Cytokines and T-cell responses in superantigen-related glomerulonephritis following methicillin-resistant Staphylococcus aureus infection

Keigyou Yoh1, Masaki Kobayashi2, Naoto Yamaguchi1, Kouichi Hirayama1, Takashi Ishizu1, Shuichi Kikuchi1, Satoshi Iwabuchi1, Kaori Muro1, Sohji Nagase1, Kazumasa Aoyagi1, Makoto Kondoh1, Katsumi Takemura3, Kunihiro Yamagata4 and Akio Koyama1,

1 Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, 2 Department of Nephrology, Tokyo Medical University Kasumigaura Hospital, Ibaraki, 3 Department of Nephrology, Kamitsuga General Hospital, Tochigi and 4 Department of Nephrology, Hitachi General Hospital, Ibaraki, Japan



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. We have previously reported that 10 patients who developed glomerulonephritis (GN) in association with methicillin-resistant Staphylococcus aureus (MRSA) infection showed a marked increase in DR+CD4+ and DR+CD8+ subsets of T cells and in T cells expressing several T-cell receptor (TCR) Vß+cells, perhaps representing Vß-specific T-cell activation by MRSA-derived superantigens (Kidney Int 1995; 47: 207–216). In this study we examine cytokine levels, T-lymphocyte subsets, natural killer NK cells, memory T cells, and the expression of IL-2 receptors in order to better understand the role of bacterial superantigens and cytokines in the pathogenesis of MRSA-associated GN.

Methods. Twenty-two patients with MRSA infection who later developed GN caused by staphylococcal enterotoxin were evaluated immunologically in comparison with patients whose MRSA infection was not followed by GN (non-GN group) and normal individuals.

Results. Among peripheral lymphocytes, the frequency of T cells expressing several TCR Vßs, especially Vß5-family TCR, was higher in the GN group than in both the non-GN group and the normal healthy control group. GN patients also showed increased serum levels of several cytokines, including tumour necrosis factor-{alpha} (TNF-{alpha}), interleukin-1ß (IL-1ß), IL-2, IL-6, IL-8, and IL-10, which have been implicated in the onset of nephritis. Memory cells, and IL-2 receptors also were elevated in the GN group.

Conclusion. These results suggest that T cells activated by MRSA-derived staphylococcal enterotoxins and subsequent production of cytokines may play an important role in the pathogenesis of MRSA-associated GN.

Keywords: enterotoxin; glomerulonephritis; MRSA; superantigen



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Methicillin-resistant strains of Staphylococcus aureus (MRSA) were initially detected in 1961 in the United Kingdom [1] shortly after methicillin came into clinical use. Patients with MRSA infection frequently develop septicaemia or toxic shock syndrome, both caused by staphylococcal enterotoxins. Staphylococcal enterotoxins are described as ‘superantigens’ that stimulate proliferation of resting T cells [2], resulting in massive T-cell activation and release of T-cell-derived cytokines such as interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor (TNF) as well as interferon-{gamma} (INF-{gamma}) [37].

We have previously reported that patients who developed glomerulonephritis (GN) after MRSA infection showed a marked increase in both DR+CD4+ and DR+CD8+ subsets of T cells and in T cells expressing several T-cell receptor (TCR) Vß+cells, perhaps representing Vß-specific T-cell activation by MRSA-derived superantigens [8]. We have also previously reported that an MRSA-associated GN case had several elevated cytokines [9]. In the present report, we have further studied immunological characteristics of 22 patients with MRSA infection who developed GN. We assessed cytokine levels, T-lymphocyte subsets, natural killer (NK) cells, memory T cells, and the expression of IL-2 receptors in order to clarify the role of superantigens and the immune response in GN associated with MRSA infection.



   Subjects and methods
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 Subjects and methods
 Results
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Patients
We studied 32 patients who had MRSA infection, including 22 who developed GN in association with MRSA infection (GN group). The GN group included 20 men and two women aged 65.4±11.8 years (mean±SD; range 43–86). Ten other patients, six men and four women aged 59.5±13.7 years (range 29–79), were infected with MRSA but did not develop GN (non-GN group) (Table 1Go). None of the individuals was serologically positive for cytomegalovirus or Epstein–Barr virus. The healthy controls included 20 men and 20 women in whom abnormal renal function or proteinuria had never been detected. The criteria of clinical syndrome were defined by WHO standards [10].


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Table 1. Patient profiles and renal function

 

Bacteriologic analysis
Blood, urine, sputum, and various fluid samples from patients were cultured, and sensitivity of bacteria to antibiotics including methicillin was tested using an antibiotic sensitivity kit (Eiken Kagaku, Tokyo, Japan). Culture supernatants were assayed for a staphylococcal enterotoxin, using a reverse passive latex agglutination method (Denka Seiken, Tokyo, Japan). Coagulase types were determined using a commercially available detection kit (Denka Seiken).

