Morphine Withdrawal Lowers Host Defense to Enteric Bacteria: Spontaneo
http://www.100md.com
《感染与免疫杂志》
Center for Substance Abuse Research
Department of Microbiology and Immunology
Department of Pharmacology
Department of Pathology and Laboratory Medicine
Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
ABSTRACT
Understanding the consequences of drug withdrawal on immune function and host defense to infection is important. We, and others, previously demonstrated that morphine withdrawal results in immunosuppression and sensitizes to lipopolysaccharide-induced septic shock. In the present study, the effect of morphine withdrawal on spontaneous sepsis and on oral infection with Salmonella enterica serovar Typhimurium was examined. Mice were chronically exposed to morphine for 96 h by implantation of a slow-release morphine pellet. Abrupt withdrawal was induced by removal of the pellet. In the sepsis model, bacterial colonization was examined and bacterial species were identified by necropsy of various tissues. It was found that at 48 h postwithdrawal, morphine-treated mice had enteric bacteria that were detected in the Peyer's patches (4/5), mesenteric lymph nodes (4/5), spleens (4/10), livers (6/10), and peritoneal cavities (8/10). In placebo pellet-withdrawn mice, only 2/40 cultures were positive. The most frequently detected organisms in tissues of morphine-withdrawn mice were Enterococcus faecium followed by Klebsiella pneumoniae. Both organisms are part of the normal gastrointestinal flora. In the infection model, mice were orally inoculated with S. enterica 24 h post-initiation of abrupt withdrawal from morphine. Withdrawal significantly decreased the mean survival time and significantly increased the Salmonella burden in various tissues of infected mice compared to placebo-withdrawn animals. Elevated levels of the proinflammatory cytokines were observed in spleens of morphine-withdrawn mice, compared to placebo-withdrawn mice. These findings demonstrate that morphine withdrawal sensitizes to oral infection with a bacterial pathogen and predisposes mice to bacterial sepsis.
INTRODUCTION
Opioid addiction is a major public health challenge that has been reported to correlate clinically with the incidence of various infectious diseases (20, 22, 24). Importantly, intravenous drug use is one of the two most frequently reported risk behaviors for human immunodeficiency virus infection. Substantial evidence from both clinical observations and animal experiments has shown that opioids are immunosuppressive, and impaired immunity has been proposed as a major cause for the increased number of infections in drug abusers (18, 26, 32). Previous experiments from our laboratory have shown that subacute exposure to morphine promotes the translocation of intestinal bacteria to the abdominal viscera and peritoneal cavity (21) and potentiates oral Salmonella sp. infection in an animal model (25). Other pathogens whose infectivities are increased by morphine treatment in laboratory models include the following: Streptococcus pneumoniae (39), Toxoplasma gondii (10), Klebsiella pneumoniae (38), Candida albicans (38), Pasteurella multocida (33), Plasmodium berghei (37), and Leishmania donovani (36).
Development of opioid tolerance and an abstinence syndrome upon termination of drug administration are among the most important characteristics of opioid addiction. Tolerance and withdrawal have been investigated at the anatomical, biochemical, physiological, pharmacological, behavioral, and molecular levels (5, 12, 28, 29, 40). However, there are surprisingly few studies on the effects of withdrawal on immune function and infection, even though drug addicts experience frequent episodes of withdrawal as they come down from their "highs" or are between "hits." Our laboratory has recently shown that mice withdrawn from morphine are sensitized to a sublethal dose of bacterial lipopolysaccharide (LPS) (16). Opioid-withdrawn animals had elevated levels of tumor necrosis factor alpha (TNF-) in serum and were significantly protected against LPS challenge by anti-TNF- antibody. Further, we also tested the resistance of mice in withdrawal to an intraperitoneal (i.p.) infection with live, virulent Salmonella enterica serovar Typhimurium and found that abstinence increased their susceptibility (17). We hypothesized that withdrawal might lead to a condition of underlying sepsis (16), where sepsis is defined here narrowly as the presence in the blood, peritoneal fluid, tissues, or organs of bacteria normally confined to the gastrointestinal tract.
In the present study, we investigated the effect of abrupt (spontaneous) withdrawal from morphine on an enteric bacterial infection by using a spontaneous sepsis model and exogenous infection with a pathogen via the oral route. The findings show that morphine withdrawal sensitizes to oral infection with Salmonella and predisposes animals to bacterial sepsis with endogenous flora.
MATERIALS AND METHODS
Animals. Pathogen-free, female, 6-week-old C3HeB/FeJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were allowed to acclimate for at least 1 week before use. Rodent chow and fresh water were available ad libitum.
Bacterial strain. Salmonella enterica serovar Typhimurium strain W118-2 was used for experimental infection in this study. It has been used extensively by our laboratory (1, 15, 25, 35). The oral 50% lethal dose is 2 x 104 cells for C3HeB/FeJ mice. For culture growth, a lyophil was rehydrated with 5 ml of brain heart infusion (BHI) broth, incubated overnight, and then streaked onto a tryptic soy agar (TSA) plate and grown overnight at 37°C. Typical colonies were picked and inoculated into 10 ml of BHI broth. The culture was incubated for 3.5 h at 37°C, and the top 5 ml of medium was transferred into 50 ml of BHI broth and grown on a shaker at 37°C for 1.5 h to produce log-phase organisms. Bacteria were counted in a Petroff-Hauser counter and diluted to the desired concentration in sterile, pyrogen-free saline. The actual number of organisms inoculated was verified later from duplicate spread plates on TSA.
Morphine and withdrawal treatment. Mice were anesthetized by isoflurane inhalation, and an area of the back was shaved. A 1-cm incision was made in the skin of the back, and mice were implanted subcutaneously with either a 75-mg slow-release morphine pellet or a cellulose placebo pellet (National Institute on Drug Abuse, Rockville, MD) sealed in a nylon mesh bag to permit easy removal later. Slow-release pellets are a preferred method of opioid delivery in studying chronic administration (7, 8, 11), as the continuous presence of drug prevents opioid withdrawal (9). Blood levels are in the 0.6- to 2-μg/ml range (8). Pellets were removed surgically 96 h later, at which time the animals were dependent on morphine (31). Removal of the morphine pellets initiates spontaneous withdrawal (abstinence syndrome) in these animals, based on our previous work (31). Placebo pellets were also removed at 96 h with no overt symptoms.
Microbial assessment. To assess spontaneous bacterial infection, blood, peritoneal lavage fluid (PLF), Peyer's patches (PPs), mesenteric lymph nodes (MLNs), spleens, and livers were collected from individual animals at the desired time points after withdrawal. Tissues of individual animals were homogenized in 5 ml of ice-cold phosphate-buffered saline (PBS), using a Tekmar tissuemizer (Tekmar Co., Cincinnati, Ohio). Homogenates were spread onto TSA II 5% sheep blood agar plates and incubated at 37°C overnight, and the numbers of CFU were counted and recorded. Typical individual bacterial colonies were subcultured and identified by the Clinical Microbiology Laboratory at Temple University Hospital using a semiautomated microbial identification system (Biomerieux, Inc., Hazelwood, Mo.).
