T-cell Activation and Proliferation in a Case of Recurrent Menstrual Toxic Shock Syndrome

Zbl. Bakt. 284, 164-169 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

T-cell Activation and Proliferation in a Case of Recurrent Menstrual Toxic Shock Syndrome* MARDJAN ARVAND** and HELMUT HAHN Institut fur Medizinische Mikrobiologie und Infektionsepidemiologie der Freien Universitiit Berlin, Germany Received March 4, 1996 . Accepted March 25,1996

Abstract A case of menses-related toxic shock syndrome with a relapse within 9 days is presented. Cervical smear of the patient yielded growth of a Staphylococcus aureus strain which produced toxic shock syndrome toxin-I (TSST-I). Serum antibody against TSST-I were absent in the first sample which was obtained in the acute phase and did not increase significantly within 5 weeks. We studied the proliferative response of the patient's T cells during and after the acute phase of disease to evaluate whether they have been rendered unresponsive to TSST-1. We could not detect any reduction in the T cell proliferation at any time. Peripheral T cells of the patient were analysed flow-cytometrically at two different times to determine the expression of the T cell activity markers CD25 and HLA-DR. The expression of HLA-DR was increased during the acute phase and had declined significantly at the time of the second analysis; the CD25 expression remained constant. Introduction

Toxic shock syndrome (TSS) is a life-threatening disease caused by Staphylococcus au reus characterized by high fever, profound and refractory hypotension, rash, mental confusion, and multiorgan involvement (12). It is frequently observed in women, with onset mainly during menstruation (menstrual TSS) (2). Toxic shock syndrome toxin-1 (TSST-1), a member of the superantigen family (6), has been identified as being associated almost invariably with menstrual TSS. Although the prevalence of TSSTI-specific antibody is above 90 percent in the general population, practically all patients with menstrual TSS were found to have undetectable antibody titres against TSST-l on onset of the disease (13). We report here about a patient who suffered from recurrent menstrual TSS. The titre of serum antibody against TSST-1 was determined during the acute phase and 5 weeks later to monitor antibody production. * Dedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday.

* * Corresponding author.

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Previous studies with animal models have demonstrated that superantigens can induce anergy of T cells based on clonal deletion of the target T cells (7, 8, 10). Here, we studied the proliferative response of our patient's T cells, both during the shock phase and after 5 weeks, to evaluate whether a T cell anergy had been induced in this patient after two bouts of TSS. In a whole-blood lymphoproliferation assay, T cells were stimulated with TSST-l, with erythrogenic toxin A (ETA), a superantigen produced by Streptococcus pyogenes (3, 11), and with a lectin mitogen. The proliferative response of the patient's T cells was compared with the response of T cells from healthy donors. In-vitro and in-vivo studies have shown that superantigen-induced T cell activation results in increased expression of T cell activity markers, e. g. CD25 (IL-2 receptor) and/or HLA-DR (6, 8, 1). In this study, we analysed peripheral T cells of our patient by means of flow cytometry, both during the shock phase and 5 weeks later, to monitor the T cell activation.

Materials and Methods Case report. A 17-year-old previously healthy woman was admitted to a municipal hospital with fever (40.5°C), vomiting and diarrhoea. Blood pressure was 60/40 mmHg and the pulse frequency 160/min. Initial analysis of her venous blood revealed the following values: WBC, 15.3 x 10911; pH, 7.28; base excess, -6.2 mmoUL; bicarbonate, 20.0 mmoUL; CRP, 226 mglL; creatinine, 16.5 mg/L; bilirubin, 2 giL; prothrombin time, 38%. She was transferred to the intensive care unit and fluid and electrolyte resuscitation was initiated immediately. Cultures of blood, urine and stools were performed. She recovered soon under the symptomatic therapy and could be transferred to the regular care unit after 4 days. A papulomacular exanthema, which had occurred during the last days on the chest, back, flanks and limbs disappeared gradually. On day 9, however, the temperature increased and the blood pressure decreased again. At this time, the tentative diagnosis of a menstrual TSS was made, since the patient reported that she had used a tampon because of irregular menstrual bleeding the day before and was still carying this tampon. Likewise, it could be established that she also had used a tampon 9 days ago, at the onset of the first febrile periode. The tampon was removed, a cervical smear was performed and i. v. fluid substitution and an antibiotical therapy with flucloxallin (2 gld, i. v.) was initiated. A desquamation of the skin on her fingertips was observed on day 10. The patient recovered completely within a few days and was discharged on day 16. Microbiological cultures. All blood cultures remained sterile and the cultures of stools and urine did not reveal growth of pathogenic organisms. The cervical swab yielded growth of a TSST-1-producing Staphylococcus aureus strain. Toxin production was determined by means of a latex agglutination kit (TST-RPLA, Oxoid, England). The titre of TSST-1 in the supernatants of this strain was 1: 128. Blood collection was performed on day 9 and on day 45 after onset of the first shock. Heparinized blood was obtained for the flow-cytrometric analysis and for the T cell proliferation assay, and serum was collected for determination of antibody against TSST-l. One healthy laboratory worker volunteered as a control for each Iymphoproliferation assay. Measurement of serum antibodies against TSST-l was kindly performed by the Institut fur Medizinische Mikrobiologie und Hygiene der Universitat Koln, Germany (Director: Professor Dr. G. Pulverer). The whole blood lymphoproliferation assay was performed as described by Leroux et al. (4). In brief, heparinized blood was mixed 1:8 with RPMI 1640 supplemented with glutamine, penicillin and streptamycin (Gibco, Karlsruhe). 0.1 mL of the blood suspension was distributed to the wells of a round-bottomed 96-well plate (Nunc, Wiesbaden). Each well

