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Characterization of Cytokine/Chemokine Profiles of Severe Acute Respiratory Syndrome
     Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University

    Guangzhou Institute of Respiratory Diseases, Guangzhou

    Department of Biochemistry, Hong Kong University of Science and Technology, Hong Kong Special Administrative Region, China

    ABSTRACT

    Rationale: There is currently no optimal treatment or effective drug for severe acute respiratory syndrome (SARS), because the immunopathologic mechanism is poorly understood. Objectives: To explore the immune mechanism underlying the pathogenesis of SARS, we studied the expression profile of cytokines/chemokines in the blood and the immunopathology of the lung and lymphoid tissues. Methods: Fourteen cytokines/chemokines in the blood of 23 patients with SARS were dynamically screened, using a bead-based multiassay system. Reverse transcription-polymerase chain reaction was performed to amplify mRNA. Histopathology of the lung and lymphoid tissues at autopsy was examined, using methods of immunohistochemistry and double immunofluorescence staining. Main Results: Interferon-inducible protein-10 (IP-10) was markedly elevated in the blood during the early stage of SARS, and remained at a high level until convalescence. Moreover, IP-10 was highly expressed in both lung and lymphoid tissues, where monocyte-macrophage infiltration and depletion of lymphocytes were observed. The levels of interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 were concomitantly increased in the blood of the patients with superinfection, and the mRNAs for these cytokines were also increased in lung tissues. Conclusions: Induction of IP-10 is a critical event in the initiation of immune-mediated acute lung injury and lymphocyte apoptosis during the development of SARS. Superinfection after the immune injury is the main cause of death. The prompt elevation of interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 is a sign of superinfection, indicating a high risk of death.

    Key Words: chemokine coronavirus cytokine severe acute respiratory syndrome

    Severe acute respiratory syndrome (SARS) is a newly identified clinical entity that was first recognized in Guangdong Province, China in November 2002 (1eC3). SARS has become a threat to the international community, although the spread of infection has been effectively contained (4). Through the collaboration of an international laboratory network, a novel coronavirus (SARS-CoV) was identified as the causative agent for SARS (1eC3, 5, 6). Patients with SARS are clinically characterized by fever, dry cough, headache, dyspnea, hypoxemia, lymphopenia, and radiographic evidence of pneumonia (7eC10). Death may result from progressive respiratory failure due to alveolar damage or multiple organ dysfunction caused by systemic inflammation (11eC16). Although steroids and antiviral drugs were tried clinically, no treatment has clearly been confirmed to be effective (7eC9, 17, 18). The main reason for this problem is a lack of knowledge about the molecular mechanism(s) underlying the pathogenesis of SARS (8, 9, 19). To understand the mechanism of cellular and humoral immunity to SARS-CoV, we studied the expression profile of cytokines/chemokines in the blood of patients with SARS and the immunopathology of their lung and lymphoid tissues.

    METHODS

    Case Selection

    Ninety patients were diagnosed with SARS and treated between February and May 2003 at Guangzhou Institute of Respiratory Diseases in China. All of them met a modified World Health Organization case definition of SARS (7, 10) and had been confirmed retrospectively by the detection of specific antibody for SARS-CoV. Out of the total of 90 SARS cases, 23 previously healthy adults, comprising 15 males and 8 females, aged 27eC55 years (mean, 36 ± 6.1 years), all ethnic Chinese, were enrolled into the current study.

    Serum specimens were taken at intervals from the patients and distributed into four groups, according to the defined stages of the disease course: early stage (E), the first 2 days after the onset of fever; progressive stage (P), progressive symptoms and high levels of lactate dehydrogenase (LDH), 10eC20 days after onset; end stage (D), the day before death; and convalescent stage (C), 15eC30 days after discharge from hospital. Ten serum samples from healthy adults, aged 22eC51 years (mean, 32 ± 6.8 years), were obtained from the Health Examination Center and were distributed to Group N as normal controls.

    Twenty-five blood samples taken from non-SARS patients with atypical pneumonia at the early stage (2 days after onset) of illness were also subjected to cytokine/chemokine assay.

    Detailed information about the patients is available in Table E1 (see the online supplement).

    Approval by the local ethics committees and informed consent were obtained.

    Cytokine/Chemokine Assay

    Quantification of 14 cytokines/chemokines was performed with the LiquiChip system (Qiagen, Hilden, Germany) (20, 21).

    Autopsy Materials

    Full autopsies were performed on five patients who died of SARS (samples from two of them were included in the cytokine/chemokine assay) for detailed pathology studies; lung tissues of the autopsy patients were also taken for reverse transcription-polymerase chain reaction (RT-PCR). The lung tissue of a patient who died in a traffic accident was taken as a control.