Oral Salmonella infection and assessment of host susceptibility. At 24 h post-removal of the pellets, animals were anesthetized using isoflurane and inoculated orally with 1 x 104 CFU of log-phase Salmonella in 0.2 ml saline. A 21-gauge needle with a fine polished blunt end was used to deliver organisms into the stomach. At desired time points after inoculation, mice were bled from the retroorbital plexus under isoflurane anesthesia before being sacrificed. PPs, MLNs, and spleens were aseptically removed from each animal, and homogenates were prepared using the protocol described above. A 0.1-ml sample of serially diluted homogenate in PBS was plated on Levine eosin-methylene blue agar plates (Becton-Dickinson, Sparks, MD) and incubated at 37°C overnight, and the number of colonies was counted and recorded. Resistance to infection was also assessed by survival rate and mean survival time (MST) over a period of 30 days.
Preparation of tissue homogenates for cytokine determination. To prepare spleen extracts used for the cytokine assessment, aliquots of spleen homogenates from individual mice were clarified by centrifugation at 13,000 rpm for 20 min at 4°C using a bench-top centrifuge (model 5415R; Eppendorf), and the supernatants were stored at –80°C.
Cytometric bead array assay. Levels of multiple cytokines in spleen homogenates of individual mice were assessed simultaneously by flow cytometry using the cytometric bead array technique (BD Biosciences, San Diego, CA). The assay was carried out according to the manufacturer's instructions. Briefly, capture beads specific for the various cytokines were mixed together to form a bead suspension. Fifty-microliter aliquots of the bead suspension were then incubated with a 50-μl sample collected from an individual mouse and 50 μl of a phycoerythrin-conjugated antimurine detection reagent for 2 h at room temperature in the dark. One ml of wash buffer was then added to each assay tube and centrifuged at 200 x g for 6 min. Supernatants were discarded, and 200 μl of wash buffer was added to resuspend the bead pellet. The prepared tubes were analyzed on a BD FACScan flow cytometer (BD PharMingen, San Diego, CA) calibrated with cytometer setup beads provided by the manufacturer. The phycoerythrin intensities of each sample were converted to concentration values of cytokines using computer software.
Statistical analysis. All experiments used a completely randomized design with each preparation evaluated once. The null hypothesis was that there would be no difference between the morphine and placebo groups. Data were tested for normality using the Shapiro-Wilk test (3). The data were significantly nonnormal for all variables. In order to apply parametric methods, a "normalized-rank" transformation was applied to the data (13, 19). Ratios of positive bacterial cultures between groups were analyzed as differences between Poisson rates. The rank-transformed data were analyzed using an independent t test with equal or unequal variances. Survival data were analyzed using the Kaplan-Meier product limit method for right-censored data followed by the log rank test to compare group survival functions. Differences between groups (rejection of the null hypothesis) were considered significant if the probability of chance occurrence was 0.05 using two-tailed tests.
RESULTS
Increased incidence of sepsis in mice undergoing withdrawal from morphine. To assess sepsis, various tissues of mice undergoing withdrawal from morphine or placebo for 48 h were cultured for bacterial growth. As shown in Fig. 1, most morphine-withdrawn mice showed bacterial growth in PPs (4/5), MLNs (4/5), spleens (4/10), livers (6/10), and PLF (8/10). In contrast, no placebo-withdrawn mice showed bacterial growth in the MLNs, spleens, or peritoneal cavity. Samples of PP and liver tissue from only one placebo-withdrawn mouse tested positive for bacteria. The most frequently detected bacterial species in the cultures were identified as Enterococcus faecium followed by Klebsiella pneumoniae (Table 1), both being part of the normal gastrointestinal flora. These results clearly demonstrate that mice undergoing withdrawal for a period of 48 h from a morphine pellet, but not a placebo pellet, become septic.
Additional experiments were carried out to ascertain the time course of extraintestinal growth of enteric bacteria in organs of mice in withdrawal from morphine. Table 2 presents data on microbial burdens in various tissues on days 1, 3, and 6 after withdrawal. On day 1, bacterial growth was observed in MLNs (5/5) and livers (3/5), and one mouse (1/5) was bacteremic. No bacterial growth was found in placebo-withdrawn mice in the liver or blood, and only one placebo mouse showed bacteria in the MLN. On days 3 and 6, bacterial growth was still observed in livers, MLNs, and blood from morphine-withdrawn mice. No bacteria or bacteremia was found in placebo-withdrawn mice. Overall, only 1/45 placebo-pelleted mice had positive bacterial cultures, compared to 20/45 morphine-pelleted mice (P < 0.01). These data clearly demonstrate that morphine withdrawal markedly increased bacterial colonization in a variety of tissues from 1 day to as long as 6 days postwithdrawal. Withdrawal from a placebo pellet did not result in the same effect.
Effect of morphine withdrawal on survival of mice orally infected with Salmonella. In order to ascertain if morphine withdrawal could sensitize to an enteric pathogen, mice were orally infected with virulent Salmonella enterica serovar Typhimurium 24 h after initiation of morphine withdrawal, and survival was scored for 30 days. As shown in Fig. 2, the survival rate of infected, placebo-withdrawn mice was 47%, with death commencing on day 14 and a mean survival time of 24.0 ± 1.6 days (mean ± standard error). In contrast, in the morphine-withdrawn group death started on day 4, with a survival rate of 23%. The difference in survival rates approached statistical significance (P = 0.07). There was a statistically significant difference in mean survival time (P < 0.05). Our previous studies showed that morphine withdrawal followed by injection of saline 24 h later resulted in no mortality (0/25 mice) (16).
The more rapid mortality of morphine-withdrawn mice infected with Salmonella compared to infected, placebo-withdrawn animals was associated with markedly increased bacterial burdens in PPs, MLNs, and spleens (Fig. 3). At 24 h post-withdrawal from morphine, mice were inoculated orally with 1 x 104 CFU of Salmonella, and bacterial numbers in organs were assayed on days 2, 6, and 12 postinfection. There were no detectable Salmonella organisms in the PPs of any mice in the placebo groups over the period of 12 days. In contrast, Salmonella organisms were cultured from PPs of all morphine-withdrawn mice on day 12. For the MLNs in the placebo group, there was no bacterial growth on days 2 and 6 and only one out of five mice showed a low level of bacteria on day 12, whereas morphine-withdrawn animals had a thousandfold-greater bacterial burden at these time points. On day 12, morphine-withdrawn mice also had a significantly higher Salmonella burden in spleens than placebo-withdrawn animals. These results demonstrate that morphine withdrawal sensitizes to infection at a mucosal surface for this murine pathogen.
