Cytokine Production by Murine Cells Activated by Erythrogenic Toxin Type A Superantigen of Streptococcus pyogenes

Immunobiol., vol. 186, pp. 435-448 (1992) 1 Unite des Toxines Microbiennes (URA 557 du CNRS), 2 Unite d'Immuno-Allergie, Institut Pasteur, Paris, France, and 3 Institut fur experimentelle Mikrobiologie, Jena, Germany

Cytokine Production by Murine Cells Activated by Erythrogenic Toxin Type A Superantigen of Streptococcus pyogenes HEIDE MULLER-ALOUp l, JOSEPH E. ALOUP1, DIETER GERLACH 3 , CATHERINE FITTING 2 , and JEAN-MARC CAVAILLON 2 Received July 8, 1992 . Accepted July 20, 1992

Abstract The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.

Introduction The mechanism of action in the host of the erythrogenic toxins (ETs) produced by Streptococcus pyogenes (1, 2), also named streptococcal pyrogenic exotoxins (SPEs) (3), is still poorly understood. These protein toxins which comprise three different serotypes (ETA, ETB, ETC) are Abbreviations: ET = erythrogenic toxin; LPS = lipopolysaccharide; TNF = tumor necrosis = toxic shock-like syndrome, TSS = toxic shock syndrome

factor; TSLS

436 .

HEIDE MOLLER-ALOUF

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thought to be the major factors involved in the pathogenesis of scarlet fever and streptococcal toxic shock-like syndrome (TSLS) in humans infected with group A streptococci (4-6). Since the first report in 1983 of this severe disease (7) which has much in common with the toxic (malignant) scarlet fever reported in the first half of this century (4, 6-8), several hundreds of cases (and very likely a greater number) have been recorded so far (5, 9). The mechanisms by which ETs mediate the disease remain unclear (10). This is also the case of its epidemiological characteristics (11). TSLS shares many clinical features with the staphylococcal toxic shock syndrome (TSS). Both are characterized by fever, skin rash and desquamation, hypotension, central nervous system impairment and multi-organ dysfunctions. They differ however by their epidemiological backgrounds and mortality rates which are much higher for TSLS (20-30 %) (6) as compared to TSS (6 %) (12, 13). Several lines of evidence point to the involvement of Staphylococcus aureus toxic shock syndrome toxin-1 (TSST -1) and lor enterotoxins in the pathogenesis of TSS (14, 15). These toxins and ETs show various degrees of structural and antigenic relatedness. They also share many major biological properties such as pyrogenicity, suppression of immune responses, susceptibility to lethal endotoxin shock in experimental animals and induction of polyclonal proliferation of human, murine and other animal species T lymphocytes. The mitogenic activity of these toxins results from their behavior as superantigens through the stimulation of T cell subpopulations expressing appropriate VB (and in some cases Vy) elements of the T cell receptor after binding to MHC class II antigens at the surface of accessory cells (16-19). Staphylococcal enterotoxins and TSST-1 have been shown to elicit in vivo and in vitro the release of high amounts of various cytokines by human, murine or other animal species monocytes/macrophages and T lymphocytes (for references see ref. 2). These cytokines are thought to be directly responsible for many of TSS clinical symptoms (2, 20). This might also be the case for streptococcal TSLS. However, little information has been provided so far with respect to the cytokine inducing capacity of erythrogenic toxins (21-25). We report here a study on the production of tumor necrosis factor (TNF), interleukin-3 (IL-3) and IL-6 by peritoneal macrophages and I or spleen cells of BALB/c and the endotoxin low-responder C3H/HeJ mice. The cells were challenged with highly purified ETA preparations and Neisseria meningitidis LPS for comparative purposes. Although the natural host of group A streptococci are humans, valuable information could be obtained in murine models due to the availability of inbred mice strains and to the sensitivity of their immune cells to streptococcal erythrogenic toxins (26-28). The two mice strains used in this work possess the appropriate ETA-reactive VB elements of the TCR of their respective T lymphocytes (29, 30).