    Immunostaining

    Immunostaining of the lung and lymph node was performed with a monoclonal antibody to interferon-inducible protein-10 (IP-10) (Abcam, Cambridge, UK), as well as with monoclonal antibodies (DakoCytomation, Carpinteria, CA) to CD20, CD3, CD4, CD8, and CD68. To identify the type of cell expressing IP-10, double immunofluorescence staining was performed with primary polyclonal antibody to either CD3 or CD68 (Santa Cruz Biotechnology, Santa Cruz, CA) plus a monoclonal antibody to IP-10, and secondary antibodies of goat anti-rabbit IgG conjugated with tetramethylrhodamine isothiocyanate and goat anti-mouse IgG conjugated with fluorescein isothiocyanate (Sigma, St. Louis, MO) (22).

    RT-PCR

    Total RNA was extracted from lung tissues, using an RNA extraction kit (Monotest; Amplimedical Bioline, Turin, Italy). PCR amplification was performed with specific primers (Table E2) on the resulting cDNA at 94°C for 30 seconds, 56°C for 15 seconds, 72°C for 2 minutes, for 30 cycles.

    Measurement of Receptor Expression on Cells

    Peripheral blood mononuclear cells from healthy control samples were purified on density gradients for identification of the receptor for SARS-CoV. The isolated cells were incubated with a mouse monoclonal antibody against human angiotensin-converting enzyme-2 (ACE2) (R&D Systems, Minneapolis, MN), followed by fluorescein isothiocyanate-labeled goat anti-mouse IgG Fc (Sigma), and analyzed by flow cytometry.

    Statistical Analysis

    Data were expressed as means ± SD. Mean values were compared by one-way analysis of variance and multicomparison was done. The ManneCWhitney rank sum test was used to assess differences between patients with SARS with and without superinfection. All the data were analyzed with the Statistical Package for Social Sciences (version 11.0; SPSS, Chicago, IL). p < 0.05 was considered statistically significant.

    RESULTS

    Death Rate of Patients with SARS

    Twenty of the 90 patients with SARS developed secondary infection during the course of their illness, with a death rate of 50% (10 of 20); the other 70 cases had no secondary infection and none of them died.

    Of the 23 patients enrolled in this study, 11 cases were selected from among the 20 patients with SARS who developed secondary infections, with a death rate of 45% (5 of 11), and the other 12 were selected from the 70 cases of nonsecondary infection. Routine microbiological investigation showed no cross-infections with bacteria, chlamydia, mycoplasma, and respiratory virus on admission of the 23 patients. All of them manifested typical characteristics of SARS, even though the severity of their clinical presentation differed from mild to severe disease as shown in Table E3.

    Cytokines/Chemokines in the Blood of Patients with SARS at Different Stages of Illness

    To check the expression profiles of cytokines/chemokines in response to SARS-CoV infection, 14 cytokines/chemokines were assayed in serum samples taken from patients with SARS at different stages of the illness. The levels of interleukin (IL)-1, IL-2, IL-4, IL-10, IL-12, tumor necrosis factor- (TNF-), granulocyte-macrophage colony-stimulating factor, interferon- (IFN-), and regulated on activation, normal T cell expressed and secreted (RANTES) showed no significant changes compared with those in normal control samples. Unexpectedly, we found that the level of IP-10 was significantly increased in Groups E, P, and D (p < 0.001) but not in Group C, compared with that in Group N. IL-6 was elevated significantly only in Group D (p = 0.006), but not in Groups E, P, and C, compared with normal control samples. IL-8 and monocyte chemoattractant protein-1 (MCP-1) increased significantly in Group P (p = 0.024 and 0.038, respectively) and Group D (p = 0.023 and 0.025, respectively), compared with Group N. Macrophage inflammatory protein-1 appeared to increase in Group P compared with the other groups, but no statistical significance could be detected (p = 0.34) (Figure 1).

    Comparison of Cytokine/Chemokine Profiles between Patients with SARS and non-SARS Patients with Atypical Pneumonia

    To address the specificity of cytokine/chemokine profiles of SARS, 25 non-SARS patients with atypical pneumonia were selected for the measurement of cytokines/chemokines. It was found that 17 of the 25 non-SARS patients with atypical pneumonia (68%) had no increase in IP-10 after the onset of illness; in contrast, all of the patients with SARS had a significant increase in IP-10. Although 8 of the 25 non-SARS patients with pneumonia (32%) had an elevated level of IP-10, all of them had a concomitant increase in other cytokines/chemokines. Of the 25 non-SARS patients with pneumonia, 13 had a concomitant increase in IFN-, IL-6, IL-8, MCP-1, IL-1, and RANTES and 12 had an elevation in two or three of these cytokines/chemokines to above normal values. In comparison, 22 of the 23 patients with SARS (96%) had an increase in IP-10 alone at the early stage of their illness; only 1 of the 23 patients (4%) had a high level of IP-10 with a concomitant increase in other cytokines/chemokines. So, of all the 14 cytokines/chemokines examined, the increase in IP-10 in the patients with SARS during the early stage of their illness was the unique finding.