Lymphocyte subsets
Peripheral blood cells were obtained from GN patients, non-GN patients, and healthy controls. Peripheral blood samples from GN patients were obtained within 6 weeks of GN onset, which were collected at the maximum level of proteinuria or serum creatinine. Mononuclear cells were isolated by density centrifugation. Lymphocyte subsets were analysed on a FACScan flow cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, CA). The following biotin-, phyco-erythrin (PE)- or FITC-labelled monoclonal antibodies were used: anti-HLA-DR-biotin, anti-CD4-biotin, anti-CD3-PE, anti-CD4-PE, anti-CD8-PE, anti-CD16-PE, anti-CD45RO-PE, anti-CD56-PE, anti-CD3-FITC, anti-CD4-FITC, anti-CD25-FITC (obtained from Beckton Dickinson) and anti-Vß-FITC including Vß5.1, Vß5.2+5.3, Vß5.3, Vß6, Vß8, Vß12 (obtained from Endogen, Woburn, MA). CD4+ antibodies were used as follows: anti-CD4-PE was used for DR+CD4+T cells and IL-2 receptor (DR+CD4+CD25+), anti-CD4-biotin for TCR Vß+T cell, and anti-CD4-FITC for memory cells (DR+CD45RO+CD4+).

Detection of cytokines
Serum levels of IL-2, IL-4, IL-6, TNF-{alpha}, and INF-{gamma} were measured by ELISA using the Cytoscreen Immunoassay Kit, (BioSource International Camarillo, Camarillo, CA). Serum levels of IL-1-{alpha}, IL-1-ß, IL-8, IL-10, and TNF-ß were also measured by Quantikine ELISA kits (R&D Systems; Minneapolis, MN).

Statistical analyses
Statistical analysis for T-cell subsets was performed by analysis of variance (ANOVA) and by the Mann–Whitney test. Differences in enterotoxins between patients with GN and without GN were analysed by Fisher's exact probability test. Statistical significance was assumed at P<0.05.



   Results
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 Subjects and methods
 Results
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Patient characteristics
GN occurred either acutely or subacutely during MRSA infection. Eighteen patients showed features of rapidly progressive GN; of whom eleven had nephrotic syndrome. Three other patients had nephrotic syndrome, and one patient had acute nephritic syndrome. Proteinuria associated with haematuria appeared within 6 weeks of the onset of MRSA infection (20.1±11.6 days) in all patients with GN. Cutaneous purpura was observed in three patients.

Bacteriological analysis
MRSA infection was detected in all GN patients; Streptococcus pyogenes was additionally detected in a single patient. Type II coagulase appeared in all patients tested (16/16). Staphylococcal enterotoxin C was detected in 14 of 18, staphylococcal enterotoxin A in eight of 18, and toxic shock syndrome toxin (TSST)-1 in nine of 18. Staphylococcal enterotoxin B was detected in three patients, and enterotoxin D was detected in one patient. The relationship between type of staphylococcus enterotoxin and elevated subsets of TCR is presented in Table 2Go. For example, of the eight cases with enterotoxin A, six cases had significantly increased levels of Vß5.1, while seven cases had significantly elevated levels of Vß8. For enterotoxin C, the ratios were as follows: Vß8 (11/14), Vß5.2+5.3 (10/14), Vß5.3 (7/14), Vß5.1 (14/14). Of the nine cases with TSST-1, eight had increased concentrations of Vß8 (Table 2Go). Since multiple enterotoxins were detected in some patients, we could not determine which enterotoxin was involved in elevating TCRVß.


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Table 2. Staphylococcus enterotoxins and TCR subsets in the GN group

 

Lymphocyte subsets
Percentages of DR+CD4+ and DR+CD8+ T cells were markedly increased in the GN group compared with healthy controls and the non-GN group (Table 3Go). TCR Vß subsets Vß5.1 and Vß5.2+5.3 were significantly elevated in the GN group relative to healthy controls and the non-GN group. TCR Vß5.3, Vß6, and Vß8 levels were higher in the GN group than in healthy controls. NK cells (DR+CD16+CD56+CD3-), memory cells (DR+CD45RO+CD4+), and cells with the IL-2 receptor (DR+CD4+CD25+) were significantly higher in the GN group than in control subsets. The NK cell subset was significantly increased in the non-GN group compared with the GN group. NK and memory cells in the non-GN group were also significantly higher than in controls.


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Table 3. Comparison of lymphocytes subsets with the GN group (GN), the non-GN group (non-GN), and healthy controls (controls)

 

Cytokine analysis
Serum levels of TNF-{alpha} and IL-8 were significantly higher in the GN group than in the non-GN group. Serum levels of TNF-{alpha}, IL-2, IL-6, IL-8, and IL-10 were significantly higher in the GN group than in healthy controls (Table 4Go). In the GN group, INF-{gamma} and IL-1ß were higher than in the other groups, but differences were not statistically significant. TNF-ß and IL-4 were not detected in any group.


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Table 4. Cytokine profiles in the GN group (GN), the non-GN group (non-GN), and healthy controls (controls)

 



   Discussion
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 Subjects and methods
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 References
 
We have previously reported that 10 patients who developed GN after MRSA infection exhibited bacterial infection producing staphylococcal enterotoxins which act as superantigens [8]. In that report, we also described polyclonal increases of IgA and IgG, with high levels of circulating immune complexes in those GN patients. In the present study we found several elevated cytokines, and these cytokines might play some role in the pathogenesis of GN.

