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And Anti-Inffammatory Cytokine Patterns Induced by Streptococcus INFECTION AND IMMUNITY, Apr. 1996, p. 1450–1453 Vol. 64, No. 4 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology Human Pro- and Anti-Inflammatory Cytokine Patterns Induced by Streptococcus pyogenes Erythrogenic (Pyrogenic) Exotoxin A and C Superantigens HEIDE MU¨ LLER-ALOUF,1* JOSEPH E. ALOUF,1 DIETER GERLACH,2 JO¨ RG-HERMANN 2 3 3 OZEGOWSKI, CATHERINE FITTING, AND JEAN-MARC CAVAILLON Unite´ des Toxines Microbiennes (URA 1858 Centre National de la Recherche Scientifique)1 and Unite´ d’Immuno-Allergie,3 Institut Pasteur, 75724 Paris Cedex 15, France, and Institut fu¨r Experimentelle Mikrobiologie, 07745 Jena, Germany2 Received 18 August 1995/Returned for modification 12 October 1995/Accepted 9 January 1996 The superantigenic streptococcal erythrogenic toxins A and C (ETA/SPEA and ETC/SPEC) elicit the pro- duction by human peripheral blood mononuclear cells of substantial amounts of Th1-derived cytokines (inter- leukin-2 [IL-2] and gamma interferon) as well as anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist). In contrast, very low levels of IL-4 and no alpha interferon were induced. The production of these cytokines after stimulation with Streptococcus pyogenes heat-killed bacteria and lipopolysaccharide from gram- negative bacteria differed qualitatively and quantitatively from that elicited by the superantigens. Group A streptococci (Streptococcus pyogenes) provoke se- IL-2 and gamma interferon (IFN-g) and Th2-derived IL-4 and vere invasive infections in humans including streptococcal toxic IL-10 was investigated. Because IL-4 and IL-10 possess the shock syndrome. Several lines of evidence suggest that eryth- potential ability to act as anti-inflammatory cytokines, we fur- rogenic (pyrogenic) exotoxins A and C (ETA and ETC or ther extended our investigation to other well-known anti-in- SPEA and SPEC, respectively), which belong to the bacterial flammatory cytokines, namely, IL-1 receptor antagonist (IL- superantigen family, are the major effectors involved in the 1ra) (9) and IFN-a (3, 4). development of multiorgan failure and other clinical aspects of Human PBMC (2.5 3 106) from healthy donors were chal- streptococcal toxic shock syndrome (2, 12, 14, 17, 19, 20, 24, 29, lenged with 1 ng to 10 mg of erythrogenic toxins A (natural and 30, 32, 33). Clinical and experimental observations strongly recombinant) and C (13, 18, 22), 100 ng of Neisseria meningi- suggest that the pathophysiological features of the syndrome tidis LPS (polymyxin B resistant) per ml (6), and heat-killed (30 to 50% mortality) as well as staphylococcal toxic shock group A streptococci (strain S 84) as previously described (18). syndrome are mainly due to massive release of monocyte- and The enzyme-linked immunosorbent assay (ELISA) of IL-2, T-lymphocyte-derived cytokines and other inflammatory fac- IL-4, and soluble IL-1ra in PBMC supernatants was performed tors in infected patients resulting essentially from the extensive with commercially available test kits (Quantikine;R&DSys- polyclonal stimulation of T-lymphocyte populations bearing tems Europe, Abington, United Kingdom). IL-10 was analyzed the appropriate Vb motifs of T-cell receptor recognized by by a sandwich ELISA with monoclonal anti-human IL-10 an- toxin molecules (1, 12, 17, 26, 32, 33). Cytokine release is also tibody and a biotinylated monoclonal rat anti-human IL-10 thought to play a pivotal role in lipopolysaccharide (LPS)- antibody (Pharmingen, San Diego, Calif.) according to the mediated septic shock induced by gram-negative bacterial in- manufacturer recommendations. IFN-g ELISA was developed fections. As previously reported, human peripheral blood by using two monoclonal antibodies provided by Pharmingen. mononuclear cells (PBMC) exposed in vitro to ETA and ETC IFN-a was titrated by specific ELISA provided by Biosource released significant amounts of interleukin-1a (IL-1a), IL-1b, (Camarillo, Calif.). All data were expressed as mean values 6 IL-6, IL-8, tumor necrosis factor alpha (TNF-a), and TNF-b standard errors of the mean (SEM) of three experiments for (18). In contrast to PBMC, IL-1, IL-6, and TNF-a were prac- tically not produced by the isolated monocytes challenged with each cell sample from at least 3 to 10 donors. the toxins, indicating the necessity of lymphocyte cooperation IFN-g was released in a dose-dependent and time-depen- for the release of these cytokines. However, monocyte suspen- dent manner by the PBMC from all donors (n 5 7) stimulated sions stimulated with LPS or heat-killed group A streptococcal with the superantigens. As little as 10 ng of ETA per ml elicited cells released significant amounts of IL-1, IL-6, and