocorticoid receptor (Mahajan and London, 1997). After RU486 binds to target receptors, receptor interaction with heat-shock protein 90 and p53 is strengthened, but there is no interaction with DNA at the hormone response element (Mahajan and London, 1997). The phenyl-aminodimethyl group at the 11-? and 17- position of the steroidal skeleton and the carboxyl side chain of RU486 correlated with an antagonist activity of progesterone (Leonhardt and Edwards, 2002). In other words, RU486, an 11-? substituted nor-steroid containing a 17- propylnyl group, is used as an antiprogestin agent (He et al., 1999). RU486, an antiprogesterone and antiglucocorticoid, antagonizes not only PR-mediated transactivation but also ER transactivation via PR inhibition (Papaconstantinou et al., 2001). Because EDs were expected to have the properties of both estrogenic and weak progestogenic activities, the effect of EDs in the induction of CaBP-9k expression has not been elucidated yet in a mouse in vivo model. Thus, we investigated the effect of EDs on the expression of CaBP-9k mRNA and protein compared to that of endogenous E2 and P4 in the uterus of immature mice. To specify an estrogenicity or progestogenicity of EDs, both RU486, a PR antagonist, and ICI 182,780, an ER antagonist, were employed. This study describes a novel in vivo model of immature mice used for the first time to detect estrogenicity and progestogenicity. MATERIALS AND METHODS Chemicals. The chemicals were purchased from the following sources: E2, (98% pure), P4, NP, BPA (97 % pure), and Mifepristone (RU486) were obtained from Sigma-Aldrich Corp (St. Louis, MO). OP (98% pure) and ICI 182,780 were purchased from Fluka Chemical (Seoul, Korea) and Tocris (Avonmouth, UK), respectively, and used in the present study (Fig. 1). Animals and treatments. Immature female Crj:CD-1 mice (10 days old) were obtained from Daehan Biolink Co (Eumsung, Korea) and delivered early in the morning with dams, to minimize shipping stress. They were then adapted for 4 days in the Animal Facility, College of Veterinary Medicine, Chungbuk National University. All experimental procedures and animal care were approved by the Ethics Committee of the Chungbuk National University. Animals were housed in polycarbonate cages, and used after they had become acclimated in an environmentally controlled room (temperature: 23°±2°C, relative humidity: 50 ± 10%, frequent ventilation and 12 h light cycle) for 4 days. For all experiments, mice were injected at 09:00 A.M. daily. To investigate the effective dose of EDs in the uterus, the immature mice were treated with increasing doses of OP, NP, and BPA in a dose-dependent manner (Fig. 2). In the first experiment, nine groups of five animals (14 days old) were injected subcutaneously (s.c., 0.1 ml per mouse) with OP, NP, or BPA at three different doses: 100, 250, and 500 mg/kg body weight (BW) per day. These EDs were administered in a corn oil vehicle, once per day for 3 days. The positive groups of mice (5 of each treatment) were given s.c. injections with E2 (10 μg/kg), P4 (200 mg/kg), E2 plus P4, or corn oil alone as a vehicle. All mice were euthanized at 24 h after the final injection. To examine the expression level of CaBP-9k, the immature mice were treated with EDs for 3 days and euthanized in a time-dependent fashion (Fig. 2). In the second experiment, four groups of 30 animals (14 days old) were injected s.c. with corn oil, OP, NP, or BPA (500 mg/kg BW) daily for 3 days. Five animals from each group were euthanized at 3, 6, 12, 24, 48, and 72 h after the final injection. In addition, to specify steroid hormone–like activity of EDs, the third experiment was performed as shown in Figure 2. After treatment with RU486 (300 mg/kg BW) or ICI 182,780 (10 mg/kg BW) 1 h before injection, 10 groups of five animals (14 days old) were injected s.c. with E2 (10 μg/kg), P4 (200 mg/kg), OP, NP, or BPA (500 mg/kg BW) daily for 3 days. The mice were euthanized 24 h after final injection. Northern blot analysis. Northern blot analysis was performed to determine CaBP-9k mRNA in immature mice as described previously (Lee et al., 2003). Total RNA was extracted from tissues using Trizol Reagent (Invitrogen Life Technologies, Inc, Carlsbad, CA) according to the manufacturer's suggested protocol. The concentration of RNA was determined by measuring absorbance at 260 nm. RNA was denatured by heating at 85°C for 10 min. Total RNA (15 μg) was electrophoresed on a 1% formaldehyde denaturing agarose gel for 70 min at 110 V, and 28S rRNA served as an indicator of the quantity of total RNA. RNA was transferred from the agarose gel to a nylon membrane by the capillary method according to the manufacturer's suggested protocol (GeneScreen, NEN Life Science Products, Boston, MA). RNA was UV cross-linked to the membrane using the GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA). Membranes were prehybridized in 50% formamide, 5 x SSPE, 5x Denhardt's, 0.1% SDS, 0.1 mg/ml salmon sperm DNA for 2 h at 42°C. Radiolabeled probes were prepared using the Random Primer DNA Labeling Kit Ver 2 (TaKaRa Bio Inc, Shiga, Japan) according to the manufacturer's suggested protocol. The 32P-labeled probes were denatured by heating to 95°C for 3 min and added to the hybridization solution. Membranes were allowed to hybridize for 16 h at 42°C and were washed 3 times at 42°C in 2x SSC, 0.1% SDS, once at 53°C in 1x SSC, 0.1% SDS, and 3 times at 68°C in 0.1x SSC, 0.1% SDS. Membranes were exposed to X-ray film (Eastman Kodak Co, Rochester, NY), and the films were scanned and analyzed using the Molecular Analysis Program version 1.5 (Gel Doc 1000, Bio-Rad Laboratories). Real-time polymerase chain reaction (PCR). The standard curve was generated for a standard RNA preparation by serial dilution (1, 1/10, 1/100, 1/1,000, 0). The Real-Time PCR reaction was carried out in a 25 μl final volume containing 5 μl of 5x Taq DNA polymerase (TaKaRa Bio Inc.), 2.5 μl of diluted (1:30,000) SYBR Green (TaKaRa Bio Inc.), 0.5 μl of each of forward and reverse primers, 2 μl of cDNA, and distilled water up to 25 μl. The oligonucleotide sequences of primers employed to detect mouse CaBP-9k mRNA, were 5'-GCA AAA TGT GTG CTG AGA A-3' (sense) and 5'-GGA ACT CCT TCT TCC TGA CT-3' (antisense). Primers for the 1A gene were: 5'-GAT ATA GCA TTC CCA CGA ATA-3' (sense) and 5'-GGG CTT TTG CTC ATG TGT CAT-3' (antisense). Polymerase chain reaction amplification using the Smart Cycle System (TaKaRa Bio Inc.) began with an initial denaturation at 95°C for 30 s. Each of the 35 amplification cycles consisted of denaturation at 95°C for 5 s, annealing at 55°C for 7 s, and extension at 72°C for 12 s. Relative expression levels of each sample were calculated based on the Cycle threshold (Ct) and monitored for amplification curve. The PCR amplification curves were evaluated by fluorescence of the double-stranded DNA-specific dye, SYBR Green, versus the amount of standardized PCR product. Expression of CaBP-9k was normalized to IA mRNA. Western blot analysis. To measure the expression level of CaBP-9k protein by EDs, we performed Western blot analysis as described previously (Hong et al., 2003). Briefly, immature female mice were euthanized, the uteri were isolated, and protein was extracted with Pro-prep (Intron Co, Seoul, South Korea) according to the manufacturer's instructions. The amount of protein was determined using a Bradford assay (Bio-Rad Laboratories) (Bradford, 1976). Cytosolic protein (40 μg) was separated by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Rockville, MD) with a Semi-dry Transfer Cell (Bio-Rad Laboratories) according to the manufacturer's protocol. Membranes were incubated in 4% (w/v) skim milk dissolved in PBS-T buffer (137 mM NaCl, 2 mM KCl, 10 mM phosphate buffer and 5% (v/v) Tween-20) for 4 h at room temperature to block nonspecific binding. Membranes were then incubated with a rabbit polyclonal antibody (Swant, Switzerland, 1:3,000) specific for mouse CaBP-9k, and washed in PBS-T buffer. Incubation with the secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:3,000 in PBS-T containing 3% (w/v) bovine serum albumin) was for 1 h at room temperature. Protein bands were visualized with the ECL chemiluminescent system (Amersham Pharmacia Biotech) and autoradiography. The expression of CaBP-9k was quantitated by densitometry (NIH image beta 3). Data analysis. Data are presented as the mean ± S.D. Data were analyzed by the nonparametric Kruskal-Wallis test, followed by Dunnett's test for five-pair comparisons. Each value of Dunnett's test was converted to rank for statistical analysis. All statistical analyses were performed with the Statistical Analysis System (SAS Institute, Cary, NC); p < 0.05 was considered statistically significant. RESULTS Dose-Dependent Effects of EDs on the Expression of CaBP-9k mRNA and Protein The effects of environmental compounds and steroid hormones on expression levels of CaBP-9k mRNA and protein were assessed using increasing doses of EDs. The expression of CaBP-9k mRNA increased significantly in the uterus of immature mice after 3 days of treatment with a high dose (500 mg/kg BW per day) of OP (9-fold vs. vehicle), NP (10-fold vs. vehicle), or BPA (8-fold vs. vehicle), as shown in Figure 3. In addition, treatment with a mid dose (250 mg/kg BW per day) of OP, NP, and BPA increased 7-fold, 8-fold, and 6-fold in CaBP-9k mRNA, respectively (Fig. 3). Treatment with a low dose of EDs (100 mg/kg BW per day) appeared to increase the level of CaBP-9k mRNA, but the increase was not significant. In addition, treatment with P4 alone resulted in significant induction of CaBP-9k mRNA, whereas E2 did not have a significant effect in the uterus of immature mice. Although E2 appeared to increase CaBP-9k mRNA, the increase was not significant. However, it is of interest that E2 plus P4 enhanced P4-induced CaBP-9k mRNA compared to P4 alone. In an agreement with its mRNA levels, treatment with OP, NP, or BPA (250 and 500 mg/kg BW per day) induced a dramatic increase in CaBP-9k protein in the uterus of immature mice (Fig. 4). Most notably, CaBP-9k protein was significantly increased by treatment with the highest dose of OP (11-fold vs. vehicle), NP (11-fold vs. vehicle), or BPA (9-fold vs. vehicle). In addition, treatment with a mid dose (250 mg/kg BW per day) of OP (6-fold vs. vehicle), NP (6-fold vs. vehicle), or BPA (5-fold vs. vehicle) induced CaBP-9k protein in this tissue as shown in Figure 4. It is of interest that treatment with a low dose (100 mg/kg BW per day) of OP resulted in a significant CaBP-9k protein increase in the uterus (4-fold vs. vehicle). As described in its mRNA level, E2 plus P4 also enhanced P4-induced CaBP-9k protein level compared to P4 alone. Time-Dependent Effects of EDs on the Expression of CaBP-9k mRNA and Protein The levels of CaBP-9k mRNA and protein in the uterus of immature mice were examined in a time-dependent manner after 3 days of treatment with OP, NP, or BPA (500 mg/kg BW per day). Mice were euthanized at 3, 6, 12, 24, 48, and 72 h after final injections of EDs. Treatment with a high dose of ED resulted in induction of CaBP-9k mRNA as early as 3 h (OP and NP) and 6 h (BPA) after final injection (Fig. 5A and 5B). This increase was sustained until 48 h and 72 h after treatment with OP and NP. Treatment with BPA induced a significant increase in CaBP-9k mRNA expression at 6, 12, and 24 h in this tissue, but no significant induction was observed at 48 h and 72 h after treatment with BPA (Fig. 5A and 5B). The expression of CaBP-9k protein in the uterus was also monitored by immunoblot analysis. As shown in Figure 6A and 6B, a significant increase in CaBP-9k protein was observed in OP-, NP-, and BPA-treated mice at all time points tested. The ED-induced increase of CaBP-9k was greater at the protein level than at the mRNA level. Effect of Steroid Antagonists on Steroid Hormone–Induced or EDs-Induced CaBP-9k mRNA and Protein Expression As described above, treatments with OP, NP, and BPA resulted in dose-dependent and time-dependent increases in CaBP-9k mRNA and protein in the uterus of immature mice. To elucidate an involvement of steroid hormone receptors, ER and PR; ICI 182,780, an antagonist for ER; and RU486, a PR antagonist, were employed for further study. Treatment with ICI 182,780 (3 mg/kg BW per day) or RU486 (300 mg/kg BW per day) significantly blocked P4-induced CaBP-9k mRNA in the tissue (Fig. 7). It is of interest that treatment with ICI 182,780 (10 mg/kg BW per day) significantly blocked OP-, NP-, and BPA-induced CaBP-9k mRNA expression in the uterus of immature mice, whereas treatment with RU486 (300 mg/kg BW per day) reversed the EDs-induced CaBP-9k mRNA in part (Fig. 8A). The relative levels of CaBP-9k mRNA in the mice treated with RU486 plus OP, NP, or BPA were 51% that of OP alone, 53% that of NP alone, and 58% that of BPA alone, respectively. The relative levels of CaBP-9k mRNA in the mice treated with ICI 182,780 plus OP, NP, or BPA were 21% that of OP alone, 27% that of NP alone, and 19% that of BPA alone, respectively. The result in which RU486 reversed the EDs-induced CaBP-9k mRNA in part suggests that EDs may have a progestogenic effect through PR in this tissue. In agreement with the results by Northern blot analysis, the expression of CaBP-9k mRNA measured by a real-time PCR was significantly induced when immature mice were treated with EDs and P4 (Fig. 8B). Furthermore, treatments with ICI 182,780 or RU486 reversed ED-induced CaBP-9k mRNA. The protein level of CaBP-9k expression was further examined by immunoblot analysis as shown in Figure 9. In parallel with its mRNA level, pretreatments with ICI 182,780 or RU486 reversed EDs-induced CaBP-9k protein completely or in part. The relative levels of CaBP-9k protein when treated with a combination of RU486 and OP, NP, or BPA were 36% that of OP alone, 37% that of NP alone, and 25% that of BPA alone, respectively. In addition, the relative levels of CaBP-9k protein treated with a combination of ICI 182,780 and OP, NP, or BPA were 22% that of OP alone, 27% that of NP alone, and 17% that of BPA alone, respectively (Fig. 9). DISCUSSION In female reproductive tissues, CaBP-9k may play an important role in the regulation of reproductive functions related to calcium transfer (Li et al., 2001), but the nature of its action remains to be elucidated. It has been demonstrated that in the uterus, CaBP-9k expression is under the control of the sex steroid hormones (L'Horset et al., 1993; Nie et al., 2000). In the present study, uterine CaBP-9k mRNA and protein were significantly induced when immature mice were treated with estrogenic compounds and P4, and the increases were dose-dependent and time-dependent. To demonstrate an involvement of EDs through steroid hormone receptors, ICI 182,780, an antagonist for ER, and RU486, a PR antagonist, were employed. The expression of CaBP-9k mRNA and protein in the immature uterus was induced by estrogenic compounds and completely blocked by ICI 182,780 to the vehicle level. In addition, RU486 significantly reversed ED-induced expression of CaBP-9k, in part. In the rat uterus, CaBP-9k expression is completely repressed during diestrus when E2 concentration is low, and increased at proestrus in response to the rise in plasma E2 (Krisinger et al., 1992; L'Horset et al., 1994). Treatment of rats with E2 significantly increased CaBP-9k mRNA and protein in the uterus (An et al., 2002; 2003a; Elger et al., 2000), indicating that E2 is an important factor in the regulation of CaBP-9k in the uterus of rats. It is interesting that the regulation of CaBP-9k by P4 has been demonstrated in the mouse uterus, but its regulation by E2 has not (Nie et al., 2000). It has been shown that E2 treatment alone did not affect CaBP-9k mRNA expression in the uteri of ovariectomized mice (Nie et al., 2000; Tatsumi et al., 1999). This difference between E2 and P4 suggests that distinct mechanisms exist in the regulation of the CaBP-9k gene during the estrous cycles of mice and rats (Nie et al., 2000). Taken together, these previous findings indicate that P4 is a major factor in the regulation of CaBP-9k gene during the estrous cycle in mice, and CaBP-9k is mainly regulated by P4, not E2, in the uterus of mice (Tatsumi et al., 1999). In the present study, it is not surprising that treatment with P4 only, but not E2, resulted in the induction of CaBP-9k mRNA and protein and a combination of E2 and P4 enhanced P4-induced CaBP-9k expression in the uterus of immature mice, suggesting that P4 is a dominant factor in the regulation of CaBP-9k and that E2 may enhance P4 effect on its gene expression through activation of PR. There is increasing evidence that man-made chemicals in the environment may interfere with the human body's complex and carefully regulated hormonal system (Crisp et al., 1998). These synthetic chemicals can disrupt the endocrine system in diverse ways; for example, they not only mimic or block endogenous hormones but also alter hormonal levels, affecting the functions of various tissues (Crisp et al., 1998; Gaido et al., 1997). Examples of suspected environmental estrogenic chemicals include polychlorinated hydroxybiphenyls, DDT, kepine, methoxychlor, and BPA. Alkylphenolic compounds are environmentally persistent and have an estrogenic activity (Laws et al., 2000; Sumpter and Jobling, 1993; White et al., 1994). In our previous studies, we elucidated the effect of estrogenic compounds, OP, NP, and BPA on the expression of CaBP-9k mRNA and protein as a biomarker in the uterus of immature rats (An et al., 2002; 2003a). EDs can replace endogenous hormones in the uterus, so a high dose of EDs resulted in the induction of CaBP-9k mRNA and protein. Using an immature rat model, we demonstrated for the first time that maternally injected estrogenic compounds (OP, NP, and BPA) caused an increased CaBP-9k mRNA and/or protein in the maternal tissues (uterus and placenta) and the fetal uterus during late pregnancy, suggesting that for fetal health, the placenta may not be a reliable barrier against some estrogenic compounds in a rat model (Hong et al., 2003, 2004a). In addition, maternally injected estrogenic compounds may be transferred to neonates through breast milk, thus affecting uterine function, as shown by the induction of CaBP-9k gene expression in the neonatal uterus (Hong et al., 2004b). Estrogenic compounds exhibit affinities to ER and PR and bind to both steroid receptors in vitro binding assay (Laws et al., 2000). In our previous studies, estrogenic induction of uterine CaBP-9k mRNA and protein by environmental phenol products, OP, NP, and BPA were analyzed using