Morphine withdrawal increases proinflammatory cytokine production in Salmonella-infected mice. To examine the mechanism underlying the increased susceptibility to oral Salmonella infection in mice undergoing morphine withdrawal, the dynamics of production of TNF-, gamma interferon, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1) in the spleen were assessed in comparison to infected, placebo-withdrawn mice. As shown in Fig. 4, there were no changes in cytokine levels on day 2 after Salmonella inoculation in either group. On day 6 postinfection, the levels of all four proinflammatory cytokines were increased in morphine-withdrawn animals and IL-6 and MCP-1 concentrations peaked, but no change was observed in the cytokine levels of placebo-withdrawn mice. On day 12, the production of TNF-, gamma interferon, and IL-6 in both groups was increased, but the levels in morphine-withdrawn mice were higher than those in placebo-withdrawn animals. The levels of the chemokine MCP-1 were the same in morphine- and placebo-withdrawn mice on day 12. The increase in the proinflammatory cytokines is probably due to the higher bacterial burdens in the morphine-treated mice compared to controls. IL-12 and IL-10 levels were also tested but were undetectable.
DISCUSSION
The results presented in this report support the conclusion that withdrawal from morphine in an experimental mouse model results in sepsis, as organisms such as Enterococcus faecium and Klebsiella pneumoniae, which are part of the normal gut flora, are found in peripheral sites, including the PPs, MLNs, spleen, liver, and peritoneal lavage fluid. Appearance of these organisms extraintestinally is not due to administration of anesthesia or to surgical manipulation for removal of the morphine pellets, as the majority of animals that received placebo pellets and that were subjected to similar procedures were sterile at these sites. Detection of sepsis is consonant with our previous observation that mice in withdrawal from morphine are sensitized to a sublethal dose of LPS, produce more TNF- in response to LPS than mice which are withdrawn from a placebo pellet, and are protected from LPS-induced mortality by antibodies to TNF- (16). It is noteworthy that we have previously reported that mice implanted with a similar slow-release morphine pellet were septic at 48 h after pellet implantation and were also hypersensitive to sublethal doses of LPS (21). Sepsis induced by subacute administration of morphine has been confirmed by three other laboratories, using both mice and rats (6, 27, 34). Exposure of rats to morphine for 5 days was also shown to result in measurable endotoxin in serum, as well as other signs of sepsis, such as coagulopathy in the microvasculature and diminished arterial blood pressure (27). The sepsis observed following withdrawal could be the result of additive outcomes from morphine implantation and morphine withdrawal.
In contrast to studies on the effects of morphine administered acutely, subacutely, or chronically, there is a paucity of research in the area on effects of withdrawal from opioids on resistance to infection. There are only three papers in the literature in this field. Donahoe et al. (14) reported that if simian immunodeficiency virus-infected rhesus monkeys were treated chronically with morphine and withdrawn from the drug, the viral titer increased. In contrast, cats infected with feline immunodeficiency virus given morphine chronically and then withdrawn had no alteration in their levels of feline immunodeficiency virus (4). The third paper is from our laboratory and showed that mice withdrawn from morphine are sensitized to systemic (intraperitoneal) infection with Salmonella (17). The present results expand this limited data to test the effect of withdrawal on an oral bacterial infection. We have shown that withdrawal from morphine potentiates growth of Salmonella administered orally 24 h after the initiation of abstinence. This time point was chosen because it coincides with the onset of maximal systemic immunosuppression (31). When the results are compared, withdrawal from morphine produces less robust effects on oral Salmonella infection (present study) than on intraperitoneal Salmonella infection, the subject of a previous paper from our laboratory (17). The effects of withdrawal on oral Salmonella infection are also several orders of magnitude less robust than those observed when Salmonella is given orally coincident with initiation of morphine administration (25). A significant aspect of the contrast between the previous i.p. and oral challenge experiments with Salmonella relates to levels of cytokines. Using i.p. challenge, we reported in the previous paper on cytokine levels measured in plasma. In the current experiments cytokine levels were analyzed in splenic homogenates. The results of the two studies are not consonant, but the reason for this lack of agreement is not certain, since there are two variables that were different between the studies, the route of challenge and the tissue used to analyze cytokine levels. Thus, following i.p. challenge with Salmonella, IL-12 levels were suppressed in plasma but the IL-12 levels were undetectable in spleen homogenates in either placebo- or morphine-treated mice orally infected with Salmonella. The data on sensitization to Salmonella infection suggest that morphine itself and withdrawal from morphine may each affect different aspects of the immune system and gastrointestinal function that alter host resistance to Salmonella infection. For example, morphine has been shown to inhibit the mucosal immunoglobulin A response to cholera toxin (30), raising the possibility that increased sensitivity to oral Salmonella infection might be due to impaired mucosal immune responses.
The present studies are important because sepsis in the United States is a major medical problem, with an estimated 750,000 cases per year, and more than 210,000 of the cases die (mortality ranging from 30 to 50%) (2). In many cases, a precipitating cause of the septic condition cannot be identified, but sepsis primarily occurs in a hospital setting. Demonstration that mice in withdrawal from morphine are septic adds to evidence from two previous publications showing that withdrawal increases proinflammatory cytokines (16, 23) and sensitizes to the lethal effects of LPS, as measured by mortality (16). These data, along with previous observations that sepsis ensues after subacute administration of morphine (21), support the hypothesis that morphine, and possibly other opioids, administered to acutely ill patients may be a cofactor in induction of sepsis.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institute on Drug Abuse, DA14223 and DA13429.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. Phone: (215) 707-3585. Fax: (215) 707-7920. E-mail: tke@temple.edu.
REFERENCES
1. Angerman, C. R., and T. K. Eisenstein. 1980. Correlation of the duration and magnitude of protection against Salmonella infection afforded by various vaccines with antibody titers. Infect. Immun. 27:435-443.
2. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29:1303-1310.
3. Armitage, P., and G. Berry. 1994. Statistical methods in medical research, 3rd ed. Blackwell Science, Cambridge, Mass.
4. Barr, M. C., S. Huitron-Resendiz, M. Sanchez-Alavez, S. J. Henriksen, and T. R. Phillips. 2003. Escalating morphine exposures followed by withdrawal in feline immunodeficiency virus-infected cats: a model for HIV infection in chronic opiate abusers. Drug Alcohol Depend. 72:141-149.
5. Bergstrom, L., T. Sakurada, and L. Terenius. 1984. Substance P levels in various regions of the rat central nervous system after acute and chronic morphine treatment. Life Sci. 35:2375-2382.
6. Bhaskaran, M., K. Reddy, S. Sharma, J. Singh, N. Radhakrishnan, A. Kapasi, and P. C. Singhal. 2001. Morphine-induced degradation of the host defense barrier: role of macrophage injury. J. Infect. Dis. 184:1524-1531.
7. Bryant, H. U., E. W. Bernton, and J. W. Holaday. 1988. Morphine pellet-induced immunomodulation in mice: temporal relationships. J. Pharmacol. Exp. Ther. 245:913-920.
8. Bryant, H. U., B. C. Yoburn, C. E. Inturrisi, E. W. Bernton, and J. W. Holaday. 1988. Morphine-induced immunomodulation is not related to serum morphine concentrations. Eur. J. Pharmacol. 149:165-169.