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contained already 0.1 mL of a superantigen or mitogen diluted to appropriate concentrations in RPMI 1640 (see below). Wells containing only RPMI medium served as negative controls. After 5 days of incubation, cultures were labelled with 1 mClwell (specific activity 25 Cilmmol) (3H) thymidine (Amersham, Braunschwieg) and incorporated radioactivity was determined after transfer to a filter paper in a liquid scintillation counter. Mitogen and superantigens preparation. Phytohaemagglutinin (PHA, Burroughs Wellcome, Grosswedel), a lectin mitogen, was used in the following concentrations: 25, 12.5, and 6.25 mg/L. TSST-l and ETA were utilized as crude supernatants of toxin-producing bacterial strains isolated in our laboratory. TSST-1 production of the Staphylococcus aureus strain was evaluated by means of the toxin detection kit TST-RPLA (titre 1 :64). The ETA-producing strain of Streptococcus pyogenes has been described elsewhere (1). The supernatants were obtained from a log-phase bacterial culture grown in RPMI 1640, were passed through a filter (mean pore size: 0.2 mm, Millipore, Bedford, Massachusetts) and kept frozen at -70°C until use. A serial 1: 10 dilution of the supernatants in RPMI 1640 was used in the entire blood lymphoproliferation assay. Flow cytometry. Peripheral blood lymphocytes were isolated by means of Ficol-Isopaque centrifugation (4) and were stained with phycoerythrin (PE)-conjugated monoclonal antibodies specific for CD4 (Clone MT310, Dako, Denmark), CD8 mAb (Clones B9.11 and B9.2, Dianova, Hamburg), CD25 (Clone ACT-I, Dako), and HLA-DR (Clone CR3/43, Dako). Cell sorting was performed on an FACScan (Becton Dickinson, USA). Results Serum antibodies against TSST-l. The titre was 1 :25 (negative) in the first serum specimen, which was obtained during the second febrile period (i. e. 9 days after the onset of the first shock), and 1: 100 (= cut-off) in the second specimen drawn after 5 weeks. Whole blood lymphoproliferation assay. The T cells of our patient responded to stimulation with the superantigens TSST-1 and ETA, and with the mitogen PHA, being similar to the T cells of healthy donors. Figure 1 shows the proliferative response of the patient's T cells during the second shock (day 9) and the response of the T cells of a healthy donor. We could not detect any significant alteration in the proliferative response of the patient's T cells in the second lymphoproliferation assay, which was performed 5 weeks later (data not shown). Flow cytometry. The expression of the T cell activity marker molecules CD25 and HLA-DR in the peripheral blood lymphocytes of this patient was determined during and after the acute episode of the disease via FACS-analysis (Table 1). HLA-DR+ T cells had a share of about 17% among the circulating T cells on day 9 (vs. 2 % by a healthy donor), indicating a massive T-cell activation. They had dropped to 2.3% in the second analysis, which was performed after 5 weeks. The percentage of CD25+ T cells, however, remained constant.

Discussion We report on a young patient who suffered from two bouts of menstrual TSS within 9 days. A TSST-l-producing strain of Straphylococcus aureus could be isolated from the cervical swab obtained during the second febrile episode. We concluded that these staphylococci might have also induced the first shock and persisted in the cervix of the patient.

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Fig. 1. Proliferation response of peripheral T cells of the patient (full bars) and of a healthy donor (hatched bars) after stimulation with different stimuli as depicted on the abscissa. The cultures were performed in triplicates and the results are presented as the mean values of thymidine uptake per minute (SD < 15%).