an immature rat model; however, no model system was available to detect a progestogenic effect of EDs on the induction of CaBP-9k mRNA and protein (An et al., 2003a; Hong et al., 2003). An immature mouse model is an excellent system because CaBP-9k expression in the uterus is regulated in a P4-dependent manner (Nie et al., 2000). The present study demonstrated that estrogenic chemicals resulted in the induction of CaBP-9k mRNA and protein in a dose-dependent and time-dependent manner as only P4 did. These results indicate that these estrogenic compounds OP, NP, and BPA have a progestogenic activity in the uterus of immature mice, implicating the risk assessment of these chemicals. It is of interest that RU486 significantly reversed EDs-induced CaBP-9k mRNA expression, in part, indicating that EDs may have a specific progestogenic effect through PR in this tissue. On the other hand, treatment of immature mice with ICI 182,780 completely blocked OP-, NP-, and BPA-induced CaBP-9k expression in the uterus compared to the vehicle level. In addition, the effect of P4 on the expression of CaBP-9k was completely blocked by ICI 182,780, although E2 itself did not have any effect on the expression of CaBP-9k. These results suggest that ICI 182,780 may block both P4-induced CaBP-9k mRNA and induction of CaBP-9k mRNA itself in the uterus of immature mice. RU486 (Mifepristone) is an 11-beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity to PR and glucocorticoid receptors. Because RU486 has a higher binding affinity to PR than does P4, this antagonist effectively blocks the action of P4 (Koide, 1998). ICI 182,780 binds to ER with high affinity and completely inhibits the effects of E2 on the growth of rat uterus, on the growth of MCF-7 cells in vitro, and on E2-stimulated breast tumor and endometrial tumor growth in the nude mouse (Koide, 1998; Wakeling and Bowler, 1988a, 1988b; Wakeling et al., 1991). In addition, ICI 182,780 alone has no estrogenic activity in the induction of CaBP-9k gene expression (Blin et al., 1995). In the present study, ED-induced CaBP-9k mRNA and protein was reversed in part or completely by treatments with RU486, a P4 antagonist, or ICI 182,780, an E2 antagonist, suggesting that these synthetic chemicals may have both progestogenic and estrogenic properties. The previous work showed that treatment of pregnant mice with RU486 decreased uterine CaBP-9k mRNA expression, suggesting that P4 upregulated CaBP-9k gene in this tissue (An et al., 2003b). Taken together, these results demonstrated a novel finding that OP, NP, and BPA may have progestogenic activity in the uterus of immature mice and that the expression of CaBP-9k mRNA may be completely blocked by ICI 182,780 in this tissue. However, it is not clear how ICI 182,780 blocks EDs-induced CaBP-9k mRNA expression in this tissue because ICI 182,780 is a complete antagonist of ER, which warrants further study. In conclusion, treatment of immature mice with OP-, NP-, and BPA-induced CaBP-9k mRNA and protein in a dose-dependent and time-dependent manner in the uterus of immature mice. Treatment of the mice with ICI 182,780 and EDs or RU486 and EDs reversed EDs-induced CaBP-9k mRNA and protein levels in this tissue, suggesting that EDs may have both progestogenic and estrogenic properties through PR and ER in this in vivo model. These results validate a novel in vivo model of immature mice for the first time to detect activities of estrogenic and progestogenic EDs through the induction of uterine CaBP-9k mRNA and protein. NOTES 1 Y.-W. J. and E.-J. H. contributed equally this work and should be considered as first authors. ACKNOWLEDGMENTS This work was supported by Korea Research Foundation Grant (KRF 2003–041-E00238). We are grateful to Dr. Barb Conway at the British Columbia Research Institute for Children's and Women's Health, University of British Columbia, for a critical review of the manuscript. REFERENCES An, B. S., Choi, K. C., Kang, S. K., Hwang, W. S., and Jeung, E. B. (2003a). Novel calbindin-D(9k) protein as a useful biomarker for environmental estrogenic compounds in the uterus of immature rats. Reprod. Toxicol. 17, 311–319. Hu, J. Y., and Aizawa, T. (2003). Quantitative structure-activity relationships for estrogen receptor binding affinity of phenolic chemicals. Water Res. 37, 1213–1222. Jeung, E. B., Krisinger, J., Dann, J. L., and Leung, P. C. (1992). Cloning of the porcine calbindin-D9k complementary deoxyribonucleic acid by anchored polymerase chain reaction technique. Biol. Reprod. 47, 503–508. Jorgensen, M., Vendelbo, B., Skakkebaek, N. E., and Leffers, H. (2000). Assaying estrogenicity by quantitating the expression levels of endogenous estrogen-regulated genes. Environ. Health Perspect. 108, 403–412. White, R., Jobling, S., Hoare, S. A., Sumpter, J. P., and Parker, M. G. (1994). Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135, 175–182. Witorsch, R. J. (2002). Low-dose in utero effects of xenoestrogens in mice and their relevance to humans: An analytical review of the literature. Food Chem. Toxicol. 40, 905–912...查看详细 (31066字节)

☉ 11120231:Modeling and Predicting Stress-Induced Immunosuppr
ABSTRACT Previous studies have shown that the area under the corticosterone concentration vs. time curve (AUC) can be used to model and predict the effects of restraint stress and chemical stressors on a variety of immunological parameters in the mouse spleen and thymus. In order to complete a risk assessment parallelogram, similar data are needed with blood as the source of immune system cells, because this is the only tissue routinely available from human subjects. Therefore, studies were conducted using treatments for which the corticosterone AUC values are already known: exogenous corticosterone, restraint, propanil, atrazine, and ethanol. Immunological parameters were measured using peripheral blood from mice treated with a series of dosages of each of these agents. Flow cytometry was used to quantify MHC II, B220, CD4, and CD8 cells. Leukocyte and differential counts were done. Spleen cell number and NK cell activity were evaluated to confirm similarity to previous studies. Immune parameter data from mouse blood indicate that MHC II expression has consistent quantitative relationships to corticosterone AUC values, similar to but less consistent than those observed in the spleen. Other immune parameters tended to have greater variability in the blood than in the spleen. The pattern observed in the spleen in which the chemical stressors generally produced very similar effects as noted for restraint stress (at the same corticosterone AUC values) was not observed for blood leukocytes. Nevertheless, MHC class II expression seems to provide a reasonably consistent indication of stress exposure in blood and spleen. Key Words: modeling; stress; biomarker; ethanol; atrazine; propanil. INTRODUCTION Guidance documents have been released by the U.S. Environmental Protection Agency and Food and Drug Administration for immunotoxicity safety testing of chemicals and drugs in rodents (Anonymous, 1998, 1999). In assessing the safety of chemicals or drugs, it is recommended that some animals receive a relatively high dosage of the agent being tested. Immunotoxicity can then be quantitatively assessed on the basis of changes in histological characteristics, blood parameters, or a few immune parameters that have previously been demonstrated to be good indicators of immunotoxicity (Luster et al., 1987, 1992, 1993). However, the guidance documents do not include specific recommendations for distinguishing genuine immunotoxicity from immunotoxicity that is secondary to a generalized stress response. Because many drugs and chemicals administered at high dosages induce immunosuppressive stress responses (Pruett et al., 1999, 2000a,b, 2003), methods are needed to allow identification of stress-induced immunosuppression, preferably without the assessment of multiple additional parameters or the use of more rodents. If consistent quantitative patterns of change are seen in immune parameters as stressor dosages increase, then these patterns could be used to suggest the presence of a stress effect. It has been established that stress can affect immune function through the activation of the hypothalamic pituitary adrenal axis resulting in the production of a number of neuroendocrine mediators (Riley, 1981; Zwilling et al., 1993). Some of these mediators, such as corticosterone (in rodents) or cortisol (in humans), have been shown to be immunosuppressive in both rodents and humans (Dhabhar et al., 1994). Plasma corticosterone levels have been used for many years as an indicator of stress in mice. The effect that a stress response has on immunological parameters can be quantified by relating immunosuppression to the quantity and duration of the stress response represented by the area under the corticosterone concentration vs. time curve (AUC) (Pruett et al., 1999). Linear regression analysis can then be used to indicate correlations between AUC and immunosuppression of each immunological parameter examined. Quantitatively consistent results showing similar effects on several parameters by both chemical and physical stressors, at comparable corticosterone AUC values have been demonstrated in a series of studies using spleen and thymus (Pruett et al., 1999, 2000a,b, 2003). In the present study, similar procedures were used to determine if an immunological biomarker for stress can be identified using blood samples instead of spleen or thymus. Finding such a biomarker would improve the accuracy of risk assessment in humans because blood is the only tissue routinely available for such comparative studies. MATERIALS AND METHODS Animal care. Female B6C3F1 mice, obtained through the National Cancer Institute's Animal Program, were housed and treated according to NIH and LSUHSC-S guidelines in a facility accredited by the American Association for Accreditation of Laboratory Animal care. Mice were allowed to acclimate to the facility and recover from shipping stress for at least two weeks. Mice were given food (Purina Lab Chow) and water ad libitum and were maintained on a 12-h light/dark cycle. They were used in experiments at 8–12 weeks of age. General dosing. Five cages of five mice were used for each chemical or restraint stressor. Each cage comprised one of five dosage groups. One of these cages of mice was used as a control group that was either naive (untreated) or treated with the vehicle for the chemical that was administered. All doses were administered at 2130–2200 h to allow for corticosterone circadian rhythms to be similar with those used to calculate AUC values (Pruett et al., 1999, 2000a,b, 2003). The treated groups were placed in 50 ml conical tubes for 2, 4, 6, or 8 h. These conical tubes were ventilated by a longitudinal slit used to carefully pull each mouse into the tube. After placing each mouse in its tube, the tubes were placed back in the cage for the duration of the restraint period. The mice in the groups receiving the two highest dosages of restraint (6 and 8 h) were allowed access to food and water for 10 min at the 4-h time point. Corticosterone dosing. A corticosterone (Sigma, St. Louis, MO) suspension was made in a vehicle of phosphate buffered saline (Sigma, St. Louis, MO) containing 2% ?-cyclodextrin (Sigma, St. Louis, MO). Corticosterone doses were administered via sc injections at concentrations of 9 mg/kg, one dose at 18 mg/kg, two doses at 18 mg/kg (2 h apart), or three doses at 18 mg/kg (2 h apart). Propanil dosing. A propanil (Chem Service, West Chester, PA) suspension was made by mixing the chemical in corn oil (Mazola). Propanil was given by ip injection at dosages of 50, 75, 100, or 150 mg/kg (in 0.2 ml). Atrazine dosing. Atrazine (Chem Service, West Chester, PA) was mixed in corn oil (Mazola) and administered by ip injection at dosages of 75, 150, 225, or 300 mg/kg (in 0.2 ml). Ethanol dosing. Ethanol (AAPER Alcohol Chemical Co., Shelbyville, KY) used for dosing was diluted in sterile tissue culture-grade water (Sigma, St. Louis, MO) to 32% by volume. Ethanol was given by po gavage at dosages of 4, 5, 6, or 7 g/kg. Blood cell harvesting. Mice were placed under halothane anesthesia. Their blood was collected in heparin coated tubes (Becton Dickinson and Company, Franklin Lakes, NJ) by bleeding from the retrorbital plexus 12 h after dosing. Flow cytometric analysis. Blood from each mouse (0.15 ml) was labeled with anti-MHC II FITC (BD Pharmingen, San Diego, CA) and anti-CD45R/B220 (BD Pharmingen, San Diego, CA) by adding 5 μl of each antibody diluted 1/10 in FACS buffer (phosphate buffered saline with 0.1% bovine serum albumin and 0.1% sodium azide pH 7.4). Another blood sample from each mouse was labeled with 5 μl anti-CD4 PE (BD Pharmingen, San Diego, CA) and 5 μl anti-CD8 Cychrome (BD Pharmingen, San Diego, CA) diluted 1/10 in FACS buffer. These samples were allowed to incubate at 4°C in the dark for 30 min. After incubation, RBCs were lysed by adding 8 ml of ammonium chloride buffer (4.13 g NH4Cl, 0.5 g NaHCO3, 0.03 g EDTA per 500 ml water, pH 7.0) warmed to 37°C. The samples were allowed to incubate at 37°C for 10 min. They were then washed with FACS buffer. Following one wash the samples were resuspended in 1% paraformaldehyde (in PBS) and incubated in the dark for 10 min at room temperature. The samples were washed twice using FACS buffer, resuspended, and stored at 4°C in FACS buffer until analyzed by flow cytometry (FAC Calibor BD, Franklin Lakes, NJ) no more than five days after fixation. Spleen cell counts. Each spleen was removed and placed in 3 ml of RPMI (Invitrogen, Carlsbad, CA), which was kept on ice. Frosted slides were then used to press the spleens yielding single cell suspensions in RPMI. The cells were centrifuged at 300 x g for 7 min, resuspended in 3 ml of RPMI 1640, and 20 μl of each cell suspension was added to 10 ml of isoton (Beckman Coulter, Miami, FL) in a counting vial. Three drops of Manual Lyse reagent (J&S Medial Associates Inc., Framingham, MA) were added, and the samples were then counted using a Coulter Z1 counter (Coulter Corporation, Miami, FL). hite blood cell counts. White blood cells were counted by placing 20 μl of whole blood in 10 ml of isoton (Beckman Coulter, Miami, FL). Manual Lyse Reagent (J&S Medial Associates Inc., Framingham, MA) was then added to lyse erythrocytes. The samples were counted using a Coulter Z1 counter (Coulter Corporation, Miami, FL). NK assay. Spleen cells were diluted to 1.0 x 107 cells/ml and were plated in a 96 well v-bottom plate using triplicate samples for at least three effector/target ratios. YAC-1 target cells were labeled with 51Cr (ICN Biomedicals, Irvine, CA), then diluted to 1 x 105 cells/ml and plated in a 96 well plate, and incubated for 4 h at 37°C. A gamma counter (Perkin-Elmer, Wellesley, MA) was then used to measure release of 51CR into culture supernatants, as an indication of NK cell activity. The assay and calculation of lytic units were carried out as described previously (Pruett et al., 1999). Differential counts. Manual differential counts were made using blood smears for each animal. These slide were then stained using a Diff-Quik three step staining kit (Dade Behring Inc., Newark, DE). Cells were then counted by observation under the microscope. At least 100 cells were counted for each sample. Statistical analysis. Statistical analysis was carried out by using Microsoft Excel to first normalize data to control values to facilitate comparison of results from different experiments. Data were then transferred to Prism Graph Pad 4.0 (San Diego, CA) where linear regression models were generated by comparing the change in each immune parameter to the area under the corticosterone AUC value corresponding to the dose that caused the change. These AUC values were obtained in our previously published studies (Pruett et al., 1999, 2000a,b, 2003), which indicate that the values are quite reproducible (Pruett et al., 1999, 2000a,b, 2003). Each regression generated was analyzed using the "runs test" to determine if there was a significant nonlinear component in the regression models. None was detected for any of the data shown. The linear regression models were then compared by evaluating overlap in their 83.7% confidence intervals. These confidence intervals were calculated using StatView software (v4.5 for Macintosh). Overlap of the 83.7% confidence intervals for linear regression models indicate that the values are not significantly different at the p = 0.05 level: lack of overlap indicates significance at the 0.05 level (Barr, 1969; Nelson, 1989). RESULTS Effects of Stress on Blood Lymphocyte and Neutrophil Populations Differential cell counts shown in Figures 1 and 2 demonstrate that increases in plasma corticosterone are associated with a decrease in the number of lymphocytes and an increase in the number of neutrophils present in the blood. These results show that restraint had a smaller effect on the lymphocyte to neutrophil ratio than did atrazine, propanil, exogenous corticosterone, or ethanol. Direct comparisons of the regression lines using Prism 4.0 software indicated that the lines for restraint were significantly different from the corresponding lines for all other treatments. However, there was also considerable variability among these other treatments. For example, ethanol induced a much greater increase in neutrophils than did exogenous corticosterone at similar AUC levels (Fig. 2). Since the percentage of lymphocytes and neutrophils had been examined separately, it was important to assess the effect stress had on the leukocyte population as a whole. It has been reported that the total number of white blood cells in animals decreases when they are subjected to stress (Li et al., 2001). This decrease has been attributed to increases in adrenal hormones including plasma corticosterone levels (Cunnick et al., 1990). Figure 3 shows the relationship between white blood cell number and increasing dosages of each stressor. The slope indicates decreasing WBC counts with increasing dosage of ethanol, atrazine, and corticosterone. However, there were increases in WBC counts with increasing dosages of the stressors restraint and propanil. The slopes for ethanol and corticosterone were similar indicating a similar decrease in WBC count at similar corticosterone AUC values. Effects of Stress on CD4+ and CD8+ T Cells in the Blood Increases in plasma corticosterone levels have been shown to effect T-cell mediated immunity (DePasquale-Jardieu and Fraker, 1980). In these blood studies both helper T-cell (CD4+) and cytotoxic T-cell (CD8+) populations decreased in association with an increase in corticosterone AUC values for all treatments. In Figures 4 and 5 the responses of both CD4+ and CD8+ cells to propanil, atrazine, and ethanol are more similar to exogenous corticosterone than to restraint stress. Restraint had very little effect. These results are quite different from the effects of these agents on CD4+ and CD8+ T cells in the spleen and thymus studies (Pruett et al., 1999, 2000a,b, 2003). Only atrazine had a substantial effect in the spleen, and the percentage of both CD4+ and CD8+ cells increased substantially, concomitant with a decrease in the percentage of B cells (Pruett et al., 1999, 2000a,b, 2003). In the present study all agents except restraint caused substantial decreases in CD4+ and CD8+ T cells in the blood. The decreases were generally similar for similar corticosterone AUC values, but the slope for ethanol was significantly greater than for any other agent, indicating greater effects at comparable corticosterone AUC values and suggesting the possibility of direct effects on these cells or effects caused by neuroendocrine mediators other than corticosterone. Effects of Stress on B-Cell Expression of the MHC II Phenotype in the Blood Expression of major histocompatibility complex class II proteins shown in Figure 6 decreases in association with an increase in corticosterone AUC values for all treatments. MHC II expression is shown as percent MHC II positive cells divided by the percentage of B cells (B220+) multiplied by 100. This function was used to insure that decreases in B-cell numbers, the most abundant MHC II expressing cell type in blood or spleen, were not directly causing the observed decreases in MHC II. Unlike the T-cell population, expression of MHC II proteins shows reasonably consistent quantitative relationships for all stressors indicating that their expression might be a good indicator of stress-induced immunological changes. Before this parameter can be implemented in a predictive model, it was important to examine the MHC II response to cyclophosphamide (Anonymous, 1998). Cyclophosphamide is widely used as a positive control in immunotoxicity studies, and it seemed an ideal agent to confirm that the MHC II/B cell parameter is not suppressed indiscriminately by all immunotoxicants but that it could be relatively specific for chemical stressors. Effects of Cyclophosphamide Compared to Stressors on Plasma Corticosterone Levels and MHC II Expression in the Blood Figure 7 presents plasma corticosterone levels in mice 2 h after treatment with 200 mg/kg of cyclophosphamide. This figure shows that plasma corticosterone levels do significantly increase with this large dosage of CyP. However, as compared with corticosterone levels in mice dosed with high dosages of chemical stressors used in this study, the increase in corticosterone after CyP exposure is quite small (91.58 ± 25.97 ng/ml). The effect that 150 mg/kg and 200 mg/kg of cyclophosphamide had on MHC II expression was determined to be insignificant (p > 0.05). These results are shown in Figure 8. These results indicate that MHC II expression is not suppressed by all immunotoxicants MHC II Expression in the Blood as a Predictor of Suppression of Other Immune Parameters and MHC II Expression To examine MHC II and its viability as a predictor of stress induced immunosuppression, regression lines for the MHC II/B cell parameter in the blood generated from restraint and exogenous corticosterone treated mice were used to develop a predictive model. Numbers predicted by using these two regression equations were compared to actual data points collected throughout the course of the experiments. The ability of the corticosterone and restraint regression equations to predict experimental values was tested by first using the area under the corticosterone curve value at which 50% suppression of MHC II occurred in the restraint regression equation (AUC value of 5471.77 ng/ml?h) and the corticosterone regression equation (AUC value of 3841.87 ng/ml?h). Experimentally observed values for each immune parameter were determined by calculating them from the linear regression equations for propanil, atrazine, and ethanol. The observed values for each immune parameter were determined at AUC values of 5471.77 or 3841.87 ng/ml?h. If the 83.7% confidence interval of the observed values for a parameter overlapped with the 83.7% confidence interval of the value predicted by the restraint or corticosterone regression equation for a parameter at the aforementioned AUC values, this indicated no significant difference (p > 0.05), and the model was deemed predictive for that particular parameter (Barr, 1969; Nelson, 1989). It is commonly assumed that if the 95% confidence intervals for points on different lines do not overlap, this means that they are significantly different at the p 0.05) (Barr, 1969; Nelson, 1989). T he results shown in Table 2 demonstrate that this approach provides an accurate estimate of only one parameter in the blood, WBC number. The restraint and exogenous corticosterone models accurately predict the effect that all the chemicals tested had on WBC number. The corticosterone model predicts the change in the neutrophil and lymphocyte subpopulations after stress induced immunosuppression for two of the three chemicals. The confidence intervals for the third chemicals effect, propanil, on the subpopulations is within 10–20% of overlapping those predicted for the treatment. Quality Control To examine consistency in the amount of stress induced by the treatment used in this study as compared to previous studies, NK cell activity in the spleen was examined using a chromium 51-release assay. This allowed for a comparison of responses seen in these studies to those seen in previous studies of chemical stressor effects on immune parameters in the spleen (Pruett et al., 1999, 2000a,b, 2003). Linear regressions for NK cell activity generated in previous experimentation were compared to those generated in this study. Comparison of NK cell activity linear regressions generated in the present study and in previous studies indicated that there was no significant difference (p > 0.05) between the slopes of the linear regressions generated for each individual stressor. No significant difference in the slopes of the linear regressions indicates that the effects of these stressors are sufficiently consistent to suggest that prediction of stressor effects on immune parameters is feasible. DISCUSSION The system investigated here would provide a tool that would give some insight into whether or not observed immunosuppression was caused by stress or direct immunotoxicity. Normally, determining the role of stress in chemical-induced immunosuppression requires sets of animals dedicated to specialized experiments that are not typically included in safety assessment (Weiss et al., 1996; Wu and Pruett, 1997). The goal of the series of studies, which includes the present one, was to identify patterns of change in particular immune parameters that are predictably associated with stress and which can be easily measured in the same animals used for safety testing. In both the present and past reports one parameter examined, MHC II, had a consistent quantitative relationship to corticosterone AUC values (Pruett et al., 1999, 2000a,b, 2003). Increased plasma corticosterone levels have also been associated with decreases in MHC II expression in studies conducted using peritoneal macrophages and splenic B cells (Weiss et al., 1996; Zwilling et al., 1990). These findings further suggest that corticosterone plays a major role in the regulation of B cells and their immunological capabilities. In addition, there is now substantial evidence that decreased MHC II expression following trauma or surgery is remarkably predictive of risk of infection in humans (Lekkou et al., 2004). Although the ratio of lymphocytes to neutrophils in humans is the inverse of what is seen in mice the MHC II/B220 parameter could still be applicable to a human model since there are still plenty of B cells present for such a calculation. As a result, MHC II expression could be considered a possible biomarker for stress-induced immunosuppression. Such a biomarker would be helpful in risk assessment analysis by indicating that immunosuppression is stress related. The expression of a suggested biomarker for stress-induced immunosuppression would also need to be unaffected by immunotoxicants that do not cause dramatic increases in plasma corticosterone levels. A review of literature indicates that there are no reports of MHC class II suppression by immunotoxicants that are not stressors (indicated by a Medline search using the terms immunotoxic and MHC class II). This was further investigated in this study by using a well-known broad-spectrum immunotoxicant, cyclophosphamide, which had no effect on MHC II expression. Even though MHC II expression seems to correlate well with stress-induced immunosuppression, the present study did reveal limitations to predictive models based on the blood parameters. Previous studies showed that chemical stressors affected immune parameters in spleen and thymus more like restraint stress than exogenous corticosterone (Pruett et al., 1999, 2000a,b, 2003). Differences seen in these studies indicate that the same endpoints in blood are affected differently by the stressors than in the spleen. As compared to restraint stress, exogenous corticosterone and chemical stressors caused more dramatic decreases and increases in the percentages of lymphocytes and neutrophils, respectively. It is conjectured that increases in neutrophil number may act to counter balance some of the immunosuppressive effects of corticosterone (Dhabhar, 2000). Increases in neutrophil number have been associated with an increase in the amount of glucocorticoid in circulation (Miller et al., 1994). It has also been suggested that an increase in glucocorticoid, such as corticosterone, increases both the longevity and rate of production of neutrophils (Fauci and Dale, 1974; Friedman et al., 1995; Mishler, 1977). Since chemical stressors and exogenous corticosterone seem to affect the leukocyte population to a greater extent and plasma corticosterone levels are similar in restrained mice, it can be suggested that restraint stress elicits either the production of other stress mediators or different amounts of these mediators, which may counteract the effects of corticosterone (Ader and Cohen, 1993). Exogenous corticosterone and chemical stressors also cause a more dramatic change in CD4+ cells and CD 8+ cells as compared to restraint stress. Decreases in both CD4+ and CD8+ T-cell subpopulations with increasing plasma glucocorticoid levels did correspond to previously observed decreases in thymus weight and cellularity (Pruett et al., 1999, 2000a,b, 2003). However, the relationship between these decreases and the corticosterone AUC values were more variable among the three chemicals than in previous studies in which these cells were evaluated in the thymus. The data presented here suggest that the blood parameters are more sensitive to exogenous corticosterone and chemical stressors than to restraint stress. The chemicals themselves may also have some direct effect on the cell types examined in the blood. Testing the suitability of MHC II expression on blood leukocytes as a predictive parameter was accomplished by implementing the MHC II/B220 parameter in a predictive model. Comparison of the predictive ability of the blood models showed that using the particular dosage of the stressor at which 50% suppression of MHC II/B220 was observed was more accurate in predicting experimental values for each parameter measured than the corticosterone AUC based model, but it was not quite as effective as models based on decreased MHC II expression in the spleen (Pruett et al., 1999, 2000a,b, 2003). No data yet collected completely explains the differences in predictive value of spleen and thymus parameters as compared to the same parameters in blood. However, results from other studies suggest some of the factors that may be involved. Blood seems to show a greater sensitivity to the differences in stressors. One explanation of difference in the sensitivity of blood, as compared to spleen, may be the extremely dynamic nature of change in blood leukocyte populations in response to stress. Previous studies have shown that both catecholamines and glucocorticoids are involved in these changes (Dhabhar, 2002; Shephard, 2003). The relative amounts of norepinephrine and glucocorticoids may vary following treatment with different chemicals (Pacák et al., 1998), causing substantial changes in leukocyte trafficking (Richter et al., 1996) which may not be reflected in the spleen or thymus. Although it is now clear that stressors can significantly decrease resistance to infection (Cohen et al., 1998, 1999; Kiecolt-Glaser et al., 1996; Vedhara et al., 1999; Zhang et al., 1998), quantitative estimations are not yet possible. To obtain such quantitative predictions of the effect of stressors on host resistance to infection in humans one could employ the parallelogram approach (Loveren et al., 1998). The parallelogram approach is based upon the idea that if the effects of a chemical on immune functions and host resistance are known for an animal model and its effects are known for the same immune functions in humans, then using these three corners of the parallelogram it is possible to extrapolate the fourth, host resistance to infection in humans. However, it is not usually possible to obtain immune function data from humans exposed to chemicals under controlled conditions. If the chemical is immunosuppressive primarily because it induces a stress response, it should be possible to model at least some of the immunotoxic effects of the chemical in humans by administering the major immunosuppressive stress hormone, cortisol, to attain stress inducible cortisol levels (Blazar et al., 1986; Davis et al., 1991; Tonnesen et al., 1987; Vedhara et al., 1999). This would allow extrapolation of an endpoint that would otherwise be unattainable without a predictive model, the effect of chemical-induced stress on resistance to infection. Although the differences between blood, spleen, and thymus are distinct in their reactions to stress, they still share one parameter that is effected similarly, MHC II. Using MHC II expression coupled with observing for a pattern of change in other blood parameters affected by stress (e.g., >the ratio of neutrophils to lymphocytes and the number of WBC) could indicate a stress effect of a chemical. Predicting the stressor's effect on other parameters would be less reliable than hoped (Tables 1 and 2). Nevertheless, identifying MHC II as a reliable biomarker for stress induced immunosuppression in mouse blood has brought the first corner of the parallelogram to completion. REFERENCES Ader, R., and Cohen, N. (1993). Psychoneuroimmunology: Conditioning and stress. Annu. Rev. Psychol. 44, 53–85. Anonymous (1998). Immunotoxicity. EPA: Heath Effects Test Guidelines, 1–11. Dhabhar, F. S. (2002). Stress-induced augmentation of immune function—the role of stress hormones, leukocyte trafficking, and cytokines. Brain Behav. Immun. 16, 785–798. Dhabhar, F. S., Miller, A. H., Stein, M., McEwen, B. S., and Spencer, R. L. (1994). Diurnal and acute stress-induced changes in distribution of peripheral blood leukocyte subpopulations. Brain Behav. Immun. 8, 66–79. Fauci, A. S., and Dale, D. C. (1974). The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Invest. 53, 240–246. Riley, V. (1981). Psychoneuroendocrine influences on immunocompetence and neoplasia. Science 212, 1100–1109. Shephard, R. J. (2003). Adhesion molecules, catecholamines and leucocyte redistribution during and following exercise. Sports Med. 33, 261–284.[ISI] Tonnesen, E., Christensen, N. J., and Brinklov, M. M. (1987). Natural killer cell activity during cortisol and adrenaline infusion in healthy volunteers. Eur. J. Clin. Invest. 17, 497–503....查看详细 (33271字节)
☉ 11120232:Association between baseline radiographic damage a