9. Cerletti, C., S. H. Keinath, M. M. Reidenbery, and M. W. Adler. 1976. Chronic morphine administration: plasma levels and withdrawal syndrome in rats. Pharmacol. Biochem. Behav. 4:323-327.
10. Chao, C. C., B. M. Sharp, C. Pomeroy, G. A. Filice, and P. K. Peterson. 1990. Lethality of morphine in mice infected with Toxoplasma gondii. J. Pharmacol. Exp. Ther. 252:605-609.
11. Cheney, D. L., and A. Goldstein. 1971. Tolerance to opioid narcotics: time course and reversibility of physical dependence in mice. Nature 232:477-478.
12. Cicero, T. J., and E. R. Meyer. 1973. Morphine pellet implantation in rats: quantitative assessment of tolerance and dependence. J. Pharmacol. Exp. Ther. 184:404-408.
13. Conover, W. J., and R. L. Iman. 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35:124-129.
14. Donahoe, R. M., L. D. Byrd, H. M. McClure, P. Fultz, M. Brantley, F. Marsteller, A. A. Ansari, D. Wenzel, and M. Aceto. 1993. Consequences of opiate-dependency in a monkey model of AIDS. Adv. Exp. Med. Biol. 335:21-28.
15. Eisenstein, T. K., L. M. Killar, and B. M. Sultzer. 1984. Immunity to infection with Salmonella typhimurium: mouse-strain differences in vaccine- and serum-mediated protection. J. Infect. Dis. 150:425-435.
16. Feng, P., J. J. Meissler, Jr., M. W. Adler, and T. K. Eisenstein. 2005. Morphine withdrawal sensitizes mice to lipopolysaccharide: elevated TNF-alpha and nitric oxide with decreased IL-12. J. Neuroimmunol. 164:57-65.
17. Feng, P., Q. M. Wilson, J. J. Meissler, Jr., M. W. Adler, and T. K. Eisenstein. 2005. Increased sensitivity to Salmonella enterica serovar Typhimurium infection in mice undergoing withdrawal from morphine is associated with suppression of interleukin-12. Infect. Immun. 73:7953-7959.
18. Friedman, H., C. Newton, and T. W. Klein. 2003. Microbial infections, immunomodulation, and drugs of abuse. Clin. Microbiol. Rev. 16:209-219.
19. Harter, H. L. 1961. Expected values of normal order statistics. Biomtrika 48:151-165.
20. Haverkos, H. W., and W. R. Lange. 1990. Serious infections other than human immunodeficiency virus among intravenous drug abusers. J. Infect. Dis. 161:894-902.
21. Hilburger, M. E., M. W. Adler, A. L. Truant, J. J. Meissler, Jr., V. Satishchandran, T. J. Rogers, and T. K. Eisenstein. 1997. Morphine induces sepsis in mice. J. Infect. Dis. 176:183-188.
22. Horsburgh, C. R., Jr., J. R. Anderson, and E. J. Boyko. 1989. Increased incidence of infections in intravenous drug users. Infect. Control Hosp. Epidemiol. 10:211-215.
23. Kelschenbach, J., R. A. Barke, and S. Roy. 2005. Morphine withdrawal contributes to Th cell differentiation by biasing cells toward the Th2 lineage. J. Immunol. 175:2655-2665.
24. Louria, D. B., T. Hensle, and J. Rose. 1967. The major medical complications of heroin addiction. Ann. Intern. Med. 67:1-22.
25. MacFarlane, A. S., X. Peng, J. J. Meissler, Jr., T. J. Rogers, E. B. Geller, M. W. Adler, and T. K. Eisenstein. 2000. Morphine increases susceptibility to oral Salmonella typhimurium infection. J. Infect. Dis. 181:1350-1358.
26. McCarthy, L., M. Wetzel, J. K. Sliker, T. K. Eisenstein, and T. J. Rogers. 2001. Opioids, opioid receptors, and the immune response. Drug Alcohol Depend. 62:111-123.
27. Ocasio, F. M., Y. Jiang, S. D. House, and S. L. Chang. 2004. Chronic morphine accelerates the progression of lipopolysaccharide-induced sepsis to septic shock. J. Neuroimmunol. 149:90-100.
28. Paronis, C. A., and S. G. Holtzman. 1992. Development of tolerance to the analgesic activity of mu agonists after continuous infusion of morphine, meperidine or fentanyl in rats. J. Pharmacol. Exp. Ther. 262:1-9.
29. Patrick, G. A., W. L. Dewey, F. P. Huger, E. D. Daves, and L. S. Harris. 1978. Disposition of morphine in chronically infused rats: relationship to antinociception and tolerance. J. Pharmacol. Exp. Ther. 205:556-562.
30. Peng, X., J. J. Cebra, M. W. Adler, J. J. Meissler, Jr., A. Cowan, P. Feng, and T. K. Eisenstein. 2001. Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice. J. Immunol. 167:3677-3681.
31. Rahim, R. T., M. W. Adler, J. J. Meissler, Jr., A. Cowan, T. J. Rogers, E. B. Geller, and T. K. Eisenstein. 2002. Abrupt or precipitated withdrawal from morphine induces immunosuppression. J. Neuroimmunol. 127:88-95.
32. Risdahl, J. M., K. V. Khanna, P. K. Peterson, and T. W. Molitor. 1998. Opiates and infection. J. Neuroimmunol. 83:4-18.
33. Risdahl, J. M., P. K. Peterson, C. C. Chao, C. Pijoan, and T. W. Molitor. 1993. Effects of morphine dependence on the pathogenesis of swine herpesvirus infection. J. Infect. Dis. 167:1281-1287.
34. Roy, S., R. G. Charboneau, and R. A. Barke. 1999. Morphine synergizes with lipopolysaccharide in a chronic endotoxemia model. J. Neuroimmunol. 95:107-114.
35. Schwacha, M. G., J. J. Meissler, Jr., and T. K. Eisenstein. 1998. Salmonella typhimurium infection in mice induces nitric oxide-mediated immunosuppression through a natural killer cell-dependent pathway. Infect. Immun. 66:5862-5866.
36. Singal, P., A. G. Kinhikar, S. Singh, and P. P. Singh. 2002. Neuroimmunomodulatory effects of morphine in Leishmania donovani-infected hamsters. Neuroimmunomodulation 10:261-269.
37. Singh, P. P., S. Singh, G. P. Dutta, and R. C. Srimal. 1994. Immunomodulation by morphine in Plasmodium berghei-infected mice. Life Sci. 54:331-339.
38. Tubaro, E., G. Borelli, C. Croce, G. Cavallo, and C. Santiangeli. 1983. Effect of morphine on resistance to infection. J. Infect. Dis. 148:656-666.
39. Wang, J., R. A. Barke, R. Charboneau, and S. Roy. 2005. Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection. J. Immunol. 174:426-434.