Cytokine Induction by Erythrogenic Toxin Type A . 437

Materials and Methods Erythrogenic toxins and other cytokine inducers Two erythrogenic toxin A preparations purified to apparent homogeneity as assessed by SDS-PAGE were used. One preparation was fractionated from S. pyogenes NY-S strain culture supernatants (31) and the other was recombinant ETA purified from the culture filtrate of Streptococcus sanguis (Challis strain) containing the speA gene (32). Both ETA preparations were comparable in regard to their mitogenic and cytokine inducing capacities and molecular weight (25 kDa). They were used without discrimination in this work. Both preparations did not contain endotoxin contaminants greater than 340 pg/mg as assessed by the Limulus colorimetric reagent (Whittaker, Walkersville, MD, USA). N. meningitidis LPS was kindly provided by Dr. MARTINE CAROFF (Paris XI University, Orsay, France). At the LPS concentrations used, the cytokine inducing capacity of this preparation was not inhibitable by polymyxin B as for other endotoxins (33).

Mice strains BALB/c (H-2d, Mls-1b), nude BALB/c, their heterozygous littermate and C3H/HeJ mice (H-2k, Mls-1b) bred in the animal facility of Pasteur Institute (Paris, France) were used as the sources of peritoneal macrophages and spleen cells.

Peritoneal macrophages BALB/c and C3H/HeJ mice of either sex, 6 to 12 weeks old, were injected i.p. with 1.5 ml of thioglycolate medium (Pasteur Diagnostics, Marnes-Ia-Coquette, France). Five days later, the peritoneal cavities were washed three times with 2 ml of RPM I 1640 medium (Gibco, Paisley, U.K.) containing 10 IU/ml heparin (Roche Laboratories, Paris, France). The macrophages were purified from exudate cells by surface adherence in 24-well plates (Costar, Cambridge, MA, USA) at a concentration of 6 x lOS peritoneal cells in 0,5 ml RPMI supplemented with glutamine and antibiotics (penicillin, 100 IU/ml, and streptomycin, 100 ltg/ml). After incubation for 1.S h at 3rc and 7 % CO 2 , the cells were washed vigorously and adherent macrophages were further cultured at 37°C without serum in an incubator (7 % CO 2 - 93 % air). The incubation medium contained 1 ltg/ml indomethacin to avoid a possible interference with prostaglandin synthesis. All cytokine inducing experiments were performed in the presence of 2 ltg/ml polymyxin B.

Spleen cells Spleens of BALB/c, C3H/HeJ, BALB/c nude and their heterozygous littermate mice were transferred into RPM I 1640 medium. Single cells suspensions were obtained by teasing the spleens and membraneous debris were removed by passing the cell suspensions through a nylon filter. After two washings, 3 x 106 spleen cells were suspended in a total volume of 1 ml RPMI 1640 containing 1.5 % foetal calf serum (Boehringer, Mannheim, Germany), 10-6 M 2mercaptoethanol and 2 ltg/ml polymyxin B. The stimulators were added at the beginning of culture. The cells were incubated as indicated for macrophages. At the end of t\1e culture period, the supernatants of macrophages and splenocytes were collected, clarified by centrifugation and stored at -20°C until assessed for cytokine content.

TNF assay This assay was determined as described previously (34). It is based on the determination of the cytotoxic activity on mouse fibroblasts L929 in the supernatants of ETA or LPS stimulated macrophages and splenocytes of the above-mentioned mice strains. Briefly 3 X 104 L929 cells in 0.1 ml of RPMI 1640 supplemented with antibiotics and 10 % FCS were cultured overnight in 96-well flat-bottomed microtiter plates (Costar). The next day, serial dilutions of test supernatants were applied in triplicate to the cells in the presence of 2 ltg/ml actinomycin D. The plates were incubated at 37°C for 18 h. The culture medium was removed by inverting the

438 . HEIDE MULLER-ALOUF et al. plates. The remaining cells were stained with crystal violet (50 [-II, 0.1 % in 20 % methanol! water) for 20 min at room temperature. The microtiter plates were gently rinsed with water and the remaining dye was solubilized in 1 % SDS. Absorbance was read in an automated micro ELISA auto reader at 550 nm. One unit of TNF activity was defined as the amount required to lyse 50 % L929 cells. The assay was standardized with a human recombinant TNFa preparation. Murine TNF-a was determined by radioimmmunoassay using 125I-TNF_a assay system generously provided by Amersham (Amersham France, Les Ulis, France). The test kit was used according to the instructions of the producer.