ABSTRACT Objectives: To identify factors associated with poor physical function in rheumatoid arthritis and to assess whether baseline joint damage has an impact on improvement in physical function during infliximab treatment. Methods: 428 patients with active rheumatoid arthritis despite methotrexate treatment received methotrexate alone or with infliximab (3 mg/kg or 10 mg/kg every four or eight weeks) for 54 weeks (the ATTRACT trial). Data on clinical outcomes and physical function (assessed by the health assessment questionnaire (HAQ)) were collected. Structural damage was assessed using the van der Heijde modification of the Sharp score. Odds ratios (OR) for factors associated with severe functional disability (HAQ 2.0) at baseline were estimated using multiple logistic regression analyses, and baseline factors related to the change in physical function after treatment at week 54 were determined. Results: Baseline radiographic scores were correlated with baseline HAQ scores. After adjustment for demographic characteristics in the logistic regression model, baseline disease activity scores, radiological joint damage, fatigue, and morning stiffness were found to be associated with severe functional disability (HAQ >2.0), with OR values of 2.00 (1.53 to 2.63), 1.82 (1.15 to 2.87), 1.19 (1.05 to 1.34), and 1.07 (1.01 to 1.13), respectively. In multiple linear regression analysis, physical disability, joint damage, and fatigue at baseline were correlated with less improvement in physical function after treatment. Infliximab treatment was associated with greater improvement in physical function. Conclusions: Greater joint damage at baseline was associated with poorer physical function at baseline and less improvement in physical function after treatment, underlining the importance of early intervention to slow the progression of joint destruction. Abbreviations: ATTRACT, anti-TNF trial in rheumatoid arthritis with concomitant therapy; HAQ, health assessment questionnaire Keywords: health assessment questionnaire; physical disability; radiographic damage; rheumatoid arthritis Physical function in patients with rheumatoid arthritis deteriorates progressively throughout the course of the disease, starting with functional limitations and progressing to physical disability if no effective treatment intervention is introduced in a timely manner.1 Approximately 50% of patients with rheumatoid arthritis are expected to experience enough loss of function to cause work disability within 10 years after disease onset.2 Previous studies have shown that multiple factors are associated with functional disability in patients with rheumatoid arthritis, including disease activity, radiographic damage, co-morbidities, and socioeconomic and psychological factors.3–6 The association of radiographic joint damage with physical function may vary with disease phase. In the early stages of rheumatoid arthritis, the link between joint damage and physical function is not well established.7,8,9,10,11 However, in established disease, joint damage is known to be a major determinant of functional disability.4–6 It is not clear whether pre-existing joint damage affects the extent of improvement in physical function that could be achieved with highly effective treatment. Understanding the factors associated with improvement in physical function may have implications for appropriate disease management of this disease. In this post-hoc analysis, data collected from ATTRACT (the anti-TNF trial in rheumatoid arthritis with concomitant therapy) were used to evaluate the association between radiological joint damage and physical function at baseline, as well as the association of baseline radiological damage with improvement in physical function after one year of treatment. METHODS Patient eligibility Patient enrolment criteria and the design of the study have been described in detail previously.12,13 Briefly, patients with rheumatoid arthritis who had active disease despite the use of concomitant methotrexate were randomly assigned to receive methotrexate alone or methotrexate plus one of four infliximab treatment regimens (3 mg/kg or 10 mg/kg every four or eight weeks, after a three dose induction phase with infusions at weeks 0, 2, and 6). Treatment outcomes were collected at four week intervals from baseline to week 54. Clinical and radiographic evaluation Outcomes, including the number of swollen and tender joints and the erythrocyte sedimentation rate (ESR), were collected from baseline to week 54. Patient and evaluator global assessments of disease activity and patient assessments of fatigue were made using a visual analogue scale (ranging from 0 to 10). The disease activity score based on 28 joints (DAS 28), calculated as 0.56*(tender joint count) +0.28*(swollen joint count) +0.7ln(ESR) +0.014*(patient global assessment of disease activity),14 was used as a measure of rheumatoid arthritis disease activity. Physical function was measured using the health assessment questionnaire (HAQ).15 Structural damage of the hands and feet was assessed using the van der Heijde modification of the Sharp score (vdH-Sharp score, ranging from 0 to 440).16 The total vdH-Sharp score was the average of the scores from two blinded readers and is referred to in this paper as the total radiographic score. Statistical analysis Patients with radiographs of both the hands and feet at baseline and week 54 were included in the analysis. Data from patients in both the placebo and infliximab groups were used in the analysis, with the assumption that the impact of baseline factors on improvement in physical function was similar for the two treatment groups. This assumption was tested by including treatment group as a covariate in the regression model. In the statistical model exploring the factors associated with baseline HAQ, multivariate linear regression and logistic regression analyses were employed to adjust for the confounding effect of correlated variables. In the logistic regression analysis, patients were classified into two groups based on the HAQ scores at baseline: HAQ 113) had a significantly higher HAQ score (1.86 v 1.57, p0.05), and more severe disease activity (6.7 v 6.5, p50 (yes or no), morning stiffness (minutes), and fatigue score were used as independent variables. In the final model using stepwise selection, baseline disease activity and radiographic joint damage were the most significant risk factors for severe functional disability at baseline. The odds ratio for severe functional disability for patients with severe joint damage (radiographic score >50) was 1.82 (95% confidence interval (CI), 1.15 to 2.87) (p = 0.01). The odds ratio for severe functional disability for patients with higher disease activity (per unit increase) was 2.0 (1.53 to 2.63) (p<0.01). Fatigue and morning stiffness were also significantly associated with severe functional disability. Men were less likely to have severe functional disability than women. Disease duration was not a significant factor in the model after adjusting for the other factors; therefore it was not incorporated into the model (table 4). Association of baseline joint damage and treatment outcomes The mean per cent improvement in physical function was significantly associated with baseline radiographic scores, with patients in the highest radiographic score quartile having significantly smaller improvements in HAQ than those in the lowest radiographic score quartile (16% v 32%, p<0.01). In contrast, the mean per cent improvement in disease activity, the number of tender joints, and the number of swollen joints was not significantly different among groups defined by radiographic score quartiles (fig 1). As patients in the infliximab plus methotrexate group had greater improvement in physical function in each radiographic score quartile than those in the placebo plus methotrexate group, the correlation of improvement in physical function at week 54 with baseline factors was further analysed using a multiple linear regression analysis, with treatment as one of the covariates. In the final regression model using stepwise model selection, infliximab treatment was significantly correlated with improvement in HAQ score at week 54. Age, baseline HAQ scores, baseline joint damage, and baseline fatigue were also independently associated with the change (improvement) in physical function, regardless of the treatment group assignment (table 5). Greater radiographic joint damage and more severe fatigue at baseline were associated with less improvement in physical function at week 54 (p<0.05). In addition, patients with greater disability at baseline showed a greater improvement in physical function after treatment at week 54 (p<0.01). Baseline disease activity and morning stiffness were not significant factors in predicting improvement in physical function at week 54 after adjustment for other baseline factors; therefore they were not incorporated into the model. DISCUSSION Rheumatoid arthritis is a chronic, progressive, and destructive disorder. Functional disability increases rapidly in affected individuals, such that about half will experience work disability within 10 years of diagnosis.2 Previous studies have shown that multiple clinical and non-clinical variables are associated with functional disability, the most important being disease activity and radiographic joint damage.3,6 In the early stage of the disease, functional ability may be influenced more by disease activity than by radiographic joint damage.7 However, as the disease progresses, joint damage becomes a more dominant factor in determining physical function.6 Clarke et al3 reported that the correlation between radiographic scores and HAQ scores tends to increase with disease duration, from 0.15 after 0 to 5 years of disease onset to 0.42 after 10 to 15 years. The ATTRACT baseline data showed a similar trend, with the correlation between HAQ scores and radiographic scores increasing from 0.14 to 0.20 with an increase in disease duration (<10 years v 10 years). In general, traditional disease modifying antirheumatic drug (DMARD) treatments can slow disease progression but may not prevent the bone and cartilage erosion associated with rheumatoid arthritis.19 Unchecked progression of the disease can eventually lead to functional impairment. Thus controlling only the clinical signs and symptoms may not be adequate to retain the patient’s long term physical function. Understanding and managing the risk factors related to long term functional impairment is critical for better treatment outcomes. In the past decade, joint damage as assessed by radiography has been considered one of the critical measurements in the evaluation of disease severity and treatment efficacy in clinical trials.20 An association has been observed not only between joint damage and severe functional loss, but also between joint damage and loss of employability21 and higher mortality in the rheumatoid population.22 ATTRACT data showed that radiographic joint damage was associated with unemployment, and that patients with a radiographic score of more than 50 had a much lower probability of being employed full time than those with lower radiographic scores.23 As demonstrated in this analysis, baseline radiographic joint damage is related to baseline physical function and is also a significant predictor of improvement in physical function after treatment. Patients with radiographic scores of 20.5 or less had better improvement in physical function (32%) from baseline to week 54 than those with radiographic scores of more than 113 (16%), even though both patient groups had similar improvement in disease activity scores (31% v 27%). The ATTRACT trial was ideally suited to this study, as the range of radiographic scores at baseline in ATTRACT was larger than that of the study populations in other trials. The results of this analysis strongly support the association between radiographic damage and physical disability in patients with established disease. Furthermore, the results indicate that physical disability caused by joint damage from erosions and joint space narrowing is less likely to be reversible at later disease stages. A major limitation of the study was that it was a post hoc exploratory analysis, examining the association between joint damage and physical function. Any conclusions regarding causal relations based on these results should be made with caution. Another limitation was that only patients with moderate to severe rheumatoid arthritis who were inadequately responding to methotrexate were enrolled in the ATTRACT study. Thus caution should be exercised in extrapolating these results beyond this patient population. Conclusions The results of our analysis show that the improvement in physical function after one year of treatment in patients with moderate to severe rheumatoid arthritis was correlated with the degree of radiographic joint damage already evident at baseline. If this indicates the relatively lower reversibility of functional loss resulting from structural damage, then these results underline the importance of early treatment to limit joint damage. ACKNOWLEDGEMENTS The ATTRACT study was sponsored by Centocor Inc. FCB, AFK, RNM, and DvdH have received research support from and served as consultants to Centocor Inc. CH, MB, and DB are employees of Centocor Inc. REFERENCES Kavanaugh AF, Lipsky PE. Rheumatoid arthritis. In: Rich RR, Fleisher TA, Schwartz B, Shearer W, et al, eds. Clinical immunology: principles and practice. St Louis: Mosby Year Books, 1996:1093–116. Wolfe F, Hawley DJ. The long-term outcomes of rheumatoid arthritis: work disability; a prospective 18 year study of 823 patients. J Rheumatol 1998;25:2108–17. Clarke AE, St-Pierre Y, Joseph L, Penrod JT, Sibley MH, Genant HK. Radiographic damage in rheumatoid arthritis correlates with functional disability but not direct medical costs. J Rheumatol 2001;28:2416–24. Bruce B, Fries JF. The Stanford Health Assessment Questionnaire: dimensions and practical applications. Health Qual Life Outcomes 2003;1:20–6. Snedecor GW. Statistical methods. 7th ed. Iowa City: Iowa State University Press, 1980....查看详细 (16923字节)
☉ 11120233:Assessing competencies in rheumatology
ABSTRACT Assessment of competencies in rheumatology is difficult, but possible, and is an important part of the evaluation of practising clinicians, helping to prevent poor performance. Competencies are currently assessed by the Royal College of Physicians, the General Medical Council, and the National Clinical Assessment Authority. Abbreviations: BOF, best of five; DOPS, direct observation of procedural skills; EMQs, extended matching questions; GMC, General Medical Council; MCQs, multiple choice questions; NCAA, National Clinical Assessment Authority; OSCE, objective structured clinical examination; PACES, practical assessment of clinical examination skills; PLAB, Professional and Linguistic Assessments Board Keywords: Competencies; assessment The term competence implies what a doctor should be able to do (knowledge and skills), and performance means what a doctor does in real clinical practice (knowledge, skills, and attitudes, including behaviour). In this review methods of assessment of clinical competence will be outlined, some innovations currently in use in the UK will be discussed, and the assessment of clinical performance in the UK will be reviewed. The theme is that the assessment of competencies in rheumatology is difficult, but possible. The need to assess competencies in UK rheumatologists has come from a requirement to be overt and demonstrate that we have high standards of practice. There has been an increase in public demand for good quality doctors led by problems in Bristol,1 and Alder Hey2 and because of doctors who have come to the attention of the General Medical Council (GMC) in a public manner, such as Harold Shipman and Rodney Ledward. In addition, the Calman Specialist Registrar programmes have resulted in less time for trainee rheumatologists to develop their skills.3 One of the major problems with registrar training has been the implementation of the European working time directive.4 This has meant a reduction in trainee’s working hours in rheumatology.5 Trainees gain less experience on call and in the hospital and have less continuity of care because of working in shifts, resulting in less time for teaching and learning. Can competence be acquired and maintained under these circumstances? The assessment of competence can be divided into assessment of knowledge, assessment of problem solving, assessment of skills, and assessment of attitudes. This was described by Miller, and is referred to as the Miller triangle,6 with the verbal descriptions of "knows knows how shows how does" representing the developmental stages of acquisition of knowledge problem solving skills attitudes and behaviour. To understand assessment it is necessary to have some definitions of assessment terminology. The validity of an assessment is whether the assessment measures what it is supposed to measure. The reliability of an assessment is whether it is a consistent measure and whether the sample of activity that is being assessed is large enough. The practicability of an assessment measures whether the assessment can be carried out with the available resources. TESTS OF KNOWLEDGE The commonest test of knowledge that is used is the multiple true-false multiple choice question (MCQ). These tests allow a large amount of knowledge to be assessed and are reliable7 but not valid as they do not relate to what our candidates do in practice. However, they are relatively easy to administer and require few resources. "MCQs are not valid because they do not relate to practice" Extended matching questions (EMQs) and best of five (BOF) questions are variants of the multiple true-false MCQ that allow problem solving to be assessed in addition to knowledge. In EMQs, a list of possible answers is provided and candidates match the correct answer to the clinical scenarios provided. In BOF style questions, only one of the five options is correct. These question styles reduce the cueing effect of standard MCQs. BOF questions are now the preferred format for MRCP part 1 and 2 written examinations. TESTS OF SKILL Tests of skill should reflect real practice. The traditional unobserved long case examination is highly unreliable and provides an inaccurate picture of candidate strengths, weaknesses and overall competence, especially if only one case is being assessed. Reliability can be increased by assessing more cases, using several examiners, and directly observing the candidate. The objective structured clinical examination (OSCE) was developed by Harden and Gleeson.8 This is a circuit of stations during which students perform standardised tasks and examiners mark against structured rating forms. It is flexible, reasonably valid, reliable, and quite practical. A technique such as the GALS Screen9 is easy to assess using this style of examination.10 The OSCE is a valid and reliable form of assessment provided that there are sufficient numbers of stations. However, it can be a logistical nightmare to administer. Many people are needed to make the examination run smoothly, especially if large numbers of candidates are being examined. Cost effectiveness and the space required are also problems. In postgraduate assessment the OSCE is used for the Professional and Linguistic Assessments Board examination (PLAB), run by the General Medical Council. This is a test of clinical competence in doctors who have not trained in the European Economic Community who wish to practise in the United Kingdom.11 TESTS OF COMPETENCE To assess the competencies of rheumatologists Aeschlimann and colleagues have developed the EULAR multiple choice question game,12 a knowledge and problem solving test. The reliability of this multiple choice questionnaire is high and it has been shown to be helpful in the training and assessment of competence of rheumatologists in Europe. It is feasible to extrapolate knowledge assessments to computer based methods, but there are problems with probity as the identity of candidates is difficult to verify in computer based tests. In an observed long case13 the candidate has standard instructions and takes a history from, and examines, a patient. Examiners observe and do not interact with the candidate and there is a structured marking schedule. Standardised patients can be used14 to ensure consistency in this style of assessment. The structured long case has been developed at Leicester University Medical School in the UK.15 During this procedure the candidates take a history with a standard instruction, and examiners mark according to an objective marking schedule. There is then a targeted viva with questioning relating to the case, usually involving a problem list and management. This method of skills assessment could be easily applied to patients with chronic rheumatic conditions such as rheumatoid arthritis. In postgraduate assessment the OSCE is used for the PLAB examination. It is a 14 station test which is