    Effect of Secondary Infection on the Levels of Cytokines/Chemokines in the Blood of Patients with SARS

    There are three prerequisites for the diagnosis of secondary infection in patients with SARS in our institute: (1) evidence of bacterial and fungal infection obtained by biopsy; (2) detection of the same pathogen in the cultures of both blood and tracheal specimens; and (3) recurrence of fever, the development of high absolute neutrophil counts, and yellow sputum produced with in conjunction with two positive tracheal cultures of bacteria or fungus. A patient with SARS, who presented at least one of the three prerequisites and developed a new interstitial infiltrate on chest radiograph, was identified as having secondary infection. Of the 23 cases, 11 developed secondary infection. Of these 11 patients, 6 had Pseudomonas aeruginosa infection, based on positive cultures of both blood and tracheal sputum (2 cases) or positive tracheal culture alone (4 cases); 3 had Candida albicans infection, based on tracheal culture alone; and 2 had invasive Aspergillus infection, based on a positive lung biopsy. The seven patients with SARS who had a positive tracheal culture alone met the third prerequisite for the diagnosis of secondary infection. We wondered whether the change in the levels of cytokines/chemokines was associated with secondary infection during the course of illness. The levels of 14 cytokines/chemokines were compared between patients with SARS without and with secondary infection at the progressive stage of their illness. It was found that IL-6 and IL-8 were elevated significantly in the sera of patients with SARS with secondary infection (p = 0.042 and p < 0.001, respectively), but not in those without secondary infection compared with normal control samples. Similarly, MCP-1 was also increased significantly in the patients with secondary infection (p = 0.025), although it also had a tendency to rise in those without secondary infection when compared with normal control samples (p = 0.052). In contrast, the levels of IP-10 were similarly high between patients with secondary infection and those without (Figure 2). There were no significant differences in the levels of the other 10 cytokines/chemokines between the groups.

    Dynamic Changes in IP-10, Lymphocytes, and LDH in the Blood of Patients with SARS

    A complete blood count with differential counts was done on admission and then daily until the clinical symptoms had significantly improved. The patients in convalescence were studied at a follow-up visit. Lymphopenia (fewer than 1,000 lymphocytes/mm3) was noted in 88 (of 90) patients with SARS hospitalized at our institute. Most patients had a mild decrease in lymphocyte count at the onset of disease and lymphopenia occurred progressively during the course of illness (Figure 3). In the 23 cases selected for quantification of cytokines/chemokines, a significant increase in IP-10 occurred at the onset of the illness and remained at a high level during the course of the illness. Lymphopenia and elevated LDH developed progressively after the increase in IP-10. Lymphopenia reached its lowest point when LDH in the circulation rose to its highest levels.

    Cytokine/Chemokine Expression in Lung Tissues of Patients with SARS

    The majority of patients with SARS with secondary infection had increased levels in IL-6, IL-8, and/or MCP-1 in the blood, but not all of them had the same pattern of cytokine/chemokine induction. To test whether the increase in the level of cytokines/chemokines was due to mRNA induction, we performed RT-PCR and found increased mRNA expression of IP-10 in lung tissues from all autopsy patients. Of those autopsy patients, two were found to have a concomitant increase in both MCP-1 and IL-8, two showed an increase in both IL-6 and IL-8, and one presented an increase only in MCP-1. In contrast, none of the cytokines was expressed at a detectable level in the control. Figure 4 shows the results from one of the autopsy patients with SARS, in whom it was found that levels of mRNA expression for IP-10, MCP-1, and IL-8 were elevated in the lung tissue but none of the other 11 cytokines/chemokines were increased.

    Immunohistochemical Staining

    Histopathologic examination of lung tissues obtained from patients with SARS at autopsy revealed diffuse alveolar damage, fibrin exudation, and alveolar hyaline membrane formation infiltrated with inflammatory cells, most of which were CD68+ monocytes/macrophages; some were CD3+ T cells and only a few were CD8+ T cells. IP-10eCpositive staining in alveolar epithelial cells, macrophages, and lymphocytes was also observed (Figure 5). Depletion of lymphocytes was noted in the lymphoid tissues of the patients with SARS, and IP-10eCpositive staining was found in CD3+ T lymphocytes (see Figure E1 in the online supplement).