IL-3 assay The assay was performed as described previously (35). Briefly, IL-3 activity in supernatants of cultured splenocytes was assessed by the growth of the IL-3 dependent FDC-P2 cell line. The test was set up in duplicate cultures containing 10' cells in a final volume of 100 [-II RPM I 1640 supplemented with antibiotics and 15 % inactivated horse serum (Boehringer) and containing 20 [-II of nondiluted supernatant. The cultures were incubated for 24 h at 3rc and 7 % CO 2 , Four to 5 h before the harvest, 0.25 [-ICi/20 [-II eH)-thymidine was added (2 Ci/ mmol, Amersham). The cells were collected on glass fiber filters with a Titertek cell harvester. Thymidine incorporation was determined by liquid scintillation counting. An IL-3 containing WEHI-3 supernatant served as a positive control.

IL-6 assay Interleukin-6 was determined using the IL-6 dependent lTDl mouse hybridoma which was a gift of Dr. J. VAN SNICK, (Ludwig Institute, Brussels, Belgium). IL-6 activity was estimated as described previously (34) with minor modifications. Briefly, 1200 cells/well were grown in 200 [-II RPM I 1640 supplemented with antibiotics, 2-mercaptoethanol (5 X 10-5 M) and 7.5 % FCS in the presence of serial dilutions of supernatants of stimulated macrophages or spleen cells. The IL-6 activity in the samples was monitored by a dye method using MTT (36). Twenty-five [-II of phosphate-buffered saline containing 5 mg of MTT (3,[ 4,5-dimethylthiazol2-yl]2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, USA) per ml was added to the wells, and the plates were incubated for 2 to 4 hat 37°C, then 100 [-II of extraction buffer (20 % w/v SDS was dissolved at 37°C in a solution of 50 % DMF in water, pH was adjusted to 4.7 by adding 2.5 % of an 80 % acetic acid and 2.5 % N HCI) were added to dissolve the MTTformazan precipitate. After overnight incubation at 3rc the optical density of the dissolved precipitate was read at 550 nm. One unit of IL-6 corresponds to half maximal growth of the hybridoma cell. Recombinant mouse IL-6 (kindly provided by J. VAN SNICK, Brussels) was used as a positive control. Titrations were performed in duplicates.

Results

TNF production Freshly isolated thioglycolate-elicited mouse macrophages were incubated for 24 h with increasing amounts of ETA (0.1 to 10 Ilg/well). The cellfree supernatant was assayed for the presence of TNF assessed by the L929 cytotoxicity assay. N. meningitidis LPS served as positive control (0.1 Ilg per well). Upon ETA stimulation, peritoneal macrophages of BALB/c mouse did not produce detectable amounts of TNF (Table 1); even when the culture time was extended over 96 h. TNF activity was not detected in ETA-stimulated macrophage cultures and ETA did not enhance the LPSinduced TNF production. The specific radioimmunoassay of TNF-a confirmed an absence of significant release of this cytokine. No TNF activity

Cytokine Induction by Erythrogenic Toxin Type A . 439 Table 1. Cytokine production by ET A- and LPS-stimulated BALB/c peritoneal macrophages IL-6 Units/ml

TNF Units O Inducer"· Medium ETA ETA ETA LPS

0.1 !-Ig 1.0 !-Ig 10 !-Ig 0. 1 !-Ig

BALB/c

C3HIHeJ

BALB/c

C3H/HeJ

< 10 < 10 < 10 < 10 111 ± 27

< < < < <

101 ± 37 121 ±41 148 ± 58 479 ± 233 1343 ± 777


10 10 10 10 10

". Incubation of macrophages with ETA and LPS for 24 h. ° Results are expressed as a mean of 4 independent experiments ± S.D. ".". under detection limit

could be detected in supernatants of ETA-stimulated splenocytes (data not shown).