40. Way, E. L., H. H. Loh, and F. H. Shen. 1969. Simultaneous quantitative assessment of morphine tolerance and physical dependence. J. Pharmacol. Exp. Ther. 167:1-8.(Pu Feng, Allan L. Truant, Joseph J. Meis)
Department of Microbiology and Immunology
Department of Pharmacology
Department of Pathology and Laboratory Medicine
Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
ABSTRACT
Understanding the consequences of drug withdrawal on immune function and host defense to infection is important. We, and others, previously demonstrated that morphine withdrawal results in immunosuppression and sensitizes to lipopolysaccharide-induced septic shock. In the present study, the effect of morphine withdrawal on spontaneous sepsis and on oral infection with Salmonella enterica serovar Typhimurium was examined. Mice were chronically exposed to morphine for 96 h by implantation of a slow-release morphine pellet. Abrupt withdrawal was induced by removal of the pellet. In the sepsis model, bacterial colonization was examined and bacterial species were identified by necropsy of various tissues. It was found that at 48 h postwithdrawal, morphine-treated mice had enteric bacteria that were detected in the Peyer's patches (4/5), mesenteric lymph nodes (4/5), spleens (4/10), livers (6/10), and peritoneal cavities (8/10). In placebo pellet-withdrawn mice, only 2/40 cultures were positive. The most frequently detected organisms in tissues of morphine-withdrawn mice were Enterococcus faecium followed by Klebsiella pneumoniae. Both organisms are part of the normal gastrointestinal flora. In the infection model, mice were orally inoculated with S. enterica 24 h post-initiation of abrupt withdrawal from morphine. Withdrawal significantly decreased the mean survival time and significantly increased the Salmonella burden in various tissues of infected mice compared to placebo-withdrawn animals. Elevated levels of the proinflammatory cytokines were observed in spleens of morphine-withdrawn mice, compared to placebo-withdrawn mice. These findings demonstrate that morphine withdrawal sensitizes to oral infection with a bacterial pathogen and predisposes mice to bacterial sepsis.
INTRODUCTION
Opioid addiction is a major public health challenge that has been reported to correlate clinically with the incidence of various infectious diseases (20, 22, 24). Importantly, intravenous drug use is one of the two most frequently reported risk behaviors for human immunodeficiency virus infection. Substantial evidence from both clinical observations and animal experiments has shown that opioids are immunosuppressive, and impaired immunity has been proposed as a major cause for the increased number of infections in drug abusers (18, 26, 32). Previous experiments from our laboratory have shown that subacute exposure to morphine promotes the translocation of intestinal bacteria to the abdominal viscera and peritoneal cavity (21) and potentiates oral Salmonella sp. infection in an animal model (25). Other pathogens whose infectivities are increased by morphine treatment in laboratory models include the following: Streptococcus pneumoniae (39), Toxoplasma gondii (10), Klebsiella pneumoniae (38), Candida albicans (38), Pasteurella multocida (33), Plasmodium berghei (37), and Leishmania donovani (36).
Development of opioid tolerance and an abstinence syndrome upon termination of drug administration are among the most important characteristics of opioid addiction. Tolerance and withdrawal have been investigated at the anatomical, biochemical, physiological, pharmacological, behavioral, and molecular levels (5, 12, 28, 29, 40). However, there are surprisingly few studies on the effects of withdrawal on immune function and infection, even though drug addicts experience frequent episodes of withdrawal as they come down from their "highs" or are between "hits." Our laboratory has recently shown that mice withdrawn from morphine are sensitized to a sublethal dose of bacterial lipopolysaccharide (LPS) (16). Opioid-withdrawn animals had elevated levels of tumor necrosis factor alpha (TNF-) in serum and were significantly protected against LPS challenge by anti-TNF- antibody. Further, we also tested the resistance of mice in withdrawal to an intraperitoneal (i.p.) infection with live, virulent Salmonella enterica serovar Typhimurium and found that abstinence increased their susceptibility (17). We hypothesized that withdrawal might lead to a condition of underlying sepsis (16), where sepsis is defined here narrowly as the presence in the blood, peritoneal fluid, tissues, or organs of bacteria normally confined to the gastrointestinal tract.
In the present study, we investigated the effect of abrupt (spontaneous) withdrawal from morphine on an enteric bacterial infection by using a spontaneous sepsis model and exogenous infection with a pathogen via the oral route. The findings show that morphine withdrawal sensitizes to oral infection with Salmonella and predisposes animals to bacterial sepsis with endogenous flora.
MATERIALS AND METHODS
Animals. Pathogen-free, female, 6-week-old C3HeB/FeJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were allowed to acclimate for at least 1 week before use. Rodent chow and fresh water were available ad libitum.
Bacterial strain. Salmonella enterica serovar Typhimurium strain W118-2 was used for experimental infection in this study. It has been used extensively by our laboratory (1, 15, 25, 35). The oral 50% lethal dose is 2 x 104 cells for C3HeB/FeJ mice. For culture growth, a lyophil was rehydrated with 5 ml of brain heart infusion (BHI) broth, incubated overnight, and then streaked onto a tryptic soy agar (TSA) plate and grown overnight at 37°C. Typical colonies were picked and inoculated into 10 ml of BHI broth. The culture was incubated for 3.5 h at 37°C, and the top 5 ml of medium was transferred into 50 ml of BHI broth and grown on a shaker at 37°C for 1.5 h to produce log-phase organisms. Bacteria were counted in a Petroff-Hauser counter and diluted to the desired concentration in sterile, pyrogen-free saline. The actual number of organisms inoculated was verified later from duplicate spread plates on TSA.
Morphine and withdrawal treatment. Mice were anesthetized by isoflurane inhalation, and an area of the back was shaved. A 1-cm incision was made in the skin of the back, and mice were implanted subcutaneously with either a 75-mg slow-release morphine pellet or a cellulose placebo pellet (National Institute on Drug Abuse, Rockville, MD) sealed in a nylon mesh bag to permit easy removal later. Slow-release pellets are a preferred method of opioid delivery in studying chronic administration (7, 8, 11), as the continuous presence of drug prevents opioid withdrawal (9). Blood levels are in the 0.6- to 2-μg/ml range (8). Pellets were removed surgically 96 h later, at which time the animals were dependent on morphine (31). Removal of the morphine pellets initiates spontaneous withdrawal (abstinence syndrome) in these animals, based on our previous work (31). Placebo pellets were also removed at 96 h with no overt symptoms.
Microbial assessment. To assess spontaneous bacterial infection, blood, peritoneal lavage fluid (PLF), Peyer's patches (PPs), mesenteric lymph nodes (MLNs), spleens, and livers were collected from individual animals at the desired time points after withdrawal. Tissues of individual animals were homogenized in 5 ml of ice-cold phosphate-buffered saline (PBS), using a Tekmar tissuemizer (Tekmar Co., Cincinnati, Ohio). Homogenates were spread onto TSA II 5% sheep blood agar plates and incubated at 37°C overnight, and the numbers of CFU were counted and recorded. Typical individual bacterial colonies were subcultured and identified by the Clinical Microbiology Laboratory at Temple University Hospital using a semiautomated microbial identification system (Biomerieux, Inc., Hazelwood, Mo.).