written to a blueprint and integrated between clinical specialties. More than 1000 candidates take it each year16; it includes some rheumatology stations such as examination of a hip. The practical assessment of clinical and examination skills (PACES)17 is the new clinical examination for the MRCP. It is a circuit of five stations and includes observed history taking and observed communication skills, on a standard marking schedule and in addition to clinical system examinations; the examination is computer marked. Detailed feedback is given to candidates who fail. This examination was implemented in June 2001 and has been well received. Other new methods being evaluated by the Royal College of Physicians include 360 degree appraisal, DOPS, and the mini-CEX. DOPS is direct observations of procedural skills—observation of a skill against a standard marking schedule. The mini-CEX is a series of approximately six observed real clinical encounters, which are observed and assessed against standard criteria.18 360 degree assessment is a method whereby a number of colleagues (12 or more) complete a rating form giving an assessment on a verbal scale (for example, 1–10) to describe aspects of communication skills, clinical skills, and behaviour.19 The colleagues included can come from an allied health profession or clinical background. These tests are useful summative methods to assess trainees while they are working in real time in real clinical situations. In addition to assessment, evaluation of rheumatologists’ competence has also been done by appraisal.20 Since December 2000, the Department of Health, BMA, and GMC have recommended that all consultants should be appraised. Appraisal comprises the compilation of a portfolio of activity to include such information as patient satisfaction questionnaires, hospital statistics on outcomes, and evidence of participation in educational activity. Each consultant rheumatologist is expected to have an annual interview where these issues are discussed and recorded and to document a personal and professional development plan. Hospital trusts in the UK are now penalised for not achieving appraisal targets. WHAT COMPETENCIES ARE REQUIRED? How should we decide what specific competencies a rheumatologist needs? There appears to be no clear consensus. In a survey of 173 training centres in Europe,21 there was some harmony in the requirements for a rheumatologist in areas such as the acquisition of clinical experience and knowledge, the ability to manage patients in a cost effective way, and the ability to promote shared decision making. However there was still considerable diversity in areas such as the inclusion of training in electrophysiology techniques, and some more complex procedures such as epidural injection. This should be improved with the development of a European curriculum for rheumatology22 accessible on the EULAR website. To sample the curriculum by assessment, rheumatologists need to devise a blueprint (or a grid of subject areas and competencies) to define which components of competence need to be tested. CURRENT ASSESSMENTS OF PERFORMANCE IN THE UK The most well established assessment of performance in the UK was designed by the GMC. The GMC performance procedures were set up after an Act of Parliament in 1997.23 Before this time doctors could only be removed from the medical register because of poor health or misconduct. Since 1997 poor performance has been used as a reason to remove registration. Performance assessment is by peer review in a two phase process. Phase 1 includes a portfolio of activity, a workplace visit with interviews with colleagues to establish what the doctor is like in his/her own practice, a case note review, and an observation of practice. Phase 2 is a knowledge and skills test, which comprises an extended matching questionnaire (EMQ) covering general medicine and rheumatology, with a section devoted to case histories specifically about rheumatology patients. In the UK one rheumatologist so far has been assessed for performance after a conduct problem. He was removed from the medical register. Peer review failed him in a test of knowledge and peer observation failed him in a skills test. CAN WE DETECT LESS COMPETENT DOCTORS? The GMC has been compiling validation data which suggest that most doctors practise to a similar standard so it is possible to detect the less competent doctors—fig 1 shows box plots demonstrating that the majority of our colleagues’ scores are within the box, with a very small number of outliers. Support for less competent rheumatologists should be provided by a new special health authority the National Clinical Assessment Authority (NCAA).24 This has been set up by the Department of Health to support poorly performing doctors whose problems are not severe enough to be picked up by the GMC. They aim to protect patients by helping the NHS deal with concerns about doctors. The NCAA’s assessment tools are under development and currently being implemented nationally. The authority expects that most referrals to it will be resolved by their support of local procedures. A small number of cases will proceed to an assessment. Assessment includes a knowledge test, a psychological profile of the doctor by occupational psychologists, an occupational health review, a workplace visit, observation of practice, a notes audit, and a clinical skills test. These assessments have been piloted so far, but as yet there is no published information on real cases as numbers are small. "Less competent doctors can be detected" Clinical competence and performance may be assessed most effectively only by repeated direct observation of clinical practice.25 A study in Holland has already implemented this as a research tool. In 2001, eight incognito standardised patients visited 27 Dutch rheumatologists who had agreed to the study, but did not know when the patients would be attending or their clinical diagnosis. Results showed a variation in use of resources—those doctors with longer experience in practice ordered fewer tests. This study has shown that it would be possible to observe actual practice of rheumatologists dealing with patients. This method of assessment could be extrapolated to test the competence of those of us in practice in the UK.26 CONCLUSIONS Assessment is likely to improve a rheumatologist’s competence. Competences can be assessed in rheumatology; they are already being assessed by the College of Physicians, the General Medical Council, and the National Clinical Assessment Authority (NCAA). Assessment of actual practice is feasible, but has not yet happened on a large scale. It is important that we, as a group of professionals, keep abreast of the myriad of regulatory and other bodies that will continue to affect our practice. Prevention of poor performance is clearly our aim. This means that in the UK we should be active participants in keeping up to date. The supposed "no blame" culture developing in the NHS will facilitate more open use of supportive organisations like the NCAA prevent disciplinary procedures or suspension, and referral to the GMC performance procedures. REFERENCES Dyer C. Bristol inquiry condemns hospital’s "club culture". BMJ 2001;323:181. Hunter M. GMC suspends former Alder Hey pathologist. BMJ 2001;322:320. DOH. A guide to specialist registrar training 1998. www.doh.gov.uk/medicaltrainingintheuk/ (accessed 20 October 2004). EC. European Working Time Directive No 93/104/EC of 23 November 1993 concerning certain aspects of the organisation of working time. http://www.incomesdata.co.uk/information/worktime directive.htm (accessed 20 October 2004). Armour B. What competence does a rheumatologist need? Ann Rheum Dis 2000;59:662–7. EULAR. http://www.eular.org/documents/uems_Rheumatology_Specialist_Core_Curriculum_2003.pdf (accessed 20 October 2004)....查看详细 (15066字节)
☉ 11120234:A multicentre, randomised, double blind, placebo c
ABSTRACT Objective: To assess the efficacy of interferon beta (IFN?) in combination with methotrexate in treatment of patients with rheumatoid arthritis. Methods: 209 patients with active rheumatoid arthritis, who had been on methotrexate for at least six months and at a stable dose for four weeks before study entry, were randomised in double blind fashion to receive placebo (0.05 ml or 0.5 ml), IFN? 2.2 μg (0.05 ml), or IFN? 44 μg (0.5 ml), given subcutaneously three times weekly for 24 weeks. The primary efficacy measure was a change in radiological scores at week 24. The secondary endpoint was the proportion of patients who met the ACR 20% improvement criteria at the end of the study. Synovial biopsy specimens were obtained before and after treatment from a subset of patients. Immunohistochemistry was used to detect the presence of inflammatory cells and the results were measured by digital image analysis. Collagen crosslinks were measured in urine at different times throughout the study. Results: Analysis of radiological scores and clinical variable showed no changes in any of the groups, and there were no differences between the groups. On microscopic analysis of synovial tissue there was no significant change in the scores for infiltration by inflammatory cells after IFN? treatment. Urinary levels of collagen crosslinks were unchanged between the treatment groups. C onclusions: At the doses tested, treatment with IFN? three times weekly in combination with methotrexate did not have a clinical or radiological effect in patients with rheumatoid arthritis. Abbreviations: ACR, American College of Rheumatology; CCP, cyclic citrullinated peptide; CIA, collagen induced arthritis; DMARD, disease modifying antirheumatic drug; IFN, interferon; IL, interleukin; ITT, intention to treat; mAb, monoclonal antibody; TNF, tumour necrosis factor Keywords: immunohistochemistry; interferon beta; rheumatoid arthritis Rheumatoid arthritis is a chronic inflammatory disease affecting synovial tissue in multiple joints, leading in most patients to bone destruction and severe morbidity and disability. Studies have shown the need for early diagnosis and early aggressive treatment to help prevent irreversible joint damage.1–3 The prevention of cartilage and bone erosions is an important therapeutic challenge in the treatment of this disease. At present, various disease modifying antirheumatic drugs (DMARDs) are used to control arthritis activity.4 However, DMARDs are only moderately effective and not all patients tolerate them. Therapeutic possibilities have improved with the introduction of agents that block tumour necrosis factor (TNF), although not all patients are responsive to these drugs.5,6 This has motivated the search for additional effective treatments that can reduce inflammation as well as limit bone destruction. Several studies have shown beneficial effects of interferon beta (IFN?) on clinical and MRI measures in relapsing-remitting multiple sclerosis.7–9 The effects of IFN? on the cytokine profile and cell trafficking in patients and models of multiple sclerosis have stimulated studies on its potential for treatment of patients with rheumatoid arthritis, which is also considered to be an immune mediated disease. Immunological functions of IFN? include inhibition of TNF and interleukin (IL) 1? secretion, and enhancement of IL10 and IL1RA production.10–13 Conceivably, concurrent targeting of TNF, IL1?, and other proinflammatory cytokines by the use of a counterregulatory cytokine such as IFN? might be effective in suppressing arthritis activity. Indeed, several studies in animal models of collagen induced arthritis (CIA) have shown a markedly beneficial effect of IFN? treatment. A small study done in four rhesus monkeys with CIA demonstrated clinical improvement and decreased serum levels of C reactive protein after seven days of daily IFN? treatment.14 Furthermore, constitutive expression of IFN? by gene therapy resulted in reduced paw swelling and histological improvement in CIA mice,15 and CIA mice treated with daily injections of murine IFN? had a significant reduction in inflammation and an inhibition of the development of bone erosions.16 The favourable effects on CIA in animal models and the well known safety profile of IFN? treatment in humans motivated us to conduct an open label phase I study in 12 patients with active rheumatoid arthritis.14 Patients were treated with IFN? subcutaneously three times weekly for three months with one of three different doses of fibroblast derived natural IFN? (22 μg, 44 μg, and 66 μg) (Frone?, Serono International). The treatment was generally well tolerated and there was, on average, a significant improvement in clinical outcome measurements. We undertook the present double blind, randomised, placebo controlled phase II study of treatment with IFN?-1a (Serono International) in rheumatoid patients with active disease while on methotrexate to determine whether IFN?-1a is effective in reducing radiological damage and arthritis activity. METHODS Patients Patients over 18 years of age with a diagnosis of rheumatoid arthritis according to the American College of Rheumatology (ACR) criteria17 were eligible for inclusion in the study if the duration of their active disease was more than six months and less than eight years. Patients were also required to have at least eight swollen joints, and to fulfil no less than three of the following criteria: at least eight tender joints; physician’s global assessment of disease activity between 2 and 4 on a five point scale; patient’s global assessment of disease activity between 2 and 4 on a five point scale; serum C reactive protein above 15 mg/dl. Patients were required to have used methotrexate for six months or more, and to have followed a stable regimen (7.5 mg/week) for at least four weeks before study entry. Patients who were on oral corticosteroids (<10 mg/day) or non-steroidal anti-inflammatory drugs (NSAIDs) were required to have been on a stable dose for at least four weeks before enrolment. Patients were required to have adequate bone marrow reserve, liver function, and renal function (haemoglobin 5.5 mmol/l, white blood cell count 3.5x109/l, platelet count 100x109/l; serum bilirubin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase 1.5 times the upper limit of normal values, and serum creatinine 150 μmol/l). Women who were pregnant or lactating were excluded from the study, as were patients who had received biological agents or ciclosporin within the previous six months, had used a DMARD other than methotrexate within 28 days, or had used leflunomide within eight weeks of enrolment. All study participants were hospital outpatients at time of enrolment but were excluded if they were wheelchair bound or bed ridden, had major surgery or joint replacement planned, had a history of cancer in the preceding five years, or had a positive test for anti-dsDNA antibodies. Patients were excluded if clinically significant serious abnormalities were apparent on electrocardiography or chest x ray; if they had had an active severe infection or an opportunistic infection in the three months preceding study entry; had a history of allergy to paracetamol or human serum albumin; or had a history of alcohol abuse. Study protocol Patients were randomised to one of four treatment groups: 2.2 μg (0.05 ml) IFN?-1a, 44 μg (0.5 ml) IFN?-1a, placebo (0.05 ml), or placebo (0.5 ml). All treatments were given by subcutaneous injection three times weekly for 24 weeks. Patients remained on a stable dose of methotrexate. Both the patients and the assessors were blinded to the treatment given. As IFN? side effects are easily recognised, physicians responsible for general patient management, including safety assessments, were different from those responsible for efficacy assessments. Patients gave written informed consent before entering the study and the study protocol was approved by the local medical ethics committee at each of the participating sites. Safety and efficacy analyses The primary efficacy measure was defined as the change from baseline radiological score at 24 weeks of treatment, based on the presumed mechanism of action of IFN? on osteoclasts15,16,18 and in line with previous observations showing that even modestly effective treatments may have a demonstrable protective effect on radiological joint damage after only 24 weeks of treatment.19,20 Anteroposterior radiographs of the hands and feet were scored using the Van der Heijde modified Sharp score21 by two independent blinded observers. Erosions in the feet were scored from 1 to 10, erosions in hands were scored from 1 to 5, and joint space narrowing was scored from 1 to 4. Scores were combined from hand and foot radiographs to provide a total score ranging from 0 to 448, with joint space narrowing scores ranging from 0 to 168 and erosion scores ranging from 0 to 280, with a maximum of 160 for the hands and 120 for the feet. The change in Van der Heijde x ray scores was calculated by the differences between the scores at the end of treatment and the scores on the baseline radiographs Secondary efficacy end points were defined as a 20% improvement in the ACR criteria (ACR20) and a decrease in C reactive protein concentrations. Clinical assessment for disease activity was repeated at baseline, day 15, day 29, and then every four weeks until week 28. This included a 68 joint count for joint swelling and tenderness; physician’s and patient’s assessment of disease activity on a scale from 0 (asymptomatic) to 5 (severe symptoms); assessment of pain by visual analogue scale from 0 (no pain) to 10 (severe pain); quality of life (health assessment questionnaire) from 1 (no disability) to 3 (severe disability); and erythrocyte sedimentation rate (ESR) and C reactive protein measurement. In addition, rheumatoid factor, antibodies against cyclic citrullinated peptide (CCP),22 and anti-IFN? antibodies were measured by enzyme linked immunosorbent assay (ELISA) at screening, at week 12, and at week 24. Safety assessments were completed at every visit by an independent observer, and included an interview, examination of vital signs, inspection of injection sites, and evaluation of current laboratory data. The use of concomitant drug treatment was recorded throughout the study. Arthroscopy Some patients underwent arthroscopy of an inflamed knee joint under local anaesthesia at baseline and at week 24. Patients gave separate written informed consent for this procedure. Arthroscopies, tissue sampling, and storage were carried out as described previously in detail.23 All tissue samples were sent to the AMC, Amsterdam for immunohistochemical staining and digital image analysis. Immunohistochemical analysis Serial sections were stained with the following monoclonal antibodies (mAb): anti-CD68 (EBM11, Dako, Glostrup, Denmark), anti-CD55 (Clone-67, Serotec, Oxford, UK), and anti-CD3 (SK7, Becton-Dickinson, San Jose, California, USA). Sections with non-assessable tissue—defined by the absence of an intimal lining layer—were omitted before analysis. For control sections, the primary antibodies were omitted or irrelevant isotype matched mouse antibodies were applied. Staining was done according to a three step immunoperoxidase method as previously described.24 Digital image analysis The slides were evaluated by digital image analysis. All sections were coded and analysed in random order by an independent observer, who was blinded to the clinical data as described previously.25 Urinary analysis of hydroxypyridinium collagen crosslinks The presence of the collagen hydroxypyridinium crosslinks pyridinoline and deoxypyridinoline in urine is an indication of the breakdown of mature collagen. It has recently been shown that the total amount of pyridinium crosslinks excreted correlates with disease activity in rheumatoid arthritis.26 The urinary excretion of pyridinoline (released primarily from collagens type I and II of bone and cartilage) and deoxypyridinoline (released primarily from collagens type I and II of bone and dentin) was measured at baseline, at week 12, and at week 24. Urinary crosslink levels were investigated using gradient ion-paired reversed phase high performance liquid chromatography.26 Statistical analysis The primary efficacy dataset was defined as all randomised patients for whom there were two sets of evaluable hand and foot x rays (one set for baseline and one set for week 24; patients who withdrew from study between week 12 and week 24 had x rays as soon as possible after the last injection of study drug) and who were not major protocol violators. The intention to treat (ITT) population comprised 208 patients who received at least one dose of study drug. For statistical analysis, the two placebo groups were combined and compared with the groups having IFN?