Table 1. Alterations in the T cell subsets of the patient during and after the acute phase of disease. Peripheral T cells were analysed by means of flow cytometry T cell marker

CD4 CD8 CD25 HLA-DR

% of peripheral T lymphocytes at the indicated time after onset of first shock

day 9

day 45

53.1 40.4 4.9 17.2

60.0 30.9 5.0 1.3

Epidemiological data indicate that practically all patients with menstrual TSS have undetectable antibody titres against TSST-l at the onset of the disease. Our data are in accordance with these findings, since these antibodies were undetectable in the serum of our patient on day 9. Interestingly, we could not detect a significant increase in the TSST-l titre in the second serum specimen, which was collected on day 45. Since antibody titres of = < 1: 100 are believed not to be protective against TSS, we assume

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that this patient is still at a high risk of developing a TSS, particularly during the menstrual periods. Hitherto, an anti-TSS vaccine has not been available, therefore, the prevention of recurrences includes parenteral antibiotic treatment (e. g. flucloxacillin, 810 g/d, 10-15 days), education of patients, and avoidance of tampons. To evaluate whether the peripheral T cells of this patient had been rendered unresponsive to TSST-l, their proliferative response was determined after in-vitro stimulation with superantigens and mitogens. We could not detect any reduction in the proliferative response of the patient's T cells at any time, as compared to the T cell response of healthy donors. This finding is in contrast to the results of in-vivo experiments in mice, which demonstrated a profound unresponsiveness of T cells of staphylococcal enterotoxin B-treated animals to subsequent in-vitro restimulation with staphylococcal enterotoxin B (10). However, these investigations studied the T cells derived from the adjacent lymph nodes, whereas we investigated the circulating T cells of our patient. Therefore, this discrepancy may have been due to the different T cell populations studied. On the other hand, Miethke et al. have shown that mice are far less susceptible than humans to a challenge by TSST-l (9). One can therefore suppose that the reactivity of murine T cells will fall more rapidly below a critical threshold than the reactivity of human T cells. The flow-cytometric analysis of patient's circulating T cells during and after the acute episode of shock confirmed that a great fraction of T cells had been activated. The expression of HLA-DR, a T cell activity marker, was significantly increased on day 9 (after onset of the first shock) and dropped significantly during the following 5 weeks. We could not detect any increase in the number of CD25+ T cells. Since CD25 is an early activity marker (6), it is possible that we missed the peak of CD25 expression triggered by the first shock, and that it was too early to get hold of the CD25 peak of the second febrile phase, which had occurred a few hours prior to the FACS analysis. On the other hand, it is also possible that the second febrile phase did not last long enough to evoke a expansion of activated T cells in the peripheral blood of the patient. In summary, our data confirm that repeated bouts of TSS occur in humans and do not evoke a sufficient antibody synthesis in every individual. A general activation of peripheral T cells detected hy means of flow cytometry can support the diagnosis of TSS. Acknowledgements. We thank Dr. M. Benert (Ev. Waldkrankenhaus Spandau, Berlin) for providing the specimens and clinical information.

References

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6. Marrack, P. and J. Kappler: The staphylococcal enterotoxins and their relatives. Science 248 (1990) 705-711 7. McCormack, J. E., J. E. Callahan, J. Kapplan, and P. Marrack: Profound deletion of mature T cells in vivo by chronic exposure to exogenous superantigens. J. Immunol. 150 (1993) 3785-3792 8. Miethke, T. C. Wahl, K. Heeg, and H. Wagner: Aquired Resistence to superantigen-induced T cell shock. J. Immunol. 150 (1993) 3776-3784 9. Miethke, T. K. Duschek, C. Wahl, K. Heeg, and H. Wagner: Pathogenesis of the toxic shock syndrome: T cell mediated lethal shock caused by the superantigen TSST-1. Eur. J. Immunol. 23 (1993) 1494-1500 10. Rellahan, B. L., L. A. Jones, A. M. Kruisbeek, A. M. Fry, and L. A. Matis: In vivo induction of anergy in peripheral VB8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172(1990) 1091-1100 11. Schlievert, P. M.: Role of superantigens in human disease. J. Infect. Dis. 167 (1993) 9971002 12. Todd, J. and M. Fishaut: Toxic-shock syndrome associated with phage-group-l staphylococci. Lancet2 (1978) 1116-1118 13. Waldvogel, F. A.: Staphylococcus aureus (including toxic shock syndrome). In: Mandell, G. R., J. E. Bennett, R. Dolin, eds. Principles and praxis of infectious diseases, 4th. ed. (1995) 1754-1777

Dr. med. M. Arvand, Institut fUr Medizinische Mikrobiologie und Infektionsepidemiologie der Freien Universitiit Berlin, Hindenburgdamm 27, D-12203 Berlin, Germany