Oral Salmonella infection and assessment of host susceptibility. At 24 h post-removal of the pellets, animals were anesthetized using isoflurane and inoculated orally with 1 x 104 CFU of log-phase Salmonella in 0.2 ml saline. A 21-gauge needle with a fine polished blunt end was used to deliver organisms into the stomach. At desired time points after inoculation, mice were bled from the retroorbital plexus under isoflurane anesthesia before being sacrificed. PPs, MLNs, and spleens were aseptically removed from each animal, and homogenates were prepared using the protocol described above. A 0.1-ml sample of serially diluted homogenate in PBS was plated on Levine eosin-methylene blue agar plates (Becton-Dickinson, Sparks, MD) and incubated at 37°C overnight, and the number of colonies was counted and recorded. Resistance to infection was also assessed by survival rate and mean survival time (MST) over a period of 30 days.
Preparation of tissue homogenates for cytokine determination. To prepare spleen extracts used for the cytokine assessment, aliquots of spleen homogenates from individual mice were clarified by centrifugation at 13,000 rpm for 20 min at 4°C using a bench-top centrifuge (model 5415R; Eppendorf), and the supernatants were stored at –80°C.
Cytometric bead array assay. Levels of multiple cytokines in spleen homogenates of individual mice were assessed simultaneously by flow cytometry using the cytometric bead array technique (BD Biosciences, San Diego, CA). The assay was carried out according to the manufacturer's instructions. Briefly, capture beads specific for the various cytokines were mixed together to form a bead suspension. Fifty-microliter aliquots of the bead suspension were then incubated with a 50-μl sample collected from an individual mouse and 50 μl of a phycoerythrin-conjugated antimurine detection reagent for 2 h at room temperature in the dark. One ml of wash buffer was then added to each assay tube and centrifuged at 200 x g for 6 min. Supernatants were discarded, and 200 μl of wash buffer was added to resuspend the bead pellet. The prepared tubes were analyzed on a BD FACScan flow cytometer (BD PharMingen, San Diego, CA) calibrated with cytometer setup beads provided by the manufacturer. The phycoerythrin intensities of each sample were converted to concentration values of cytokines using computer software.
Statistical analysis. All experiments used a completely randomized design with each preparation evaluated once. The null hypothesis was that there would be no difference between the morphine and placebo groups. Data were tested for normality using the Shapiro-Wilk test (3). The data were significantly nonnormal for all variables. In order to apply parametric methods, a "normalized-rank" transformation was applied to the data (13, 19). Ratios of positive bacterial cultures between groups were analyzed as differences between Poisson rates. The rank-transformed data were analyzed using an independent t test with equal or unequal variances. Survival data were analyzed using the Kaplan-Meier product limit method for right-censored data followed by the log rank test to compare group survival functions. Differences between groups (rejection of the null hypothesis) were considered significant if the probability of chance occurrence was 0.05 using two-tailed tests.
RESULTS
Increased incidence of sepsis in mice undergoing withdrawal from morphine. To assess sepsis, various tissues of mice undergoing withdrawal from morphine or placebo for 48 h were cultured for bacterial growth. As shown in Fig. 1, most morphine-withdrawn mice showed bacterial growth in PPs (4/5), MLNs (4/5), spleens (4/10), livers (6/10), and PLF (8/10). In contrast, no placebo-withdrawn mice showed bacterial growth in the MLNs, spleens, or peritoneal cavity. Samples of PP and liver tissue from only one placebo-withdrawn mouse tested positive for bacteria. The most frequently detected bacterial species in the cultures were identified as Enterococcus faecium followed by Klebsiella pneumoniae (Table 1), both being part of the normal gastrointestinal flora. These results clearly demonstrate that mice undergoing withdrawal for a period of 48 h from a morphine pellet, but not a placebo pellet, become septic.
Additional experiments were carried out to ascertain the time course of extraintestinal growth of enteric bacteria in organs of mice in withdrawal from morphine. Table 2 presents data on microbial burdens in various tissues on days 1, 3, and 6 after withdrawal. On day 1, bacterial growth was observed in MLNs (5/5) and livers (3/5), and one mouse (1/5) was bacteremic. No bacterial growth was found in placebo-withdrawn mice in the liver or blood, and only one placebo mouse showed bacteria in the MLN. On days 3 and 6, bacterial growth was still observed in livers, MLNs, and blood from morphine-withdrawn mice. No bacteria or bacteremia was found in placebo-withdrawn mice. Overall, only 1/45 placebo-pelleted mice had positive bacterial cultures, compared to 20/45 morphine-pelleted mice (P < 0.01). These data clearly demonstrate that morphine withdrawal markedly increased bacterial colonization in a variety of tissues from 1 day to as long as 6 days postwithdrawal. Withdrawal from a placebo pellet did not result in the same effect.
Effect of morphine withdrawal on survival of mice orally infected with Salmonella. In order to ascertain if morphine withdrawal could sensitize to an enteric pathogen, mice were orally infected with virulent Salmonella enterica serovar Typhimurium 24 h after initiation of morphine withdrawal, and survival was scored for 30 days. As shown in Fig. 2, the survival rate of infected, placebo-withdrawn mice was 47%, with death commencing on day 14 and a mean survival time of 24.0 ± 1.6 days (mean ± standard error). In contrast, in the morphine-withdrawn group death started on day 4, with a survival rate of 23%. The difference in survival rates approached statistical significance (P = 0.07). There was a statistically significant difference in mean survival time (P < 0.05). Our previous studies showed that morphine withdrawal followed by injection of saline 24 h later resulted in no mortality (0/25 mice) (16).
The more rapid mortality of morphine-withdrawn mice infected with Salmonella compared to infected, placebo-withdrawn animals was associated with markedly increased bacterial burdens in PPs, MLNs, and spleens (Fig. 3). At 24 h post-withdrawal from morphine, mice were inoculated orally with 1 x 104 CFU of Salmonella, and bacterial numbers in organs were assayed on days 2, 6, and 12 postinfection. There were no detectable Salmonella organisms in the PPs of any mice in the placebo groups over the period of 12 days. In contrast, Salmonella organisms were cultured from PPs of all morphine-withdrawn mice on day 12. For the MLNs in the placebo group, there was no bacterial growth on days 2 and 6 and only one out of five mice showed a low level of bacteria on day 12, whereas morphine-withdrawn animals had a thousandfold-greater bacterial burden at these time points. On day 12, morphine-withdrawn mice also had a significantly higher Salmonella burden in spleens than placebo-withdrawn animals. These results demonstrate that morphine withdrawal sensitizes to infection at a mucosal surface for this murine pathogen.