IL-6 production The supernatants of ETA-stimulated peritoneal macrophages of BALB/c and C3H/HeJ mice were assayed for the presence of IL-6 using the IL-6 dependent 7 TDI cell line. ETA-stimulated BALB/c macrophages released significant IL-6 amounts (Table 1) only when the highest concentration was used (10 !-lg). The prolongation of the incubation time up to 96 h did not elicit more IL-6 production ( Fig. 1). In all experiments 0.1 !-lg of LPS was a much stronger inducer of IL-6 than 10 !-lg ETA. Macrophages of C3H/HeJ mice did not produce detectable amounts of IL-6.

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440 .

HEIDE MOLLER-ALOUF

et al.

In contrast, ETA induced the production of IL-6 in BALB/c mouse splenocytes in a dose-dependent manner. After an incubation time of 24 h, the release of a significant IL-6 amount was triggered by 1 f..lg of ETA. The amount of IL-6 increased with the prolongation of incubation time, reaching an optimum after 72 h. The kinetics of IL-6 production by BALB/c and C3H/He] splenocytes is shown in Figure 2A. More IL-6 was produced after stimulation of splenocytes by ETA (10 f..lg) than by LPS (0.1 f..lg). In contrast to the results for IL-6 induction by these inducers in macrophages, 1 f..lg of ETA induced the same amount of IL-6 as 0.1 f..lg of LPS. In C3HI He] splenocytes, ETA triggered the release of IL-6, but a significant amount was not found before 48 h (Fig. 2B).

IL-3 production Since IL-3 is produced by T lymphocytes, only the supernatants of stimulated spleen lymphocytes were titrated for the presence of this lymphokine. BALB/c and C3H/He] mouse splenocytes were incubated with different amounts of ETA for 24-96 h. The cell free supernatants were tested for their ability to induce the growth of the IL-3 dependent FDC-P2 8000

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.t!9!m§ Figure 3. Kinetics of IL-3 producti on upon challenging of BALB/c (A) and C3H/HeJ mouse (B) splenocytes with different stimuli. The results are expressed as 3H-thymidine uptake by FDC-P2 cells in the presence ofvarious supernatants (1:5 dilution). The data are representative of 4 ( A)and 3 (B) experiments respectively.

cell line. ETA induc~d the production of IL-3 in a dose-dependent manner. Figure 3A and B illustrate the kinetics of IL-3 induction using BALB/c and C3H/ HeJ splenocytes. After 4 8 hof incubation, significant 3H -thymidine incorporation was found for 1 and 10 Ilg ETA and after 96 h even with the lowest amount of ETA (O.lllg). The optimum of IL-3 production was reached after 72-96 h. The splenocytes of C3H/HeJ mouse produced also considerable amounts of IL-3 upon ETA stimulation. Comparable increasing amounts of IL-3 were fo und with prolongation of incubation time up to 72 to 96 h (Fig. 3B).

Enhancement by LPS of cytokine release by erythrogenic toxin stimulated cells ETA enhances the host susceptibility to lethal endotoxin shock and endotoxin mediated fever in vivo (37). The purpose was to examine, whether ETA could augment the LPS-induced cytokine production by macrophages and splenocytes of BALB/c mice in vitro. When peritoneal

442 . HEIDE MOLLER-ALOUF et al.

macrophages were simultaneously stimulated with both LPS (100 ng) and ETA (0.1, 1, 10 ~g), no synergistic effects on TNF production were found over 96 h of incubation. The production of IL-6 was slightly enhanced beyond an additive effect after 72 h in cultures with 10 ~g ETA and 100 ng LPS by a factor of 1.6 (data not shown). The simultaneous stimulation of BALB/c splenocytes did not enhance TNF production. The IL-6 production was enhanced above the additive value after 72h of incubation by a factor of 1.7 with 10~g ETA and by a factor of 2 after 96 h (data not shown). The induction of IL-3 was not enhanced in splenocyte cultures stimulated with LPS and ETA. Since there was non-convincing enhancement reaction in vitro, we injected ETA (100 ~g) intravenously into BALB/c mice 6 h before the harvest of thioglycolate elicited peritoneal macrophages. The macrophages were then cultured in the presence of ETA and LPS. After 48 h of incubation, the supernatants were harvested and titrated for the presence of TNF and IL-6. The results were compared with those of non-challenged mice. The pre-stimulation of mice with ETA rendered the macrophages more reactive and allowed enhanced production of IL-6 after in vivo stimulation with ETA as well as with LPS (Table 2). In contrast, in vivo stimulation of mice with LPS (2 ~g i.v.) had a rather suppressive effect on macrophages IL-6 production upon in vitro stimulation with ETA and LPS.

