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The Journal of Immunology, 2005, 175: 6870-6877.
Copyright © 2005 by The American Association of Immunologists

The Mechanism of Superantigen-Mediated Toxic Shock: Not a Simple Th1 Cytokine Storm1

Lee Faulkner, Anneli Cooper, Cristina Fantino, Daniel M. Altmann and Shiranee Sriskandan2

Department of Infectious Diseases, Imperial College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The profound clinical consequences of Gram-positive toxic shock are hypothesized to stem from excessive Th1 responses to superantigens. We used a new superantigen-sensitive transgenic model to explore the role of TCR{alpha}{beta} T cells in responses to staphylococcal enterotoxin B (SEB) in vitro and in two different in vivo models. The proliferative and cytokine responses of HLA-DR1 spleen cells were 100-fold more sensitive than controls and were entirely dependent on TCR{alpha}{beta} T cells. HLA-DR1 mice showed greater sensitivity in vivo to two doses of SEB with higher mortality and serum cytokines than controls. When D-galactosamine was used as a sensitizing agent with a single dose of SEB, HLA-DR1 mice died of toxic shock whereas controls did not. In this sensitized model of toxic shock there was a biphasic release of cytokines, including TNF-{alpha}, at 2 h and before death at 7 h. In both models, mortality and cytokine release at both time points were dependent on TCR{alpha}{beta} T cells. Anti-TNF-{alpha} pretreatment was protective against shock whereas anti-IFN {gamma} pretreatment and delayed anti-TNF-{alpha} treatment were not. Importantly, anti-TNF-{alpha} pretreatment inhibited the early TNF-{alpha} response but did not inhibit the later TNF-{alpha} burst, to which mortality has previously been attributed. Splenic T cells were shown definitively to be the major source of TNF-{alpha} during the acute cytokine response. Our results demonstrate unequivocally that TCR{alpha}{beta} T cells are critical for lethality in toxic shock but it is the early TNF-{alpha} response and not the later cytokine surge that mediates lethal shock.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Severe sepsis leading to shock remains a leading cause of mortality and morbidity, with a rising incidence due to increasing numbers of immunosuppressed individuals and increasing age (1, 2). Infections due to Gram-positive pathogens, including Staphylococcus aureus and Streptococcus pyogenes, account for roughly half of severe sepsis cases. The immune response to Gram-positive infection may be exacerbated by the human pathophysiological reaction to bacterial superantigens, proteins with profound immunological potency that may lead to toxic shock (3, 4, 5).

Superantigen activation of T cells is dependent on HLA class II and TCR binding but is distinct from conventional Ag-specific responses which require Ag processing, presentation in the class II groove, and clonotypic T cell recognition (6). Superantigens bind to MHC class II molecules on APCs outside the peptide-binding groove and to the TCR variable chains on the T cell. Different superantigens have distinct affinities for different HLA molecules, e.g., staphylococcal enterotoxin B (SEB)3 displays differential binding for HLA-DR>DQ>DP (7). Specific superantigens also favor interactions with particular V{beta} families (3). Thus, in the case of a large V{beta} family, such as V{beta}8 receptors, which account for ~20% of the murine T cell repertoire, activation with an appropriate superantigen may affect an enormous sector of the total repertoire, far greater than the proportion involved in a given peptide-specific response. Superantigenic stimulation results in a systemic release of proinflammatory cytokines, including IL-1, TNF-{alpha}, TNF{beta}, and IFN-{gamma} (8, 9, 10, 11) with rapid expansion of T cells followed by deletion or anergy (12).

Animal models have contributed significantly to our understanding of the mechanisms involved in toxic shock, however, mice are much less sensitive to superantigen-mediated effects than humans (13, 14). The differential sensitivity resides in the lower binding affinity of superantigens to murine MHC class II than to human HLA class II (15). HLA class II transgenic mice thus provide an opportunity to study toxic shock in a readily manipulated animal model with similar sensitivity to superantigens as humans (16, 17, 18, 19). To enhance the effects of toxins in mice, some researchers have used a "double dose" regimen in which high doses of superantigen are given twice, one essentially a priming dose and the other a challenge, or have coadministered the hepatotoxic drug, D-galactosamine. This drug increases sensitivity by up to 1000-fold through an effect on liver transcription (20, 21), although under the conditions used it has no lethal or immunostimulatory effect alone (11, 22).