-1a treatment. The results before and after treatment were compared by paired t test. Two non-parametric tests were used: the Kruskal–Wallis test for several group means (comparing clinical assessment and histological scores in more than two treatment groups), followed by the Mann–Whitney U test for comparison of two groups. For the ACR20 response, non-completers of the study were considered to be non-responders. RESULTS Patient characteristics and disposition In all, 209 patients were recruited from 30 centres in 10 countries during an eight month period. Their baseline characteristics are summarised in table 1. The mean (SD) age of the 161 women (77%) and 47 men (23%) was 53 (11.9) years. The median duration of disease was 3.6 years (range 0.5 to 12.5 years); 140 patients (67%) were rheumatoid factor positive and 117 (56%) had antibodies against CCP. All patients had active disease at study entry; 165 patients (79%) were in functional class I or II, 40 (19%) were in class III, and three (1%) were in class IV. There were no statistically significant differences between the treatment groups with regard to dose or duration of methotrexate. All 209 patients were randomised and 208 received treatment (ITT population). The disposition of the patients across the study groups is shown in fig 1. Safety and tolerability Injection site reactions were the most commonly reported adverse events during the study and affected a higher proportion of patients on active treatment than on placebo. Similarly, general disorders including flu-like symptoms, headache, and increased ESR occurred at a higher frequency in the active treatment groups than in the placebo groups (table 2). Aggravated rheumatoid arthritis and raised C reactive protein concentrations were reported in a substantial number of patients in each treatment group, and there was a comparable incidence of respiratory system and gastrointestinal disorders in all treatment groups. A marginally increased incidence of raised liver enzymes appeared to be associated with the administration of IFN? 44 μg. All events were of a mild or moderate nature, and severity was comparable between active and control groups: 10 patients in the IFN? 44 μg group developed liver enzyme elevations of mild severity and six of moderate severity; five patients in the IFN? 2.2 μg group developed elevations of mild severity and two of moderate severity; two patients in the placebo groups developed elevations of moderate severity. More withdrawals were caused by adverse events in the 44 μg group (11 patients) than in the other treatment groups (two patients in the IFN? 2.2 μg group and four in the placebo groups). In the 44 μg group, injection site reactions caused the most adverse event related withdrawals (seven patients); however, in the other groups no withdrawals were caused by injection site reactions. Flu-like symptoms, aggravated rheumatoid arthritis, and increased liver enzyme levels each caused the withdrawal of two patients from the total study population. Two patients tested positive for neutralising antibodies against IFN?-1a at the end of treatment: both were receiving IFN? 44 μg. Clinical efficacy There was no significant reduction in the progression of joint damage associated with treatment with IFN?-1a at either of the doses tested compared with the control groups, as measured by the change from baseline in Van der Heijde x ray scores of the hands and feet (table 3). The 56 evaluable patients in the control group showed a median change of 1 (range –12 to 17), 48 patients in the 2.2 μg IFN? group showed a median change of 1 (range –3 to 12), and the 37 evaluable patients in the 44 μg IFN? group showed a median change of 0 (range –5 to 47). There were no statistically significant differences between the median ACR20 and ACR50 response rates between patients on active therapy and those in the control groups (fig 2). Immunohistochemical analysis Twenty five patients underwent synovial biopsy procedures, of whom 23 had results at baseline and 20 at the end of treatment (only 19 patients had both). The results of the immunohistochemical analysis are shown in table 4. After IFN? 44 μg treatment there was a decrease in the number of CD68+ macrophages and a slight decrease in the number of intimal lining layer macrophages and CD3+ T cells, whereas CD55+ fibroblast-like synoviocytes increased slightly. None of these changes reached statistical significance. The IFN? 2.2 μg treatment group showed a different trend, with an average increase in CD68+ intimal macrophages and CD55+ fibroblast-like synoviocytes and a slight decrease in CD3+ T cells; however, no statistically significant differences were detected when compared with placebo. Collagen crosslinks analysis There were no significant differences in urinary levels of the collagen crosslinks pyridinoline and deoxypyridinoline between patients treated with IFN? and those treated with placebo. Levels of crosslinks were similar before treatment and after treatment in all groups. Thus median (range) pyridinoline concentrations, in nmol/nmol creatinine, were 69 (27 to 233) at baseline and 70 (28 to 234) at week 24 with placebo; 75 (24 to 194) and 76 (30 to 212) with IFN 2.2 μg; and 75 (35 to 254) and 67(39 to 188) with IFN 44 μg. Median (range) deoxypyridinoline concentrations, also in nmol/nmol creatinine, were 17 (7 to 170) at baseline and 16 (5 to 88) at week 24 with placebo; 19 (9 to 113) and 21 (9 to 38) with IFN 2.2 μg; and 18 (6 to 47) and 16 (9 to 65) with IFN 44 μg. DISCUSSION We report the results of a double blind, placebo controlled trial that evaluated the efficacy of subcutaneous IFN?-1a on radiological and clinical variables in patients with rheumatoid arthritis who were concomitantly receiving methotrexate. Treatment with IFN?-1a for 24 weeks was not associated with clinical or radiological improvement, neither was there a statistical change in biomarkers. The absence of improvement in radiological scores after IFN? treatment reported here is in clear contrast to previous in vitro and animal studies. In these studies, IFN? has been shown to partly inhibit osteoclastogenesis and in consequence to reduce the development of erosive disease in CIA models.15,16,18 The discrepancy between the present study and previous animal work might relate to the mode of administration and the difference in IFN?-1a dosages used. In the present study IFN? was given three times weekly, following the regular treatment regimen in multiple sclerosis patients. In contrast, successful preclinical studies were done either with gene therapy, which leads to continuous IFN? release, or with daily IFN? injections at a dose of 2.5 μg/mouse/day. Although IFN? is known to have a short half life, we chose not to use daily injections with higher IFN? concentrations because it was anticipated that this would be less tolerable to the patients. It is possible that more frequent injections, higher dosages, or the use of compounds with a longer half life is required to induce clinically meaningful effects in patients with rheumatoid arthritis. In addition, we cannot exclude the possibility that we were unable to detect a modest protective effect on joint integrity in the light of the relatively short duration of the study. There was a surprisingly high rate of discontinuation in our study. This was most pronounced in the IFN? 44 μg treatment group. The most common reason for discontinuation was lack of efficacy—all treatment groups had similar percentages of drop out for this reason. However, withdrawals caused by adverse events such as injection site reactions and flu-like symptoms were more common in the 44 μg IFN? group than in the other treatment groups. The high rate of withdrawal contrasts with results from placebo controlled trials with IFN?-1a in multiple sclerosis.8 This discrepancy may be explained by a difference in disease pathogenesis or differences in the study populations. The most likely explanation is, however, the use of drug titration at the start of treatment. In trials of IFN? in multiple sclerosis, the study drug is usually titrated over the first month from 20% to 50% and subsequently to full dose of treatment to reduce the incidence of adverse events.8 In the present study patients started treatment with their full dose of IFN?-1a. Previous work has shown that analysis of serial synovial tissue samples in patients with rheumatoid arthritis is likely to reflect the biological effects of the treatment given; for example, patients who received either placebo or unsuccessful treatment with recombinant human IL10 did not show any significant synovial changes.27 In contrast, beneficial clinical effects of anti-TNF therapy are associated with decreased infiltration of synovial tissue by inflammatory cells.28 Consistent with this, our data show that persistent disease activity is associated with unchanged synovial inflammation in serial biopsy specimens in IFN? treated rheumatoid patients. An earlier study suggested a modest reduction in CD3 positive T cells after one month of IFN? treatment in synovial tissue of 11 rheumatoid patients. However, it was noted in the same study that the number of CD3 positive T cells returned to baseline levels after three months of treatment.11 In conclusion, the results of this study show that there was no apparent reduction in the progression or activity of rheumatoid arthritis compared with placebo when methotrexate treatment was supplemented with IFN?-1a in doses of either 44 μg or 2.2 μg over 24 weeks. ACKNOWLEDGEMENTS We are pleased to acknowledge the assistance and cooperation of our colleagues. We also thank the following investigators and their colleagues: B Bresnihan, Dublin, Ireland; H Dinant, Amsterdam, Netherlands; B Dijkmans, Amsterdam, Netherlands; P Emery, Leeds, UK; M Kraan, Amsterdam, Netherlands; R Inman, Toronto, Canada; C Jackson, Salt lake City, USA; J Kaltwasser, Frankfurt, Germany; E Keystone, Toronto, Canada; P Maddison, Bath, UK; W Maksymowyc, Edmonton, Canada; B Haraoui, Montreal, Canada; M Molla, Madrid, Spain; B Kidd, London, UK; N Wei, Maryland, USA; M Molloy, Cork, Ireland; L Moreland, Birmingham, Alabama, USA; R Rau, Ratingen, Germany; R Rodriguez, Sevilla, Spain; D van Schaardenburg, Amsterdam, Netherlands; M Smith, Melbourne, Australia; K Vos, Amsterdam, Netherlands; B Williams, Cardiff, UK. REFERENCES Emery P. Therapeutic approaches for early rheumatoid arthritis. How early? How aggressive? Br J Rheumatol 1995;34 (suppl 2) :87–90. Triantaphyllopoulos KA, Williams RO, Tailor H, Chernajovsky Y. Amelioration of collagen-induced arthritis and suppression of interferon-gamma, interleukin-12, and tumor necrosis factor alpha production by interferon-beta gene therapy. Arthritis Rheum 1999;42:90–9. Van Holten J, Reedquist K, Sattonet-Roche P, Smeets TJ, Plater-Zyberk C, Vervoordeldonk MJ, et al. Treatment with recombinant interferon-beta slows cartilage destruction and reduces inflammation in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis Res Ther 2004;6:R239–49. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, et al. RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta. Nature 2002;416:744–9....查看详细 (24792字节)
☉ 11120235:A good response to early DMARD treatment of patien
ABSTRACT Objective: To describe the frequency and duration of remission in the Utrecht rheumatoid arthritis cohort of patients followed since diagnosis, and the clinical and treatment characteristics of patients with remission v those without. Methods: In 1990 the Utrecht rheumatoid arthritis cohort study group started a clinical trial in which patients with recent onset of rheumatoid arthritis (50% improvement on three of four variables) at the one year follow up were compared with non-responders. Baseline values did not differ between the responder group and the non-responder group, except for age and ESR in the methotrexate group. In the group of patients who received im gold at baseline, 55% of the responders (n = 67) and 16% of the non-responders (n = 57) were in remission at some assessment point in the subsequent three years (p<0.001). In the methotrexate group these percentages were 51% of 63 patients and 13% of 60 patients (p<0.001), respectively; in the hydroxychloroquine group, they were 52% of 48 patients and 18% of 78 patients (p<0.0001), and in the pyramid group they were 67% of 15 and 19% of 37 patients (p<0.001). This indicates that despite similar baseline characteristics and similar treatment, patients are more likely to achieve remission when they were defined as responders at one year, as one would expect. For determining predictors of remission, complete baseline data were available on 397 patients. Baseline predictors of remission were a good response to treatment, less pain, absence of rheumatoid factor, and lower joint score (table 4). DISCUSSION In this cohort of patients with rheumatoid arthritis followed since diagnosis, we show that 36% of 562 patients achieved at least one period of remission during follow up and that patients were in remission for 39% of their follow up time. The latter percentage is slightly higher than found in the study by Eberhardt and Fex,4 where 62% of the patients received DMARDs and the patients remained in remission for approximately one third of their follow up time; however, these investigators used the ACR criteria for remission. In other studies using the ACR remission criteria, or a modified version of it, the percentage of patients in remission has ranged from 15% to 20%.2–5 The higher remission rate of 36% in our study might partly be explained by the difference in the definition of remission, along with differences in study design and duration of follow up. For reasons of protocol, we used a revised definition of remission, as proposed by Scott et al,7 in which we had to include the Thompson joint score. The Thompson joint score has, however, been proven to be of similar value to other joint counts in measuring disease activity.19 Although the patients followed in this study were participating in a randomised clinical trial during the first two years after disease onset, we believe they were representative of the general rheumatoid arthritis population attending outpatient clinics, because all patients with recent onset of disease and visiting one of the participating hospitals were asked to participate. Moreover, while the baseline values of the patients who did not wish to be randomised tended to be slightly better than in the study groups, the difference was not significant except for the laboratory variables.13 The values for the percentage of remission over time and the duration of remission relative to the follow up time suggest that remission does not persist for a prolonged period. It has been found that patients in remission who continue to receive second line drug therapy have a lower cumulative incidence of flares (22%) than patients who continue to receive placebo (38%),20 and this, together with our results, indicates that it would be better to continue treatment for a while when patients are in remission. Interestingly, patients who received im gold at study onset had a shorter delay until the first remission period compared with methotrexate (15 v 18 months), though it is generally thought that gold is slower to act than methotrexate. An explanation might be that the percentage of patients receiving im gold who were then changed to the second DMARD because of adverse events was greater than the percentage receiving methotrexate, and that the time to change of medication was shorter for the im gold group. This has also been observed in other studies.21–23 Overall, the percentage of patients remaining in remission was much lower than the target figure for treating rheumatoid arthritis. Biological agents are now available and their initial efficacy appears very promising.24–28 However, treatment strategies with conventional DMARDs can be improved considerably (for example with higher doses of methotrexate, up to 30 mg/week, which has greater efficacy). Furthermore, conventional DMARDs probably remain the rheumatologists’ first choice because of the lack of availability of biological agents in certain countries and their high cost. In this study, remission was more likely to occur in patients with a good response to the initial treatment strategy, in those who were initially rheumatoid factor negative, and in those with less pain and a lower joint count at baseline. In several other studies the absence of rheumatoid factor was also found to be associated with an increased probability of remission,4,8 while rheumatoid factor positivity is associated with radiological damage.29–31 Not many studies have estimated the influence of treatment as an independent predictor of remission. In a two year follow up study comparing combination therapy with single drug therapy, the combination treatment regimen was found to be the only variable predicting remission after two years.32 Although time until the first remission period tended to differ between the four assigned treatment groups at baseline, the kind of DMARD at baseline did not predict remission. We did find that the frequency of remission after one year was significantly higher among responders than among the non-responders. Conclusions After a mean follow up duration of 62 months, only 36% of the patients had fulfilled the remission criteria at least once. Good response to treatment, less pain, a negative rheumatoid factor test, and a lower joint count at baseline were predictors of remission, but not the allocated first drug. It thus seems that a good response to treatment during the first year is linked to the likelihood of going into remission rather than to the type of initial treatment given. This suggests that treatment should be tailored to the individual patient and that we should aim for a rapid response using aggressive treatment strategies such as higher doses of methotrexate. FOOTNOTES We would like to thank all the other participating rheumatologists of the Utrecht Rheumatoid Arthritis Cohort study group; C van Booma-Frankfort (Diakonessenhuis, Utrecht), E J ter Borg (Antonius Hospital, Nieuwegein), A H M Heurkens (Meander Medical Centre, Amersfoort), D M Hofman (Hilversum Hospital, Hilversum), A A Kruize (UMC Utrecht, Utrecht), M J van der Veen (St Jansdal, Harderwijk), and C M Verhoef (Flevo hospital, Almere), Netherlands. This study was supported by a grant from The Dutch Arthritis Association REFERENCES Pinals RS, Masi AT, Larsen RA. Preliminary criteria for clinical remission in rheumatoid arthritis. Arthritis Rheum 1981;24:1308–15. Prevoo ML, van Gestel AM, van’t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL. Remission in a prospective study of patients with rheumatoid arthritis. American Rheumatism Association preliminary remission criteria in relation to the disease activity score. Br J Rheumatol 1996;35:1101–5. van Jaarsveld CH, ter Borg EJ, Jacobs JW, Schellekens GA, Gmelig-Meyling FH, Booma-Frankfort C, et al. The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis. Clin Exp Rheumatol 1999;17:689–97. M?tt?nen T, Hannonen P, Leirisalo-Repo M, Nissila M, Kautiainen H, Korpela M, et al. Comparison of combination therapy with single-drug therapy in early rheumatoid: a randomised trial. Lancet 1999;353:1568–73...