Morphine withdrawal increases proinflammatory cytokine production in Salmonella-infected mice. To examine the mechanism underlying the increased susceptibility to oral Salmonella infection in mice undergoing morphine withdrawal, the dynamics of production of TNF-, gamma interferon, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1) in the spleen were assessed in comparison to infected, placebo-withdrawn mice. As shown in Fig. 4, there were no changes in cytokine levels on day 2 after Salmonella inoculation in either group. On day 6 postinfection, the levels of all four proinflammatory cytokines were increased in morphine-withdrawn animals and IL-6 and MCP-1 concentrations peaked, but no change was observed in the cytokine levels of placebo-withdrawn mice. On day 12, the production of TNF-, gamma interferon, and IL-6 in both groups was increased, but the levels in morphine-withdrawn mice were higher than those in placebo-withdrawn animals. The levels of the chemokine MCP-1 were the same in morphine- and placebo-withdrawn mice on day 12. The increase in the proinflammatory cytokines is probably due to the higher bacterial burdens in the morphine-treated mice compared to controls. IL-12 and IL-10 levels were also tested but were undetectable.
DISCUSSION
The results presented in this report support the conclusion that withdrawal from morphine in an experimental mouse model results in sepsis, as organisms such as Enterococcus faecium and Klebsiella pneumoniae, which are part of the normal gut flora, are found in peripheral sites, including the PPs, MLNs, spleen, liver, and peritoneal lavage fluid. Appearance of these organisms extraintestinally is not due to administration of anesthesia or to surgical manipulation for removal of the morphine pellets, as the majority of animals that received placebo pellets and that were subjected to similar procedures were sterile at these sites. Detection of sepsis is consonant with our previous observation that mice in withdrawal from morphine are sensitized to a sublethal dose of LPS, produce more TNF- in response to LPS than mice which are withdrawn from a placebo pellet, and are protected from LPS-induced mortality by antibodies to TNF- (16). It is noteworthy that we have previously reported that mice implanted with a similar slow-release morphine pellet were septic at 48 h after pellet implantation and were also hypersensitive to sublethal doses of LPS (21). Sepsis induced by subacute administration of morphine has been confirmed by three other laboratories, using both mice and rats (6, 27, 34). Exposure of rats to morphine for 5 days was also shown to result in measurable endotoxin in serum, as well as other signs of sepsis, such as coagulopathy in the microvasculature and diminished arterial blood pressure (27). The sepsis observed following withdrawal could be the result of additive outcomes from morphine implantation and morphine withdrawal.
In contrast to studies on the effects of morphine administered acutely, subacutely, or chronically, there is a paucity of research in the area on effects of withdrawal from opioids on resistance to infection. There are only three papers in the literature in this field. Donahoe et al. (14) reported that if simian immunodeficiency virus-infected rhesus monkeys were treated chronically with morphine and withdrawn from the drug, the viral titer increased. In contrast, cats infected with feline immunodeficiency virus given morphine chronically and then withdrawn had no alteration in their levels of feline immunodeficiency virus (4). The third paper is from our laboratory and showed that mice withdrawn from morphine are sensitized to systemic (intraperitoneal) infection with Salmonella (17). The present results expand this limited data to test the effect of withdrawal on an oral bacterial infection. We have shown that withdrawal from morphine potentiates growth of Salmonella administered orally 24 h after the initiation of abstinence. This time point was chosen because it coincides with the onset of maximal systemic immunosuppression (31). When the results are compared, withdrawal from morphine produces less robust effects on oral Salmonella infection (present study) than on intraperitoneal Salmonella infection, the subject of a previous paper from our laboratory (17). The effects of withdrawal on oral Salmonella infection are also several orders of magnitude less robust than those observed when Salmonella is given orally coincident with initiation of morphine administration (25). A significant aspect of the contrast between the previous i.p. and oral challenge experiments with Salmonella relates to levels of cytokines. Using i.p. challenge, we reported in the previous paper on cytokine levels measured in plasma. In the current experiments cytokine levels were analyzed in splenic homogenates. The results of the two studies are not consonant, but the reason for this lack of agreement is not certain, since there are two variables that were different between the studies, the route of challenge and the tissue used to analyze cytokine levels. Thus, following i.p. challenge with Salmonella, IL-12 levels were suppressed in plasma but the IL-12 levels were undetectable in spleen homogenates in either placebo- or morphine-treated mice orally infected with Salmonella. The data on sensitization to Salmonella infection suggest that morphine itself and withdrawal from morphine may each affect different aspects of the immune system and gastrointestinal function that alter host resistance to Salmonella infection. For example, morphine has been shown to inhibit the mucosal immunoglobulin A response to cholera toxin (30), raising the possibility that increased sensitivity to oral Salmonella infection might be due to impaired mucosal immune responses.
The present studies are important because sepsis in the United States is a major medical problem, with an estimated 750,000 cases per year, and more than 210,000 of the cases die (mortality ranging from 30 to 50%) (2). In many cases, a precipitating cause of the septic condition cannot be identified, but sepsis primarily occurs in a hospital setting. Demonstration that mice in withdrawal from morphine are septic adds to evidence from two previous publications showing that withdrawal increases proinflammatory cytokines (16, 23) and sensitizes to the lethal effects of LPS, as measured by mortality (16). These data, along with previous observations that sepsis ensues after subacute administration of morphine (21), support the hypothesis that morphine, and possibly other opioids, administered to acutely ill patients may be a cofactor in induction of sepsis.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institute on Drug Abuse, DA14223 and DA13429.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. Phone: (215) 707-3585. Fax: (215) 707-7920. E-mail: tke@temple.edu.
REFERENCES
1. Angerman, C. R., and T. K. Eisenstein. 1980. Correlation of the duration and magnitude of protection against Salmonella infection afforded by various vaccines with antibody titers. Infect. Immun. 27:435-443.
2. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29:1303-1310.
3. Armitage, P., and G. Berry. 1994. Statistical methods in medical research, 3rd ed. Blackwell Science, Cambridge, Mass.
4. Barr, M. C., S. Huitron-Resendiz, M. Sanchez-Alavez, S. J. Henriksen, and T. R. Phillips. 2003. Escalating morphine exposures followed by withdrawal in feline immunodeficiency virus-infected cats: a model for HIV infection in chronic opiate abusers. Drug Alcohol Depend. 72:141-149.
5. Bergstrom, L., T. Sakurada, and L. Terenius. 1984. Substance P levels in various regions of the rat central nervous system after acute and chronic morphine treatment. Life Sci. 35:2375-2382.
6. Bhaskaran, M., K. Reddy, S. Sharma, J. Singh, N. Radhakrishnan, A. Kapasi, and P. C. Singhal. 2001. Morphine-induced degradation of the host defense barrier: role of macrophage injury. J. Infect. Dis. 184:1524-1531.
7. Bryant, H. U., E. W. Bernton, and J. W. Holaday. 1988. Morphine pellet-induced immunomodulation in mice: temporal relationships. J. Pharmacol. Exp. Ther. 245:913-920.
8. Bryant, H. U., B. C. Yoburn, C. E. Inturrisi, E. W. Bernton, and J. W. Holaday. 1988. Morphine-induced immunomodulation is not related to serum morphine concentrations. Eur. J. Pharmacol. 149:165-169.