Cytokine production in nude mice The role of T cells in cytokine production by the splenocytes of BALB/c nude mice and their littermate controls stimulated with ETA, LPS and Con A was investigated (Fig. 4 ). ETA did not stimulate IL-6 production in nude mice. In contrast, these mice and their littermate were stimulated to the same degree by LPS. Con A stimulated BALB/c littermate 3-fold stronger than the homozygous mice. These results demonstrate that the presence of T lymphocytes is required for IL-6 production upon stimulation by ETA.

Table 2. IL-6 production by BALB/c macrophages after in vivo challenge of mice with ETA and LPS

in vivoo

in vitro

% Control"·

LPS

LPS 0.1 [lg ETA 10 [lg LPS 0.1 [lg ETA 10 [lg

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(n=3) (n=4) (n=4) (n=4)

°i. v. injection of mice with 2 [lg of LPS or with 100 [lg ETA six hours before the harvest of

elicited peritoneal macrophages. Macrophages were cultured for 48 h at 3rc. "The results are expressed as percent of IL-6 values obtained in mice which were not challenged in vivo.

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Discussion The streptococcal toxic shock syndrome mediated by erythrogenic toxins (ETs) and staphylococcal toxic shock syndrome due to TSST -1 and/or the enterotoxins acting alone or in concert, share many clinical and pathological features with those observed in septic endotoxin-induced shock in humans (38). Numerous clinical investigations on endotoxic septic shock and experiments on LPS-challenged animals suggest that endogeneous toxic host factors, particularly TNF-a and 13, IL-1, and interferon-yare major effectors of the shock (39, 40). The release of these and other cytokines in patients with toxic shock syndrome (TSS) through the stimulation of their cells by TSST-1 and/or enterotoxins produced in vivo is also thought to account for the clinical manifestation of this disease (16, 41, 42). With regard to cytokine production by streptococcal ETs, few data have been provided so far as compared to the abundant data about the cytokine inducing capacity of staphylococcal TSST -1 and enterotoxins (see ref. 2 for a review). The production of cytokines by ETA-stimulated T cell was first reported by CAVAILLON et al. (21) who showed interferon-y production by spleen cells of CBA mice. An optimum production of this cytokine was obtained with 10 f,lg/ml ETA. This toxin also elicited the production of IL-2 and the expression of the IL-2 receptor on human T cells (2). TNF production was described by FAST et al. (23) after stimulation of human peripheral mononucleated blood cells with staphylococcal enterotoxins, TSST -1 and ETA. We have recently established (24) that human adherent monocytes released IL-la, IL-lj3, TNF and IL-6 after challenge with relatively high amounts of ETA (1-10flg). Optimum production occurred after nh of

444 .