Many cytokines, including IL-1, IL-2, IL-4, IL-6, IL-8, and IL-10, as well as other cytokines such as TNF and INF-{gamma}, are known to be induced by superantigens [37]. IL-1, IL-6, IL-8, and TNF may also be induced by inflammatory reactions [11]. We detected high levels of many cytokines such as TNF-{alpha}, IL-2, IL-6, IL-8, and IL-10 in the GN group, but did not detect IL-4 in serum from these patients. IL-4 may have been present at levels below the detectable range. These elevated cytokines may be induced by superantigens or infection. Interestingly, however, a significant difference was found between the infection groups and normal individuals in terms of increases in serum levels of IL-2, possibly attributable to superantigens, suggesting a role for superantigens in the MRSA infected groups.

In this study, we investigated different types of enterotoxin. Five types of enterotoxins were detected in the GN group. The relationship between the types of enterotoxins and cytokines in vitro was described by Sperber et al. [12]. They reported that (i) TSST-1 stimulated more IL-2, IFN-{gamma} and TNF production than staphylococcal enterotoxin B in peripheral blood mononuclear cells, and (ii) in contrast, staphylococcal enterotoxin B stimulated more IL-6 production than TSST-1. In our study, however, although multiple enterotoxins were detected in some patients, we could not determine which cytokines were stimulated by any enterotoxin.

TNF-{alpha}, TGF-ß, IL-1, IL-4, IL-6, IL-8, and IL-10 have been shown to be associated with nephritis [13,14]. Serum levels of TNF-{alpha}, IL-6, IL-8, and IL-10 in the GN group were even higher than in normal individuals. Serum levels of TNF-{alpha} and IL-8 in the GN group were higher than the non-GN group. Thus, TNF-{alpha} and IL-8 may play some role in the onset of GN. The relationships between Th1/Th2 cells and the roles of cytokines induced by superantigens have also been described [15,16]. IL-2 produced by Th1 cells and IL-10 produced by Th2 cells were also elevated in the GN group. We lack conclusive data as to which cytokines are elevated first. This question has not been clarified, but it may be useful to determine the behaviour of Th1 and Th2 cells in MRSA-associated GN.

Previous reports have established the involvement of superantigens in the elevation of NK cells [7], memory T cells [17], and the expression of IL-2 receptors [18]. The present study also examined the levels of these three factors. Higher percentages of memory T cells, NK cells, and IL-2 receptors were detected in the GN group than in normal controls, and presumably were induced by superantigens. Compared with the controls, the expression of IL-2 receptors was significantly higher in the GN group, but not in the non-GN group. The relationship between IL-2 and IL-2 receptors with staphylococcal enterotoxin A stimulation was described by Carlsson and Sjögren [18]. The degree of IL-2 receptor increase might be linked to the elevated IL-2 level in the MRSA-infected groups. On the other hand, the levels of NK cells in the GN group was lower than in the non-GN group. We could not precisely clarify why the NK cells were lower in the GN group. Further work is required to elucidate the precise role of NK cells, such as whether they promote or inhibit GN.

Based on the results of this study and the previous report [8], we propose a possible mechanism of the pathogenic sequence for the onset of GN. In this scenario, enterotoxins are released from ongoing MRSA infection. Acting as superantigens, these enterotoxins bind directly to the MHC class II molecule of antigen-presenting cells. The enterotoxin–MHC class II complex then binds to the TCR Vß region without MHC restriction. These processes result in massive T-cell activation. NK cells and memory T cells are also activated. Consequently, cytokines such as TNF-{alpha}, IL-6, IL-8, and IL-10 are released from T cells and monocytes. These cytokines may directly damage renal tissues. Alternatively, some cytokines may induce polyclonal activation of IgA and IgG, resulting in immune complex formation.

We were not able to clarify differences in immunogenetic characteristics between the GN group and the non-GN group, except for the amounts of IgA immune complexes, serum levels of TNF-{alpha}, IL-8, percentage of TCR Vßs, and NK cells. Several questions which remain to be resolved include the differences in HLA isotype between GN and non-GN patients.

In conclusion, our results suggest that T cells and cytokines activated by staphylococcal enterotoxins may play an important role in MRSA-associated GN. These findings may help to clarify the relationship between cytokines and other diseases linked to superantigens as well as MRSA-associated GN.



   Acknowledgments
 
This study was supported by Disease Control Division, Health Service Bureau, Ministry of Health and Welfare, Japan; a Program Project grant from the Ministry of Health and Welfare of Japan; and a research grant from University of Tsukuba. We thank Professor Guy Neild of the Middlesex Hospital, London, University College London Medical School, and Professor Yousuke Takahama, Institute for Genome Research, University of Tokushima, who provided us with invaluable guidance, encouragement and support.



   Notes
 
Correspondence and offprint requests to: Akio Koyama, MD, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki 305–8575, Japan. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 3. 5.99
Revision received 10. 3.00.