9. Cerletti, C., S. H. Keinath, M. M. Reidenbery, and M. W. Adler. 1976. Chronic morphine administration: plasma levels and withdrawal syndrome in rats. Pharmacol. Biochem. Behav. 4:323-327.
10. Chao, C. C., B. M. Sharp, C. Pomeroy, G. A. Filice, and P. K. Peterson. 1990. Lethality of morphine in mice infected with Toxoplasma gondii. J. Pharmacol. Exp. Ther. 252:605-609.
11. Cheney, D. L., and A. Goldstein. 1971. Tolerance to opioid narcotics: time course and reversibility of physical dependence in mice. Nature 232:477-478.
12. Cicero, T. J., and E. R. Meyer. 1973. Morphine pellet implantation in rats: quantitative assessment of tolerance and dependence. J. Pharmacol. Exp. Ther. 184:404-408.
13. Conover, W. J., and R. L. Iman. 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35:124-129.
14. Donahoe, R. M., L. D. Byrd, H. M. McClure, P. Fultz, M. Brantley, F. Marsteller, A. A. Ansari, D. Wenzel, and M. Aceto. 1993. Consequences of opiate-dependency in a monkey model of AIDS. Adv. Exp. Med. Biol. 335:21-28.
15. Eisenstein, T. K., L. M. Killar, and B. M. Sultzer. 1984. Immunity to infection with Salmonella typhimurium: mouse-strain differences in vaccine- and serum-mediated protection. J. Infect. Dis. 150:425-435.
16. Feng, P., J. J. Meissler, Jr., M. W. Adler, and T. K. Eisenstein. 2005. Morphine withdrawal sensitizes mice to lipopolysaccharide: elevated TNF-alpha and nitric oxide with decreased IL-12. J. Neuroimmunol. 164:57-65.
17. Feng, P., Q. M. Wilson, J. J. Meissler, Jr., M. W. Adler, and T. K. Eisenstein. 2005. Increased sensitivity to Salmonella enterica serovar Typhimurium infection in mice undergoing withdrawal from morphine is associated with suppression of interleukin-12. Infect. Immun. 73:7953-7959.
18. Friedman, H., C. Newton, and T. W. Klein. 2003. Microbial infections, immunomodulation, and drugs of abuse. Clin. Microbiol. Rev. 16:209-219.
19. Harter, H. L. 1961. Expected values of normal order statistics. Biomtrika 48:151-165.
20. Haverkos, H. W., and W. R. Lange. 1990. Serious infections other than human immunodeficiency virus among intravenous drug abusers. J. Infect. Dis. 161:894-902.
21. Hilburger, M. E., M. W. Adler, A. L. Truant, J. J. Meissler, Jr., V. Satishchandran, T. J. Rogers, and T. K. Eisenstein. 1997. Morphine induces sepsis in mice. J. Infect. Dis. 176:183-188.
22. Horsburgh, C. R., Jr., J. R. Anderson, and E. J. Boyko. 1989. Increased incidence of infections in intravenous drug users. Infect. Control Hosp. Epidemiol. 10:211-215.
23. Kelschenbach, J., R. A. Barke, and S. Roy. 2005. Morphine withdrawal contributes to Th cell differentiation by biasing cells toward the Th2 lineage. J. Immunol. 175:2655-2665.
24. Louria, D. B., T. Hensle, and J. Rose. 1967. The major medical complications of heroin addiction. Ann. Intern. Med. 67:1-22.
25. MacFarlane, A. S., X. Peng, J. J. Meissler, Jr., T. J. Rogers, E. B. Geller, M. W. Adler, and T. K. Eisenstein. 2000. Morphine increases susceptibility to oral Salmonella typhimurium infection. J. Infect. Dis. 181:1350-1358.
26. McCarthy, L., M. Wetzel, J. K. Sliker, T. K. Eisenstein, and T. J. Rogers. 2001. Opioids, opioid receptors, and the immune response. Drug Alcohol Depend. 62:111-123.
27. Ocasio, F. M., Y. Jiang, S. D. House, and S. L. Chang. 2004. Chronic morphine accelerates the progression of lipopolysaccharide-induced sepsis to septic shock. J. Neuroimmunol. 149:90-100.
28. Paronis, C. A., and S. G. Holtzman. 1992. Development of tolerance to the analgesic activity of mu agonists after continuous infusion of morphine, meperidine or fentanyl in rats. J. Pharmacol. Exp. Ther. 262:1-9.
29. Patrick, G. A., W. L. Dewey, F. P. Huger, E. D. Daves, and L. S. Harris. 1978. Disposition of morphine in chronically infused rats: relationship to antinociception and tolerance. J. Pharmacol. Exp. Ther. 205:556-562.
30. Peng, X., J. J. Cebra, M. W. Adler, J. J. Meissler, Jr., A. Cowan, P. Feng, and T. K. Eisenstein. 2001. Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice. J. Immunol. 167:3677-3681.
31. Rahim, R. T., M. W. Adler, J. J. Meissler, Jr., A. Cowan, T. J. Rogers, E. B. Geller, and T. K. Eisenstein. 2002. Abrupt or precipitated withdrawal from morphine induces immunosuppression. J. Neuroimmunol. 127:88-95.
32. Risdahl, J. M., K. V. Khanna, P. K. Peterson, and T. W. Molitor. 1998. Opiates and infection. J. Neuroimmunol. 83:4-18.
33. Risdahl, J. M., P. K. Peterson, C. C. Chao, C. Pijoan, and T. W. Molitor. 1993. Effects of morphine dependence on the pathogenesis of swine herpesvirus infection. J. Infect. Dis. 167:1281-1287.
34. Roy, S., R. G. Charboneau, and R. A. Barke. 1999. Morphine synergizes with lipopolysaccharide in a chronic endotoxemia model. J. Neuroimmunol. 95:107-114.
35. Schwacha, M. G., J. J. Meissler, Jr., and T. K. Eisenstein. 1998. Salmonella typhimurium infection in mice induces nitric oxide-mediated immunosuppression through a natural killer cell-dependent pathway. Infect. Immun. 66:5862-5866.
36. Singal, P., A. G. Kinhikar, S. Singh, and P. P. Singh. 2002. Neuroimmunomodulatory effects of morphine in Leishmania donovani-infected hamsters. Neuroimmunomodulation 10:261-269.
37. Singh, P. P., S. Singh, G. P. Dutta, and R. C. Srimal. 1994. Immunomodulation by morphine in Plasmodium berghei-infected mice. Life Sci. 54:331-339.
38. Tubaro, E., G. Borelli, C. Croce, G. Cavallo, and C. Santiangeli. 1983. Effect of morphine on resistance to infection. J. Infect. Dis. 148:656-666.
39. Wang, J., R. A. Barke, R. Charboneau, and S. Roy. 2005. Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection. J. Immunol. 174:426-434.
40. Way, E. L., H. H. Loh, and F. H. Shen. 1969. Simultaneous quantitative assessment of morphine tolerance and physical dependence. J. Pharmacol. Exp. Ther. 167:1-8.(Pu Feng, Allan L. Truant, Joseph J. Meis)