查看详细 (24156字节)
☉ 11120236:The Effect of a Brominated Flame Retardant, Tetrab
ABSTRACT This study investigates the effects of one of the most frequently used brominated flame-retardants (BFR), tetrabromobisphenol-A (TBBPA), on formation of reactive oxygen species (ROS) and calcium levels in human neutrophil granulocytes. TBBPA enhanced ROS production in a concentration-depended manner (1–12 μM), measured as 2,7-dichlorofluorescein diacetate amplified (DCF) fluorescence. The results on ROS production by TBBPA was confirmed by lucigenin-amplified chemiluminescence. The TBBPA induced formation of ROS was due to activation of respiratory burst, as shown by the NADPH oxidase inhibitor DPI (10 μM). TBBPA induced activation of respiratory burst was also inhibited by the MEK 1/2 inhibitor U0126 (10 μM), the PKC inhibitor BIM (0.25 μM), and the tyrosine kinase inhibitor erbstatin-A (25 μM). We also found a small reduction in ROS formation in the absence of extracellular calcium and when verapamil was added. The phosphorylation of ERK 1/2 was confirmed by Western blotting. TBBPA also induced a concentration dependent increase in intracellular free calcium measured with Fura-2/AM. We suggest that exposure of human neutrophil granulocytes to the brominated flame retardant TBBPA leads to an activation of the NADPH oxidase primarily by an ERK 1/2 stimulated pathway. The data also show that PKC, calcium, and tyrosine kinases may be involved in the activation Key Words: brominated flame-retardants (BFR); tetrabromobisphenol-A; neutrophil granulocytes; reactive oxygen species (ROS); MAP kinase pathway; calcium; extracellular signal-regulated kinase (ERK). INTRODUCTION Brominated flame-retardants (BFRs) are a large group of compounds widely used to protect various products, such as plastics, textiles, and electronic equipment from catching fire. Several of the BFRs are persistent and lipophilic compounds. They may bioaccumulate and are thus regarded as a potential environmental health problem (de Wit, 2002). Within this group we find compounds such as polybrominated diphenyl ethers (PBDE), tetrabromobisphenol-A (TBBPA), hexabromocyclododecane (HBCD), and polybrominated biphenyls (PBBs). The phenolic TBBPA is industrially the most important individual BFR used with an annual demand of approximately 120,000 metric ton (de Wit, 2002). TBBPA is a phenolic compound primarily used as a chemically bound flame retardant, which is supposed to limit its spread in the environment and reduce its accumulative properties. However, studies have shown that this compound may leak from treated products (Sellstrom and Jansson, 1995) and several recent reports on TBBPA in human and wildlife samples have shown the presence of this compound. Human TBBPA serum levels were measured by Thomsen et al. (2001), who found TBBPA in blood serum from electronic dismantlers, circuit board producers, and laboratory personnel. TBBPA has also been reported to be a contaminant in sediments and mussels (Saint-Louis and Pelletier, 2004; Watanabe et al., 1983) and in eggs from predatory bird species (Berger et al., 2004). The majority of studies conducted to date on BFRs, both with regards to toxicology and environmental levels, have focused on the PBDEs (de Wit, 2002). There is little knowledge about the toxicity of TBBPA, which is of concern considering its extensive use and presence in the environment. A few toxicological studies have been carried out and the TBBPA can elicit thyroidogenic and estrogenic-like activity in vitro (Darnerud, 2003; Kitamura et al., 2002; Meerts et al., 2000), and has a neurotoxic potential (Mariussen and Fonnum, 2003; Reistad et al., 2002). A recent work by Fukuda et al. (2004) showed that TBBPA induces polycystic lesions in the kidney of exposed newborn rats. Pullen et al. (2003) showed that TBBPA suppresses the induction of interleukin-2 in murine splenocytes in vitro, indicating an immunotoxic potential. Human neutrophil granulocytes play a key role in host defenses against invading pathogens and are major effectors of the acute inflammatory reactions. In response to a variety of agents, neutrophils release large quantities of superoxide anion () in a phenomenon known as respiratory burst. Neutrophil production of is dependent on the NADPH oxidase, a multicomponent membrane-bound enzyme that catalyzes NADPH-dependent reduction of oxygen to (Babior, 1999). Superoxide is rapidly converted to hydrogen peroxide (H2O2), either spontaneously or enzymatically by superoxide dismutase. H2O2 is then reduced to water by catalase or converted to hypochlorous acid (HOCl) by myeloperoxidase (MPO). H2O2 can also be converted to hydroxyl radicals in the presence of transition metal ions. Inappropriate activation of respiratory burst is associated with tissue injury and impairment of the ability to clear invading microorganisms (Labro, 2000). Previously a correlation has been found between wildlife animals' exposure to environmental contaminants, such as polychlorinated biphenyls (PCB) and methyl mercury, and effects on immune parameters. Some of these findings have been attributed to activation of neutrophil granulocytes in vitro (Duffy et al., 2002; Sweet and Zelikoff, 2001; Voie et al., 1998). Because of the high production volume of TBBPA, its presence in biotic samples and the close resemblance to other environmental contaminants, we have examined its effect on human neutrophil granulocytes. MATERIALS AND METHODS Chemicals. Tetrabromobisphenol-A (TBBPA, BA-59P, Great Lakes, Lot nr 6L16,C) was all obtained from Promochem (Stockholm, Sweden). Stock solutions were prepared by dissolving the compounds in DMSO. The final DMSO concentration in the samples was always less than 1%. Bisphenol-A, bisindolylmaleimide (BIM), bromphenol blue, ponceau S concentrate, bis-N-Methylacridinium-nitrate (Lucigenin), 2,7-dichlorofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), 2-merchaptoethanol, diphenyleneiodonium (DPI), diethyldithio-carbamic acid (DDC), EGTA, verapamil hydrochloride, cyclosporine A (CSA), methanol, phosphate-buffered saline (PBS), phorbol 12-myristate 13-acetate (PMA), SB203580, superoxide dismutase (SOD), and sodium dodecyl sulphate (SDS) were all from Sigma-Aldrich (St. Louis, MO). U0126 was obtained from Promega Corporation (Madison, WI). Hanks Balanced Salt Solution (HBSS) and HEPES buffer were purchased from GibcoBRL (U.K.). Lymphoprep was purchased from Nycomed Pharma (Oslo, Norway). Enhanced chemiluminescence (ECL) reagent was from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Monoclonal mouse anti-phospho-ERK antibody (Tyr204) and polyclonal rabbit anti-ERK2 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated rabbit-anti-mouse antibody and HRP-conjugated goat-anti-rabbit antibody were purchased from DAKO A/S (Glostrup, Denmark). 2,5-Dihydroxymethylcinnamate (Erbstatin-A), FK-506 (Tacrolimus), and Fura-2/AM were from Calbiochem Novabiochem Corp. (San Diego, CA). All other reagents used were analysis grade laboratory chemicals from standard commercial suppliers. Isolation of human neutrophil granulocytes. Human venous blood was obtained from nonsmoking healthy adult male volunteers in the morning. Neutrophil granulocytes were separated from EDTA blood by dextran sedimentation followed by a standard density-gradient centrifugation as previously described (Boyum et al., 1991). In brief, EDTA blood from individual donors (30 ml) were mixed with 3 ml 6% dextran and left for sedimentation at room temperature for 30 min. The supernatant, containing the granulocytes, was subject to Lymphoprep density gradient centrifugation at 600 x g for 15 min. The pellet was washed in 0.9% NaCl and then resuspended in 7 ml 0.83% NH4Cl in 7 min for lysis of the erythrocytes, and then centrifuged for 7 min (600 x g). This was repeated if not proper lysis was obtained. Cells were then resuspended in HBSS and the number of granulocytes was determined in an AVIDA 60 hematology system. The cells were kept on ice (approximately 4°C) until use. Lactate dehydrogenase (LDH) assay. Leakage of LDH was assessed as an index of cell injury (Koh and Choi, 1987). The measurements were performed as described elsewhere (Ring and Tanso, manuscript in preparation). In brief, cells (2 x 106/ml) were exposed to BRF for the indicated times (5 or 30 min). Cells were then spun down and supernatant from each sample was transferred to sample tubes and stored at 4°C until measured (usually within 2 h). LDH measurements were performed by transfer of 100 μl aliquots of the supernatant to the wells of a custom made 48 well microplate with glass bottom and the volume was adjusted to 425 μl with 0.1 M KPO2 buffer (pH 7.5). The reactions were started by automated injection of 50 μl of an 846 μM stock solution of NADH (final concentration 84.6 μM) followed by automated injection of 25 μl of a 13.6 mM stock solution of pyruvate (final concentration 0.68 mM). The LDH activity was measured, using a BMG FLUOstar Optima fluorimeter, from the decay rate of NADH fluorescence for 30 min at 28°C. The LDH activity was calculated off line and is given as the rate constant of the decrease in fluorescence emission at 460 nm (excitation wavelength 340 nm). The LDH activity (fluorescence units/s) is not a direct measure of the number of dead cells but it gives a qualitative measure of the relative amount of cell necrosis. 100% cell death was estimated by administration of 0.01% triton and corresponded to a NADH fluorescence decay rate of approximately 95 units/s (control values, 11 units/s). In Table 1 the values are shown as % of triton ± SEM. Assay for measuring reactive oxygen species by DCF-fluorescence. Formation of ROS was measured with use of the fluorescent probe DCFH-DA. The method is based on the incubation of the granulocytes with DCFH-DA, which diffuses passively through the cellular membrane. Intracellular esterase activity results in the formation of DCFH, which emits fluorescence when oxidized to 2', 7'-dichlorofluorescein (DCF). The fluorescence emitted by DCF reflects the oxidative status of the cell and was determined essentially as described previously (Myhre et al., 2000). Briefly, the cells (final concentration 2 x 106/ml suspension) were incubated with DCFH-DA (5 μM) in HEPES-buffered (20 mM) HBSS (CaCl2 1.26 mM, KCL 5.37 mM, KH2PO4 0.44 mM; MgCl2 0.49 mM, MgSO4 0.41 mM, NaCl 140 mM, NaHCO3 4.17 mM, Na2HPO4 0.34 mM) with glucose (5 mM) at 37°C for 15 min. Following centrifugation, the extracellular buffer with DCFH-DA was exchanged with fresh buffer and the suspension was mixed gently. The cells (2 x 106/ml, 125 μl) were transferred to 250 μl wells (microtiter plate reader, 96 wells) containing 125 μl buffer with the BFR and/or the different inhibitors. Fluorescence was recorded in a Perkin-Elmer LS50B luminescence spectrometer (excitation wavelength 485 nm, emission wavelength 530 nm) at 37°C for 60 min. Results are calculated as area under the curve (AUC) and presented as values relative to control (% of control). PMA (1 x 107 M) was included as a positive control in all experiments (n = 5–8). Assay for measuring reactive oxygen species by lucigenin-amplified chemiluminescence. Lucigenin chemiluminescence was used to detect in neutrophil granulocytes. The reaction mixture (250 μl) contained 0.1 mM lucigenin, 2 x 105 cells and different concentrations of the compounds. CL was measured by a Labsystem Luminoskan luminometer at 37°C for 60 min. PMA (1 x 10– 7 M) was included as a positive control in all experiments (n = 5–7). The cells and reagents were prepared in HEPES-buffered (20 mM) HBSS with 5 mM glucose. When calcium free buffer was used, 2 mM EGTA was also added. The reaction was started by adding 100 μl of the cell suspension to each well. Results are calculated as AUC and presented as values relative to control (% of control). Western blotting. The neutrophils were isolated as described above and incubated at 37°C for 30 min before stimulation. TBBPA was added in different concentrations (6–24 μM) and the cells were incubated for different time-spans (2–20 min). After the indicated incubation period the cells were added 500 μl ice-cold PBS and immediately lysed in sample buffer (final concentration 3% SDS, 5% glycerol, 62.5 mM Tris/HCl pH 6.9, 0.1% bromphenol blue, 6% ?-Mercaptoethanol). Total cell samples were heated for 5 min at 95°C and analyzed on a 3% stacking 12% separating SDS-PAGE gel (2 h at 90 V). The separated proteins were then electrophoretically transferred to nitrocellulose membranes (0.45 μm) overnight (30 mA), and stained with Ponceau S to confirm complete transfer. The nitrocellulose blots were incubated in blocking buffer (Tris-buffered saline containing 0.05% Tween 20 [TBST] and 5% low-fat dry milk) for 1 h and probed with monoclonal anti phospho-ERK 1/2 primary antibody (1:200 dilution in blocking buffer) for 1 h. The blots were washed in TBST (6 x 5 min) and then incubated with peroxidase-conjugated rabbit anti-mouse secondary antibody (1:1000 dilution in blocking buffer) for 1 h. After washing in TBST (6 x 5 min), the blots were developed with Amersham's ECL system according to instructions provided by the producer. The signals were visualized on X-OmatBlue XB-1 film (Kodak). The experiments were repeated at least three times. The membranes were then stripped in 100 mM ?