TNF-a.On significant amounts of this cytokine (500 pg/ml after 96 h). For the other hand, these stimulants did not elicit TNF-b release the highest concentrations (1 and 10 mg of ETA per ml), 5,000 by PBMC (18). and 8,000 pg of IFN-g per ml, respectively, were elicited. In For a better understanding of the cytokine-inducing prop- contrast to ETA, LPS was a poor inducer of IFN-g (mean, 560 erties of erythrogenic toxins by these cells, we report here a pg/ml after 96 h). Interestingly, very high amounts of this quantitative and kinetic study of the release of other essential cytokine (16,370 to 22,189 pg/ml) were released by all donor cytokines produced by PBMC challenged with ETA, ETC, cells stimulated with streptococcal cells (Fig. 1). The Th1 cy- heat-killed streptococci, and LPS. The release of Th1-derived tokine IL-2 was produced as early as 24 h by ETA-stimulated PBMC in a dose- and time-dependent manner (550 to 1,500 pg/ml) from four donors (Fig. 1). Optimal amounts were re- * Corresponding author. Mailing address: Centre d’Immunologie et leased after 48 h. For longer incubation times, the amounts de Biologie Parasitaires, Institut Pasteur, 1, rue du Professeur A. triggered by the highest doses of ETA declined. Neither LPS Calmette, 59019 Lille, France. Phone: 33.20 87 79 65. Fax: 33.20 87 78 nor streptococci induced IL-2 release (Fig. 1). The Th2 cyto- 88. kine IL-4 was produced in very low amounts (39 to 55 pg/ml) 1450 VOL. 64, 1996 NOTES 1451 FIG. 2. Th2 cytokines. (A) Dose-dependent release of IL-4 by human PBMC stimulated with ETA (micrograms per milliliter). (B) Time-dependent release of FIG. 1. Th1 cytokines. (A) Dose-dependent release of IFN-g by human IL-4 by PBMC challenged with the three stimulators mentioned in the Fig. 1 PBMC after 72 h of stimulation at 378C in RPMI medium with the streptococcal legend. (C) Dose-dependent release of IL-10 by human PBMC stimulated with superantigen erythrogenic (pyrogenic) ETA. Toxin concentrations are expressed ETA (micrograms per milliliter). (D) Time-dependent release of IL-10 by as micrograms per milliliter. Toxin solutions are added in volumes of 10 ml per PBMC challenged with the three stimulators mentioned in the Fig. 1 legend. cell suspension. (B) Comparative time-dependent release of IFN-g by human Data represent means 6 SEM for the cells from 10 donors. Culture conditions, PBMC stimulated with 1 mg of ETA per ml (——F——), 0.1 mg of LPS per ml stimulators, and symbols are given in the Fig. 1 legend. The bottom line in panel (——E——), and 10 ml of heat-killed group A streptococci per ml (107 CFU) C corresponds to the background values. (——Ç——). Data represent means 6 SEM for the cells from seven donors. (C) Dose-dependent release of IL-2 by human PBMC stimulated with ETA (micro- grams per milliliter). (D) Time-dependent release of IL-2 by PBMC stimulated with ETA, LPS, and streptococcal cells. Symbols are the same as for panel B. This study was undertaken within the scope that CD41 T Data represent means 6 SEM for the cells from four donors. lymphocytes comprise two sets (Th1 and Th2 cells) which ex- press distinct patterns of cytokines and regulate different ef- fector functions (16, 21, 23, 33). Th1 lymphocytes produce by ETA-stimulated PBMC from three donors in a dose- and IFN-g, IL-2, and TNF-b, which provide help for promoting time-dependent manner (Fig. 2). Optimal production occurred cell-mediated immunity, particularly delayed-type hypersensi- after 72 h. As little as 10 ng of ETA was sufficient to elicit this tivity. Th1-derived cytokines are directly or indirectly proin- cytokine, which was not induced by streptococci and LPS (Fig. flammatory cytokines. IFN-g is a proinflammatory cytokine 2). due to its capacity to increase IL-1 and TNF-a production by IL-10 was induced by ETA in a time- and dose-dependent activated monocytes/macrophages. Th2 cells produce IL-4, manner from the cells of 10 donors. Optimal release (1,890 to IL-5, IL-6, IL-10, and IL-13, which direct humoral immune 6,457 pg/ml) occurred after 72 h for the highest dose of ETA responses and promote allergic-type responses with activation and after 96 h for lower doses. As little as 10 ng of toxin per ml elicited significant amounts of this cytokine (mean, 500 pg/ml). LPS as well as streptococci induced moderate amounts (150 to 1,017 pg/ml) detectable after 24 h, with no further increase up to 96 h of incubation (Fig. 2). The anti-inflammatory cytokine IL-1ra was released in very high and equivalent amounts by the PBMC (three donors) stimulated with ETA and LPS (Fig. 3). The lowest dose of the former (10 ng/ml) elicited 25,120 to 27,491 pg/ml after 72 h. Higher toxin doses did not release greater amounts of monocyte-derived IL-1ra.
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