INFECTION AND IMMUNITY, Apr. 1996, p. 1450–1453 Vol. 64, No. 4 0019-9567/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Human Pro- and Anti-Inflammatory Patterns Induced by Erythrogenic (Pyrogenic) A and C 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 (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-␥) 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) 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- family, are the major effectors involved in the 1ra) (9) and IFN-␣ (3, 4). development of multiorgan failure and other clinical aspects of Human PBMC (2.5 ϫ 106) from healthy donors were chal- streptococcal (2, 12, 14, 17, 19, 20, 24, 29, lenged with 1 ng to 10 ␮g 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 V␤ 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-␥ 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-␣ was titrated by specific ELISA provided by Biosource released significant amounts of interleukin-1␣ (IL-1␣), IL-1␤, (Camarillo, Calif.). All data were expressed as mean values Ϯ IL-6, IL-8, tumor necrosis factor alpha (TNF-␣), and TNF-␤ standard errors of the mean (SEM) of three experiments for (18). In contrast to PBMC, IL-1, IL-6, and TNF-␣ 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-␥ 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 ϭ 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-␣.On significant amounts of this cytokine (500 pg/ml after 96 h). For the other hand, these stimulants did not elicit TNF-␤ release the highest concentrations (1 and 10 ␮g of ETA per ml), 5,000 by PBMC (18). and 8,000 pg of IFN-␥ 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-␥ (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-␥ by human IL-4 by PBMC challenged with the three stimulators mentioned in the Fig. 1 PBMC after 72 h of stimulation at 37ЊC 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 ␮l per PBMC challenged with the three stimulators mentioned in the Fig. 1 legend. cell suspension. (B) Comparative time-dependent release of IFN-␥ by human Data represent means Ϯ SEM for the cells from 10 donors. Culture conditions, PBMC stimulated with 1 ␮g of ETA per ml (——F——), 0.1 ␮g of LPS per ml stimulators, and symbols are given in the Fig. 1 legend. The bottom line in panel (——E——), and 10 ␮l of heat-killed group A streptococci per ml (107 CFU) C corresponds to the background values. (——Ç——). Data represent means Ϯ 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 CD4ϩ T Data represent means Ϯ 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-␥, IL-2, and TNF-␤, 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-␥ is a proinflammatory cytokine 2). due to its capacity to increase IL-1 and TNF-␣ 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. Control cells in the absence of any of the stimulators used released 15,000 to 20,000 pg/ml under the same conditions. Streptococcal cells did not trigger any production of this cytokine, but in contrast, they reduced IL-1ra release by about 50% compared with the unstimulated control PBMC (Fig. 3). IFN-␣ was not signifi- cantly elicited by ETA, ETC, or LPS, whereas substantial FIG. 3. Anti-inflammatory soluble IL-1ra. (A) Dose-dependent release of amounts were produced in response to streptococci. Optimal IL-1ra by human PBMC stimulated with ETA (micrograms per milliliter). (B) release (460 pg/ml) was observed after 72 h of incubation (data Time-dependent release of soluble IL-1ra by PBMC challenged with the three not shown). In all experiments, cytokine release in response to stimulators mentioned in the Fig. 1 legend. Data represent means Ϯ SEM for the cells from three donors. Culture conditions, stimulators, and symbols are given in ETC was quantitatively similar to that elicited by ETA (Table the Fig. 1 legend. Stimulation by RPMI medium in the absence of added stim- 1). ulators is represented by a plain line (—). 1452 NOTES INFECT.IMMUN.