-Mercaptoethanol, 2% SDS, and 62.5 mM Tris/HCl (pH 7.6) for 30 min at 50°C and proceeded again with a rabbit polyclonal anti-ERK-2 primary antibody and peroxidase-conjugated goat anti-rabbit secondary antibody to confirm equal amounts of protein in each well. Measurement of intracellular free calcium in granulocytes. Intracellular free [Ca2+] was measured by using the fluorescent Ca2+-binding probe fura-2/AM by the method previously described (Grynkiewicz et al., 1985). An increase in Ca2+ concentration is indicated by an increase in the fluorescence excitation ratio (I340/I380). Granulocytes (4.5 x 106 cells/ml) in HBSS containing 20 mM HEPES and 5 mM glucose were incubated at 37°C with 5 μM Fura-2/AM for 20 min. The cells were washed and resuspended in HEPES-buffered HBSS with glucose. Measurements of Fura-2 mediated fluorescence was performed on a computerized Shimadzu RF-5301PC Spectrofluorophotometer, using excitation wavelength ranging between 340 and 380 nm and emission wavelength 510 nm. All data are results of 5–9 separate measurements. Statistical analyses. Differences between controls and treated groups were evaluated using a two-way Student's t-test (paired, two tail distribution), or by one-way ANOVA followed by Dunnett's 2-sided Post Hoc test. The calculations were performed using SPSS 11.5. RESULTS The Effect of BFRs on Human Neutrophil Granulocytes Relatively low concentrations of the brominated flame retardant TBBPA induced a concentration dependent increase in DCF fluorescence in human neutrophil granulocytes (Fig. 1). TBBPA induced formation of ROS in the granulocytes was confirmed by lucigenin (Fig. 2). DMSO, which was used for dilution of the test compounds, reduced ROS formation in unstimulated cells to 84 ± 6% (mean ± SEM) of the control in the DCF assay and to 97 ± 7% (mean ± SEM) of the control in the lucigenin assay. The neutrophils were also exposed to bisphenol-A, which is a non-brominated analog to TBBPA. Bisphenol-A induced a concentration dependent increase in DCF-fluorescence, but had no effect on the lucigenin assay (Fig. 1). The effect of TBBPA on respiratory burst in combination with the polychlorinated biphenyl congener 153 (PCB153) was also investigated to indicate potential mixture effects. PCB153 is generally the dominating toxicants found in environmental samples and is shown to induce respiratory burst in granulocytes (Voie et al., 1998). PCB153 (10 μM) induced ROS in the lucigenin chemiluminescence assay as previously reported. The combination of 10 μM PCB153 with 2 μM TBBPA induced an additive effect (Fig. 3). PCB153 (10 μM) had no effect on the DCF assay, and a mixture of 2 μM TBBPA did not influence the effect of TBBPA on the DCF assay alone (data not shown). The cytotoxicity assay shown as LDH-leakage showed a small, but significant solvent (DMSO) effect both at 5 and 30 min of exposure. The test compounds had no effect at the concentrations tested (Table 1). The Involvement of the NADPH Oxidase, Superoxide Dismutase, Protein Kinase C, Tyrosine Kinase, and Calcium in TBBPA Induced ROS Formation For mechanistic studies we have used inhibitors of different intracellular signaling pathways. The concentration of the inhibitors is based on what is used in the literature. In each assay used in the mechanistic studies we chose the concentration that gave the highest ROS response which for lucigenin was 4 μM TBBPA and for DCF 12 μM TBBPA. Incubation of the granulocytes with 4 μM of the NADPH oxidase inhibitor DPI (O'Donnell et al., 1993) reduced TBBPA induced ROS formation by 60% with the DCF assay and gave total inhibition with the lucigenin assay. This indicates that activation of the NADPH oxidase complex is involved in the ROS formation induced by TBBPA (Figs. 2 and 4). The superoxide dismutase inhibitor DDC (Misra, 1979) attenuated the TBBPA induced DCF response almost completely, whereas the lucigenin chemiluminescence on the contrary was increased, demonstrating that TBBPA induce superoxide anion radical formation. Addition of superoxide dismutase (50 U/ml) showed an almost total inhibition of TBBPA induced ROS formation in the lucigenin assay and had no effect in the DCF assay. This shows that lucigenin measure extracellular ROS while DCF measure intracellular ROS. Incubation of the granulocytes with 0.25 μM BIM, a specific PKC inhibitor (Bit et al., 1993), reduced the DCF-fluorescence by 69%, and the lucigenin response by 28%. The TBBPA induced response was also strongly inhibited by 25 μM erbstatin-A, an inhibitor of tyrosine kinases (Kawada et al., 1993). The inhibition was 82% relative to the control in the DCF assay and total inhibition in the lucigenin assay. Calcium free buffer and the voltage-dependent calcium channel blocker verapamil (Hille, 1992) reduced TBBPA induced DCF fluorescence by 76 and 61%, respectively and the TBBPA induced lucigenin chemiluminescence by 69 and 9%, respectively. The mitochondrial transition pore blocker CSA (1 μM), used to indicate release of ROS from mitochondria (Baysal et al., 1991), had no inhibitory effect (data not shown). The Involvement of ERK 1/2 in TBBPA Induced ROS Formation To test the possible role of the MAP kinase pathway in neutrophil ROS production, the granulocytes were exposed to TBBPA in combination with the MEK 1/2 inhibitor UO126 (Favata et al., 1998). U0126 (1, 3, 6, and 10 μM) reduced TBBPA induced DCF fluorescence in a concentration-depended manner (Fig. 5), and 10 μM of this compound inhibited the respiratory burst completely. Similarly, U0126 (10 μM) also inhibited TBBPA induced lucigenin chemiluminescence by 44% (Fig. 2). We also tested inhibitors of the p38- and Jun-kinase branches of the MAP kinase pathway. The p38 mitogen activated protein kinase inhibitor SB203580 (Cuenda et al., 1995) and the Jun-kinase inhibitor FK506 (Matsuda et al., 2000) made no decrease in the DCF fluorescence when the granulocytes were exposed to 12 μM TBBPA in combination with 1 μM inhibitor. Phosphorylation of ERK 1/2 To investigate the phosphorylation state of ERK 1/2, we used phospho-specific antibodies that recognize the phosphorylated Tyr204 of ERK 1/2. Dose-response experiments showed that cells exposed for 6, 12, 18, and 24 μM TBBPA for 4 min induced a concentration depended activation of ERK 1 and 2. This is demonstrated by a large increase in the relative intensities of the immunodetectable bands (44 kDA and 42 kDA, respectively) compared to basal levels (Fig. 6A). In a parallel set of immunoblots the time-response effect of ERK 1/2 phosphorylation was determined. The granulocytes were exposed to TBBPA (12 μM) for 2, 4, 6, 12, 15, and 20 min (Fig. 6B). The phosphorylation of ERK 1/2 was most pronounced after 2–6 min. We exposed the cells to 12 μM TBBPA for 4 min in combination with 10 μM of the MEK 1/2 inhibitor U0126. The pretreatment almost completely removed the immunodetectable bands for ERK 1 and 2. Pretreatment with the tyrosine kinase inhibitor erbstatin-A had no effect on the TBBPA induced phosphorylated ERK 1 and 2, while the PKC inhibitor BIM and verapamil reduced the immunodetectable bands (Fig. 6C). Intracellular Calcium Measurements with Fura-2/AM Human neutrophil granulocytes were exposed to different concentrations of TBBPA and bisphenol-A. Changes in [Ca2+]i were measured using the membrane permeable Ca2+-binding fluorescent probe Fura-2/AM. TBBPA (1–20 μM) increased intracellular free calcium in a concentration dependent manner (Fig. 7A). At 20 μM bisphenol-A also significantly increased [Ca2+]i. Cells exposed to 10 μM TBBPA in calcium free buffer containing 2 mM EGTA induced a small but significant increase in intracellular calcium (Fig. 7B). DISCUSSION The results presented in this study demonstrate that the BFR, TBBPA, activates respiratory burst in human neutrophil granulocytes as shown with DCF fluorescence and lucigenin-amplified chemiluminescence. This is an important finding, as TBBPA are used extensively for a large variety of applications and can be detected in wildlife and humans (de Wit, 2002). Bisphenol-A, a non-brominated analog of TBBPA, also induced ROS, but with lower potency than TBBPA, both on molar and weight basis. Formation of ROS in granulocytes may be induced by different mechanisms, of which activation of the NADPH oxidase is the most important (Fig. 8). The neutrophil NADPH oxidase is a multicomponent membrane-bound enzyme that catalyses NADPH dependent reduction of oxygen to , which may be converted to H2O2, in the presence of peroxidases, OONO–, HOCl, and OH? (Babior, 1999). Superoxide anion () formed by NADPH oxidase activation can be reduced to hydrogen peroxide (H2O2), either spontaneously or by superoxide dismutase. The superoxide dismutase inhibitor DDC reduced the TBBPA induced DCF fluorescent by 92% indicating that is the precursor for the ROS measured by DCF fluorescence after TBBPA stimulation. The DCF assay is an attractive and sensitive method as an overall index for oxidative stress in biological systems. It is reported to detect several types of reactive molecules such as H2O2, in presence of cellular peroxidases, OONO– and OH?, but have no sensitivity towards (Myhre et al., 2003). Lucigenin is a sensitive probe for the detection of the superoxide anion radical, and is frequently used to demonstrate activation of respiratory burst in granulocytes (Halliwell and Gutteridge, 1999). The involvement of was confirmed in the lucigenin assay where DDC on the contrary increased the TBBPA induced ROS production. The DCF assay is primarily an indicator of intracellular formation of ROS, whereas lucigenin assay primarily measures extracellular ROS (Caldefie-Chezet et al., 2002). TBBPA appeared to induce both extracellular and intracellular formation of ROS by its activity towards both assays. The addition of SOD, which generally is nonpermeable to the cell membrane (Halliwell and Whiteman, 2004), strengthened this assumption by a total inhibition of TBBPA induced lucigenin-amplified chemiluminescence and no apparent effect towards TBBPA induced DCF fluorescence. DPI, a potent inhibitor of the activation of the NADPH oxidase complex, reduced ROS formation induced by TBBPA by 60% with use of the DCF assay and was completely abolished in the lucigenin assay. Lucigenin must undergo reduction to lucigenin cation to detect . The primary reducing agent in phagocytes is the NADPH-oxidase system (Halliwell and Gutteridge, 1999). This suggests that the effect of TBBPA is mediated mainly through activation of the NADPH oxidase complex. Phosphorylation of the cytosolic subunits, p47PHOX, p67PHOX, and p40PHOX are essential elements in activation of the NADPH oxidase complex. p47PHOX is the subunit chiefly responsible for transporting the cytosolic complex from cytosol to the membrane during oxidase activation (Babior, 1999). Several pathways may activate the NADPH oxidase of which mitogen-activated protein kinase (MAPK) and PKC seem to be most important in our experiments (Fig. 8). The MAPKs are major information pathways from the cell surface to the nucleus. The MAPK pathway includes the c-Jun N-terminal kinase (JNK) and p38 MAPK cascade, which function mainly in stress responses like inflammation and apoptosis, as well as the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), which preferentially regulate cell growth and differentiation (Lewis et al., 1998). U0126 is a selective inhibitor for MEK 1/2, the upstream activator of ERK 1/2 type of MAPK (Favata et al., 1998). The ERK 1/2 pathway participate in the phosphorylation of the NADPH oxidase component p47PHOX (Dewas et al., 2000), resulting in activation of the NADPH oxidase complex, and thereby production. We found a large reduction in ROS formation after incubation with U0126 in combination with TBBPA, indicating an involvement of ERK 1/2 in the formation of ROS. In contrast, the p38 inhibitor SB203580 (Cuenda et al., 1995) and the p38/JNK inhibitor FK506 (Matsuda et al., 2000) had no effect on the TBBPA stimulated DCF fluorescence, at the inhibitor concentration tested. Activation of ERK 1/2 was confirmed by Western blotting of proteins from TBBPA stimulated neutrophils with anti-phospho ERK antibodies. This indicates that ERK 1/2 are important activators of TBBPA induced ROS formation in neutrophil granulocytes, and that the p38 and JNK branches of the MAP kinase pathway are not involved in this activation. PKC is another kinase that phosphorylates p47PHOX, and this phosphorylation plays a major role in activation of the NADPH oxidase complex (Fig. 8) (Nauseef et al., 1991). This was also the case for TBBPA induced ROS formation since the PKC inhibitor BIM reduced the DCF fluorescence by 69% and lucigenin amplified chemiluminescence by 28%. Previously it has been shown that fMLP activates the NADPH oxidase by a co-operation between PKC and the ERK 1/2 pathway (Dewas et al., 2000). Western blot analysis indicated a similar mechanism in TBBPA induced NADPH oxidase activation since the PKC inhibitor BIM also inhibited TBBPA stimulated phosphorylation of ERK 1/2. Fontayne et al. (2002) showed that four out of five known PKC isoforms in neutrophils could induce differential phosphorylation and translocation of p47PHOX, and they suggested that different phosphorylation of p47PHOX by these PKC isoforms could be important in fine-tuning of the NADPH oxidase activity. Erbstatin-A, a selective and potent inhibitor of tyrosine protein kinases (Kawada et al., 1993), strongly inhibited the ROS formation induced by TBBPA. In previous studies it has been shown that protein tyrosine kinases are involved in the signaling pathways employed by chemotactic factors and hydrocarbons in the stimulation of superoxide production in human neutrophils (Dreiem et al., 2003; Naccache et al., 1990). The phosphorylation of ERK 1 and 2 was, however, not affected by erbstatin-A. Erbstatin-A is an analogue to the tyrphostines, which previously were shown to have anti oxidant properties (Sagara et al., 2002). Erbstatin's effect on TBBPA induced ROS formation may therefore be attributed to a similar effect. However, previously it has been shown that respiratory burst induced by fMLP and PMA is very sensitive to tyrosine kinase inhibition, an effect that has not been attributed to PKC and ERK inhibition (Mocsai et al., 1997). These results indicate that tyrosine kinases might be involved in TBBPA induced ROS formation, but must be acting in parallel to or downstream of the MAP kinase cascade and PKC, rather than upstream to it (Fig. 8). Oxidative stress is often linked to calcium uptake in cells. However, respiratory burst may be activated in both calcium dependent and independent pathways (Downey et al., 1995). TBBPA increased intracellular calcium measured with Fura-2 AM in a concentration dependent manner. An interesting effect of TBBPA was the observed elevation of intracellular calcium even though extracellular calcium was removed and EGTA added, indicating that TBBPA also elevates cytosolic free calcium through release from intracellular compartments. In the absence of extracellular calcium the ROS formation was reduced. Our findings therefore suggest an involvement of calcium dependent activation of PKC (Fig. 8). The calcium antagonist verapamil, a voltage-dependent calcium channel blocker, also attenuated the ROS formation induced by TBBPA. Verapamil also reduced ERK phosphorylation as shown by Western blot (Fig. 6C). However, voltage-dependent calcium channels have not been reported to exist in neutrophil surface membrane (Schrenzel et al., 1995). It is therefore likely that inhibition of oxidant production by verapamil is attributable to some mechanism independent of its calcium channel-blocking properties. It has been reported that verapamil reduces PMA-stimulated superoxide production in neutrophils by inhibition of protein phosphorylation, probably catalyzed by PKC (Irita et al., 1986). This indicates that TBBPA also activates respiratory burst through a calcium dependent pathway, probably via activation of calcium dependent PKC. The TBBPA induced increase in intracellular calcium may also explain the residual formation of ROS in the DCF-assay after knocking out the NADPH-oxidase with DPI. Bisphenol-A also showed a small but significant increase in intracellular calcium at relatively high concentrations (20 μM). These findings indicate that the bromine substitution seems to play a crucial role in the ability to induce elevation in intracellular calcium and ROS formation. A previous study also shows that bromine substitution on the bisphenol structure plays a crucial role for activity (Meerts et al., 2000). The present article demonstrates that one of the most frequently used BFR, TBBPA, potently activates the NADPH oxidase in granulocytes primarily through elevation of intracellular calcium, activation of PKC and the MAP kinase pathway. Although one should be careful to apply these results to an in vivo situation; this is of great concern since the levels of BFR are rapidly increasing, both in human and environmental samples. Human exposure to TBBPA has not been extensively investigated and due to its phenolic structure it is not expected to accumulate in the environment to the same degree as the more lipid-soluble environmental toxicant. Plasma levels up to a few ng g–1 lipids (low nM concentrations) have, however, been reported (Thomsen et al., 2001) and TBBPA is also found accumulated in predators eggs (Berger et al., 2004). The concentrations used in this study are higher than what is detected in biota; however, it is important to remember that we are also continuously exposed to other environmental contaminants with similar chemical and toxicological properties such as PCBs and dioxins. Some studies also show that environmental contaminants may act additive or even synergistically when combined (Bemis and Seegal, 1999; Eriksson et al., 2003). Earlier findings in our laboratory show that environmental contaminants have similar effects on human neutrophil granulocytes as shown for TBBPA in this study. Ortho substituted PCBs increase respiratory burst and elevates intracellular calcium in granulocytes at concentrations around 10 μM (Voie and Fonnum, 1998; Voie et al., 1998). A preliminary experiment in this study showed that PCB153, one of the most frequently found compound in environmental samples, and TBBPA in combination induced an additive effect as shown by lucigenin-amplified chemiluminescence. PCB 153 did not induce DCF fluorescence at this concentration indicating a PCB induced extracellular formation of ROS. PCB 153 and TBBPA in combination did not influence the TBBPA induced DCF-fluorescence making it unlikely that PCB153 inhibits SOD as previously shown for PCB47 and the PCB mixture A1242 (Narayanan et al., 1998). Human neutrophil granulocytes play a key role in host defenses against invading pathogens and are major effectors of the acute inflammatory reactions. Activation of these cells during an immune response leads to formation of reactive oxygen species used to kill microorganisms. A possible threat to the cellular homeostasis arises from these reactive oxygen species, as they are known to be involved in cellular signaling and gene regulation (Allen and Tresini, 2000; Finkel, 1998). In addition to the direct threat to own cells and tissue, Koner et al. (1997) have shown a possible connection between free radical formation and immune suppression in rabbit. ACKNOWLEDGMENTS The authors are indebted to Dr. Anne Dreiem for helpful discussions, Dr. Yngvar Gundersen for proofreading the manuscript, and Avi Ring for assistance in the LDH measurements. The authors also acknowledge The Norwegian Defence Research Establishment and Norwegian Research Council, under the PROFO program, for financial support. REFERENCES Allen, R. G., and Tresini M. (2000). Oxidative stress and gene regulation. Free Radic. Biol. Med. 28, 463–499. Babior, B. M. (1999). NADPH oxidase: An update. Blood 93, 1464–1476. Dewas, C., Fay, M., Gougerot-Pocidalo, M. A., and El-Benna, J. (2000). The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils. J. Immunol. 165, 5238–5244. Matsuda, S., Shibasaki, F., Takehana, K., Mori, H., Nishida, E., and Koyasu, S. (2000) Two distinct action mechanisms of immunophilin-ligand Meerts, I. A., van Zanden, J. J., Luijks, E. A., van Leeuwen-Bol, I., Marsh, G., Jakobsson, E., Bergman, A., and Brouwer, A. (2000). Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicol. Sci. 56, 95–104. Pullen, S., Boecker, R., and Tiegs, G. (2003). The flame retardants tetrabromobisphenol A and tetrabromobisphenol A-bisallylether suppress the induction of interleukin-2 receptor alpha chain (CD25) in murine splenocytes. Toxicology 184, 11–22. Reistad, T., Mariussen, E., and Fonnum, F. (2002). The effect of brominated flame-retardants on cell death and free radical formation in cerebellar granule cells. Organohalogen Compounds 57, 391–394....查看详细 (34932字节)
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黑龙江省哈尔滨市道里区安静街孙女士：我来月经时总是沥沥拉拉地流血不止，量不多，有时下腹胀痛。诊断为卵巢巧克力囊肿，医生建议做手术，我工作很忙，没时间住院，请问除了手术，治疗卵巢巧克力囊肿还有其他方法吗？ 哈尔滨医科大学附属第四医院超声科主任董晓秋教授：您可以选择超声介入治疗，创伤小，休养的时间短，不会影响工作。如果巧克力囊肿周围粘连严重，手术在剥离过程中极易剥破，而致使一部分囊壁残留在盆腔。超声介入治疗是经腹或经阴道穿刺抽出粘稠的血液...查看详细 (939字节)