    Receptor Expression Measurement on Peripheral Blood Mononuclear Cells

    Flow cytometry was performed to check the expression of ACE2, a functional receptor for SARS-CoV, on peripheral blood mononuclear cells. Using Vero-E6 cells as a positive control, we found that ACE2 was expressed mainly by monocytes, but not lymphocytes, among peripheral blood mononuclear cells (Figure 6).

    DISCUSSION

    Immunopathologic injury of host cells triggered by the immune response to virus plays a key role in the pathogenesis of virus infections. Many cytokines/chemokines released from activated immune cells not only take part in the process of antiviral immune response, but are also involved in cell damage and development of organ dysfunction (14eC16). Determination of those soluble factors in the blood should aid our understanding of the immunopathologic processes of SARS and enable differential diagnosis of SARS from other atypical pneumonias that require quite different approaches for the management of patients (23). Although the profiles of cytokines/chemokines in patients with SARS have been reported (24eC26), there is still some controversy or uncertainty about the expression pattern of cytokines/chemokines and the initiating factor(s) in the development of SARS.

    To better understand the role of cytokines/chemokines in the immunopathologic processes of SARS, the cytokine/chemokine profiles at defined stages of the disease were analyzed. In this study, we found no increase in levels of most inflammatory cytokines/chemokines such as IFN-, TNF-, IL-1, RANTES, and so on, which have been implicated in immune reactions against viruses (27, 28). Consistently, we could not detect the mRNAs for those cytokines in the lung tissues of patients with SARS either. Interestingly, we found, without any exception, that IP-10 was augmented markedly in the blood of patients with SARS at an early stage, remained at a high level during the course of illness, and returned to normal during convalescence.

    Immunostaining showed that IP-10 was expressed in the pneumocytes as well as CD3+ T lymphocytes and monocytes/macrophages in the lungs of patients with SARS. As a non-ELR (Glu-Leu-Arg) CXC chemokine, IP-10 had been shown to be a potent chemoattractant for activated T cells, natural killer cells, and monocytes (29eC35). After viral infection, elevated IP-10 in monocytes can attract activated cytotoxic T lymphocytes as well as natural killer cells, leading to acute immune inflammation (35eC37). We found that injury to both bronchiolar and alveolar epithelial cells occurred with marked IP-10 expression, which was correlated with the recruitment of CD68+ monocytes into the interstitial lung tissues of the autopsy patients with SARS (Figure 5). IP-10 might be responsible for the infiltration of monocytes/macrophages, which is a pathologic feature in the lungs of patients with SARS (11eC13).

    We found a few CD8+ T lymphocytes in the lung tissues and depletion of lymphocytes in lymphoid tissues of patients with SARS, which was in agreement with the previous finding of lymphopenia and apoptosis of immune cells in patients with SARS (18, 19). Dynamic analysis showed that after the elevation of IP-10 that emerged at the onset of SARS, progressive lymphopenia occurred with a concomitant increase in LDH, which was in line with the depletion of lymphocytes and IP-10 expression in lymphoid tissues (Figure 3). This result is supported by a report that the selective and extensive expression of IP-10 induced by viral infections caused a rapid recruitment of activated T lymphocytes followed by apoptosis of the cells (38). Another study found that IP-10 had direct damaging effects on endothelial cells of the vasculature (39). Together, these results suggest that the increase in IP-10 might be responsible for lymphocyte apoptosis of patients with SARS.

    IP-10 can be highly induced in a variety of cells, and its expression in primary human bronchial epithelial cells is induced specifically by IFN- (40, 41). One study has reported that IFN- is elevated in the plasma of patients with SARS within the first 12 days of disease onset (24). However, we could not detect an increase in IFN- in either the blood or lungs of patients with SARS, which is consistent with a previous study showing that numbers of IFN-eCsecreting cells induced by T cell activators were below normal (25). Our results strongly suggest that IP-10 is produced in lung epithelial cells as well as immune cells by an IFN-eCindependent mechanism after SARS-CoV infection.

    One study has demonstrated that ACE2 is a functional receptor for SARS-CoV (42). Using flow cytometry, we found that ACE2 was highly expressed by monocytes, but not lymphocytes (Figure 6), indicating that monocytes might be a target for SARS-CoV, in addition to bronchial epithelial cells. Taken together, our findings strongly indicated that high IP-10 levels at an early stage of the illness might be a key event in initiating excess immune reaction in the development of lung damage and lymphocyte apoptosis.