HEIDE MULLER-ALOUF

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culture. More efficient production of these cytokines was achieved using PBMC indicating that T lymphocytes play an essential role in monokine induction by the superantigen ETA. In contrast, LPS-induced monokines did not require the presence of T lymphocytes (43). The fever-inducing capacity of ETs in experimental animals is reflected by the in vivo production of cytokines with endogeneous pyrogenic activity. Investigations on the profile of LPS- and ET-induced fever in rabbits showed, that these two toxins act differently. LPS causes biphasic fever curve in contrast to the pattern observed with ET. No cross-tolerance was shown between endotoxin and ET-mediated fever (37). Anti-lymphocyte serum inhibited the febrile response on subsequent ET injection, whereas the course of endotoxin-mediated fever remained unaffected (44). In the present work, the TNF, IL-3 and IL-6 inducing capacity of ETA was compared with the cytokine inducing capacity of LPS in a mouse model. Mouse lymphocytes undergo blastogenic transformation upon stimulation with streptococcal mitogens indicating the presence of immunoreactive cells (26, 27). Peritoneal macrophages of BALB/c and C3H/HeJ mice stimulated by ETA did not show TNF release in the supernatants (either by bioassay or RIA). IL-6 was only produced after macrophage stimulation with the highest concentration of ETA (10 [lg) whereas IL-6 production was higher for 100 ng of LPS. ETA-stimulated C3H/HeJ macrophages did not elicit detectable IL-6 for as high as 10 [lg of toxin. In addition to peritoneal macrophages, BALB/c and C3H/HeJ mice splenocytes were challenged with ETA and LPS. High amounts of IL-6 were elicited by ETA in a dose-dependent manner in splenocyte supernatants of both mice strains. The release of IL-3 which is exclusively produced by T lymphocytes was also elicited by ETA in a dose-dependent manner by the splenocytes. The IL-3 inducing property of this toxin was more than lOa-fold stronger than the IL-3 inducing capacity of LPS. IL-3 is known to act as a macrophage-activating factor, which enhances the antigen-presenting activity ot these cells by regulating the expression of class II MHC antigens of murine peritoneal macrophages (45). The weak production of IL-6 by ETA-stimulated peritoneal macrophages and the strong production of this cytokine by splenocytes indicates that the superantigen ETA requires the presence of T cells. To support this assumption, splenocytes of nude mice and their littermates were challenged with ETA and LPS. Nude BALB/c mouse splenocytes did not produce IL-6 upon stimulation by ETA, whereas their littermates responded quite well. In contrast, LPS evoked the production of IL-6 to the same extent in both mouse strains. These results are in accordance with the finding that SEA stimulated TNF-a (46) and IL-l (47) production in human monocytes only in the presence of T lymphocytes. The T cell requirement could not be replaced by the addition of T cell derived cytokines.

Cytokine Induction by Erythrogenic Toxin Type A . 445

Two mechanisms have been proposed to explain exotoxin involvement in the induction of toxic shock: the production of toxin-induced cytokines above the physiologically required level by the immune cells and/or the ability of the toxins to enhance susceptibility to endotoxin (2). Rabbit susceptibility to the lethal endotoxin shock was shown to be greatly enhanced after previous injection of ETs (37). The enhancing effect was also reflected by the potentiation of febrile response to endotoxin suggesting an increased production of endogeneous pyrogens. At the level of cytokine induction, PARSONNET and GILLIS (48) showed that TNF-a and IL-1 were produced synergistically by human macrophages upon combined TSST-1 and LPS stimulation in vitro. BEEZHOLD et al. (49) found a synergistic production of IL-1 by murine macrophages jointly stimulated by TSST-1 and LPS. In contrast to these findings with TSST -1, no enhanced TNF production was observed in BALB/c mice macrophages and splenocytes in the simultaneous presence of ETA and LPS. IL-6 production was slightly enhanced. The IL-6 producing capacity of BALB/c mouse macrophages was increased by i. v. injection of 100!!g of ETA in these mice 6h before macrophage harvesting. Under such prestimulatory conditions, the macrophages produced 3-4-fold more IL-6 upon stimulation with ETA as well as with LPS than those from nonstimulated mice. In contrast, a decreased production of IL-6 by macrophages was observed when mice were challenged with 2 !!g LPS. A strong enhancement of the production of circulating TNF by i. p. injection of LPS was observed when animals were administered 20!!g of TSST -1 12 h before exposure to endotoxin (50). Pretreatment of mice with LPS did not elicit the synthesis of circulating TNF upon stimulation with neither LPS nor TSST -1. These results confirm that the in vivo observed enhancement reaction of ET on LPS-induced fever is reflected by in vitro enhanced cytokine production under certain circumstances. However, these results do not allow to conclude that ETA-induced TSLS is caused by the enhancing action of endogeneous LPS. The pathological effects of ETA and other related superantigens could be most likely explained as a consequence of T cell activation or might also depend on toxin-stimulated T cell help on macrophage activation. Acknowledgements This work was supported by the grant 900301 (to]. E. ALOUF) from the Institut de la Sante et de la Recherche Medicale (Paris, France). We thank Prof. BERNARD DAVID for supporting the work of one of us (H. M.-A.) in his laboratory (Unite d'Immuno-Allergie, Institut Pasteur).

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