TABLE 1. Comparative cytokine-inducing capacity of release of extracellular soluble toxins and enzymes. Conse- erythrogenic (pyrogenic) ETA and ETCa quently, the release of proinflammatory cytokines reflects very Cytokine (pg/ml) (SEM) likely the interaction of the microorganisms with immune sys- Medium tem cells as supported by the in vitro production of IL-1␣, or toxin Soluble IFN-␥ IL-2 IL-4 IL-10 b IL-1␤, IL-6, IL-8, and TNF-␣ by PBMC in response to the IL-1ra heat-killed group A streptococci (18). In the work reported RPMI 11 Ϯ 912Ϯ2 1.5 Ϯ 0 112 Ϯ 82 134 Ϯ 62 here, the profile of the Th1 and Th2 cytokines as well as IL-1ra ETC 4,745 Ϯ 2,533 387 Ϯ 74 47 Ϯ 8.1 938 Ϯ 261 231 Ϯ 35 and IFN-␣ elicited by the PBMC exposed to streptococcal cells greatly differed from that found for LPS and streptococcal a Seventy-two hours of incubation and 1 ␮g of ETC per ml. b Nanograms per milliliter. superantigens. In contrast to these toxins, no significant release of the anti-inflammatory cytokines IL-4 and IL-10 and the monocyte-derived IL-1ra was elicited by the streptococci, which in addition reduced by 50% the spontaneous release of of mast cells and eosinophils. However, IL-10 has been re- this cytokine by PBMC. IL-2 release was not observed whereas ported to be also produced by Th1 lymphocytes (8) and shown IFN-␥, which is an essential factor in host defense against to inhibit cytokine production and vascular leakage during invading microorganisms (25), was produced in large amounts Th1-cell-induced delayed-type hypersensitivity (16). Further- (up to 22,190 pg/ml) compared with those elicited (5,000 to more, IL-4, IL-10, and IL-13 possess anti-inflammatory prop- 8,000 pg/ml) by the superantigens. The streptococcal prepara- erties since they strongly limit monocyte/macrophage activa- tion OK 432 also elicited IFN-␥ production (31). The obser- tion and counteract some of the IL-1 and TNF properties (10). vation that streptococci did not induce the production of the The profiles and amounts of the cytokines investigated were two other Th1 cytokines TNF-␤ and IL-2 requires further quite different in response to the three stimulators used (su- investigation. One could hypothesize that the strong stimula- perantigens, LPS, and streptococcal cells). High amounts of tion of IFN-␥ production may be mediated by heat-killed bac- IFN-␥, IL-2, IL-10, and IL-1ra were released by PBMC in teria-induced IL-12 at the monocyte level. In this respect, it response to ETA and ETC. IL-4 release was rather low, and should be mentioned that, in contrast to the superantigens, the IFN-␣ was not induced. These results confirm our previous streptococcal cells do not induce lymphocyte proliferation. observation with mouse spleen cells, which were shown to IFN-␣ was also produced in significant amounts by the produce IFN-␥ rather than IFN-␣/␤ upon stimulation with PBMC exposed to the bacteria but not upon challenge with streptococcus-derived toxins (7). In contrast to the superanti- LPS and the superantigens. The anti-inflammatory properties gens, LPS, which was previously shown to elicit high amounts of IFN-␣ are in part due to the triggering of TNF soluble of proinflammatory cytokines (18), did not induce significant receptors and the suppression of the synthesis of proinflam- release of IL-2, IL-4, and IFN-␣ and elicited poor production matory cytokines (IL-1, TNF-␣, IL-8) (4, 28). IFN-␣ was also of IFN-␥. IL-1ra levels were similar to those found with the reported to counteract IL-1 effects by inducing IL-1ra (3). superantigens. A different T-cell-derived cytokine profile than In conclusion, this work shows that, besides the induction of that found with ETA and ETC was reported in the case of high amounts of various pro- and anti-inflammatory cytokines PBMC stimulated with Staphylococcus aureus superantigenic by the streptococcal superantigenic toxins ETA and ETC, enterotoxins A and B (15). The Th1-derived IL-2 and IFN-␥ as streptococcal cells were also very potent inducers of IFN-␥ and well as the monocyte-derived cytokines IL-1, IL-6, and TNF-␣ most proinflammatory cytokines, suggesting that, besides the were released but not the Th2-derived IL-4 and IL-10. The soluble exotoxins, group A streptococci may contribute per adding of the latter to enterotoxin-stimulated PBMC inhibited se to the elicitation of certain pathophysiological disorders the production of IFN-␥ and certain proinflammatory cyto- through cytokine release. It remains to determine to what kines. These findings support the observations that IL-10 po- extent the superantigen-induced pro- and anti-inflammatory tently inhibits the release of monokines and Th1-derived lym- cytokines are produced in vivo during acute and chronic strep- phokines in response to certain mitogens (11). tococcal infections. It appears also interesting to evaluate the The wide range of anti-inflammatory and proinflammatory balance between the two types of Th1- and Th2-derived cyto- cytokines has been recently investigated at the single-cell level kines in the various clinical contexts of these infections in the by intracellular immunofluorescent staining of PBMC or light of the cross-regulatory relationship existing between the monocytes stimulated with LPS, S. aureus toxic shock syn- cytokines released by T-helper cell subsets (10, 11, 23, 27). drome toxin 1 and enterotoxins, streptococcal ETA, ETB (cys- teine proteinase), and the newly identified MF superantigen This work was supported by a Sidaction grant provided to H. Mu¨ller- (5, 20). The lack of cellular occurrence in LPS-stimulated Alouf from the Fondation pour la Recherche Me´dicale, Paris, France, PBMC of the T-cell-derived cytokines TNF-␤, IL-2, IFN-␥, and by grant Ge 684/2-1 to D. 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Editor: V. A. Fischetti