    Lymphopenia or damage in the immune system after the development of SARS is a prerequisite for the occurrence of secondary infection. We found that, when subjected to pathogenic microorganism culture, blood and respiratory specimens from patients with SARS with secondary infection were positive for P. aeruginosa, C. albicans, and invasive Aspergillus, microorganisms that usually invade patients with a compromised immune system. One study has shown acute bronchopneumonia in five of eight autopsy patients with SARS, and infection with invasive Mucor in one patient who died with a shorter course of illness (13). These data, together with our current study, suggest that superinfection is a frequent, and the main, cause of death due to acute damage of the immune system initiated by induction of IP-10 in response to SARS-CoV.

    To understand the underlying mechanism for the swift and significant increase in some cytokines/chemokines during the course of SARS, we separated the patients complicated with superinfection from those without superinfection. It was found that there was an increase in some other cytokines/chemokines besides IP-10 in the patients with secondary infection, but not in those without secondary infection, indicating that the production of these cytokines/chemokines was induced by superinfection with other pathogens rather than SARS-CoV. Among them, only the increase in IL-6, IL-8, and MCP-1 reached statistical significance, although an increase in macrophage inflammatory protein-1, IL-1, and TNF- was detected in some individuals with secondary infection. The study performed by Wong and coworkers also detected an increase in IL-6, IL-8, and MCP-1, but there was high interindividual variation in their levels (24). Our results give a reasonable explanation for the quick shift in cytokine/chemokine profiles and suggest that both IL-8 and IL-6 are markers for superinfection of patients with SARS.

    It is well established that multiple organ dysfunction syndrome is a pivotal pathologic process in many infectious diseases (43, 44). Previous studies have revealed that high levels of cytokines/chemokines can induce cell damage and organ dysfunction (44, 45). Autopsy studies have found diffuse inflammatory damage (11eC13) in many organs during the late phase of patients with SARS with secondary infection. Our results revealed that the levels of IL-6, IL-8, and MCP-1 were elevated markedly in the patients at the end stage of their illness, suggesting that they were the main mediators involved in the death of patients with SARS with superinfection. Therefore, these cytokines/chemokines are useful markers for identifying patients with SARS at high risk of death.

    Our findings in this study are not only helpful in early diagnosis, but are also useful in judging the clinical situation of patients with SARS. Comparative analysis found that early induction of IP-10 in the blood was a prominent characteristic of the immune reaction to SARS-CoV, which is different from other, non-SARS viral infections that cause no increase in IP-10 (46). The early induction of IP-10 in the absence of other cytokines/chemokines that are usually induced in response to virus infection (27, 28) is an important feature of patients with SARS. By comparison, the "avian flu" H5N1 that also has a primary viral pneumonia presents an early induction of IP-10 with a concomitant and marked elevation of RANTES and TNF- (46, 47). Physicians should be aware of conditions mimicking SARS-CoV infection, as negative microbiologic results on SARS-CoV serology and RT-PCR become available only later (48). In the present study, we found that most patients without SARS with clinical symptoms similar to SARS could be excluded by detection of the unique profile of cytokines/chemokines during the early phase of illness. Thus, the early induction of IP-10 in the blood could be used as an alternative diagnosis marker for SARS, which should be an effective complement to the immune-based methods for the production of specific antibodies, taking 2eC3 weeks (49). On the other hand, the prompt change in cytokine/chemokine profile in the blood of patients with SARS could be used as an index of secondary infection.

    Our findings could be beneficial for the development of new anti-SARS drugs designed specifically to target IP-10, which has been used as a therapeutic target previously (50, 51). Although it is a universal reaction of immune cells to produce IFN- to control the proliferation or propagation of invading virus (52), we failed to detect any increase in IFN- in either the blood or tissues of patients with SARS. Therefore, antiviral cytokines, that is, IFN-, might be effective for patients with SARS with severe immune suppression (53, 54). This concept is supported by studies in a SARS model in which monkeys were treated with interferons (55, 56). However, the molecular mechanism for the phenomenon of IFN-eCindependent IP-10 induction remains to be investigated (57, 58).

    Acknowledgments

    The authors thank Prof. Y. Q. Ding (Department of Pathology, Southern Medical University) for providing specimens, Prof. N. S. Zhong (Guangzhou Institute of Respiratory Diseases) for providing clinical data, Prof. Jerry H. Wang (Department of Biochemistry, Hong Kong University of Science and Technology) for critical reading of this manuscript, and Dr. Donna E. Davies (Division of Infection, Inflammation and Repair, School of Medicine, Southampton General Hospital, UK) for kind help on the revision of this manuscript.

    This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

    These two authors contributed equally to this work.

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