Superantigens have a marked ability to stimulate T cells in vitro and during toxic shock trigger profound hypotension and multiorgan failure. The proposed causal link between in vitro and in vivo observations is the cumulative Th1 cytokine storm elicited by excessive T cell activation by superantigens (23, 24). In support of a simple T cell activation model, early studies from Marrack et al. (25), using SEB-induced weight loss as a correlate of shock, showed that nude or cyclosporine-treated mice were relatively protected from this effect (11, 25). Bette et al. (26) showed that early cytokine mRNA production in the spleen was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets (PALS) following SEB injection and was not affected by macrophage depletion. In addition, Tsytsykova and Goldfeld (27) showed that mice deficient in NFATp, a critical T cell transcription factor for TNF-{alpha}, are resistant to SEB-induced toxic shock. However, there is evidence that superantigens can in certain circumstances elicit inflammation in a non-T cell-dependent manner. Some recombinant mutant superantigens, which lack residues necessary for T cell activation, still retain lethality in rabbit models of toxic shock (28). Conversely, some mutant superantigen constructs that have lost lethality have unaltered ability to superantigenically stimulate T cells (29).

The evidence supporting the importance of T cells in toxic shock led to the expectation that T cell-derived cytokines would play a key role in the pathogenesis of toxic shock. IFN-{gamma}, as the hallmark cytokine of Th1 activation, is implicated by the finding that IFN-{gamma}R knockout mice are resistant to endotoxin or SEB-induced shock (30). However, in other studies, treatment with anti-IFN-{gamma} Abs appears only partially protective (10, 31, 32). Thus, the aim of the current study was, using a highly superantigen-sensitive transgenic model, to dissect the events leading to toxic shock, asking which elements of immune activation are most evidently linked to the lethal effects of the bacterial toxin. We have characterized the response of HLA-DR1 transgenic mice to SEB both in vitro and in vivo, and compared this response to control mice. Our objectives were to investigate which cytokines and which cell populations are critical for lethality in SEB-induced toxic shock. To this end, we have compared responses of HLA-DR1 mice with and without an intact TCR{alpha}{beta} T cell population.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

FVB/N wild-type controls, HLA-DR1, and HLA-DR1 TCR{alpha}–/– mice were bred and maintained in accordance with Home Office guidelines. HLA-DR1 transgenic mice were generated from FVB/N mice by insertion of HLA-DRAI*0101 and HLA-DRB1*0101 genes as described in Ref.33 . TCR transgenic mice carry a targeted disruption in the TCR {alpha} gene as described in Ref.34 . They have no TCR{alpha}{beta}-positive T cells but have normal TCR{gamma}{delta} T cells. These mice were then bred with HLA-DR1 mice and a breeding colony maintained as HLA-DR1 TCR{alpha}+/– and HLA-DR1 TCR{alpha}–/– mice. The presence of TCR{alpha}{beta}-positive T cells in HLA-DR1 TCR{alpha}+/– and their absence in HLA-DR1 TCR{alpha}–/– mice was confirmed by FACS. HLA-DR1 TCR{alpha}–/– mice had similar or proportionally higher levels of B cells, monocytes, and TCR{gamma}{delta} cells in spleen and lymph nodes compared with HLA-DR1 TCR{alpha}+/– mice (data not shown). The genotype of the transgenic mice was routinely assessed by PCR.

Treatment groups

Groups of mice received injections i.p. with 20 mg of D-galactosamine (Sigma-Aldrich) in 0.2 ml of saline and 0.2–200 µg of SEB (Toxin Technology) in 0.2 ml of saline. Mice rarely showed any adverse symptoms for the first 6 h following treatment. Thereafter mice suffering from toxic shock showed prostration, piloerection, weight loss, dehydration, and liver necrosis (21, 22), usually dying 7–8 h after treatment. Alternatively, groups of mice received injections i.p. with 2 doses of 50–100 µg of SEB in 0.2 ml of saline, 48 h apart. Survival was monitored 8–24 h after last dose.

Some groups of mice received injections i.p. with anti-cytokine Abs in addition to challenge with 20 mg of D-galactosamine and 20 µg of SEB. Mice received injections with 500 µg/mouse of hamster anti-TNF-{alpha} Ab, TN3-19.12 (35) or the isotype-matched control, L2-3D9 (CellTech) 1 h before or 4 h after challenge and 400 µg/mouse of purified rat anti-IFN-{gamma} Ab R4-6A2 (36) (provided by Dr. A. Annenkov, Kennedy Institute of Rheumatology, Imperial College, London, U.K.) or the isotype control, 337.217.7 1 h before challenge. Ab efficiency in removing cytokine from the serum was established by ELISA of serum samples (data not shown).

Blood samples were taken by tail bleed or cardiac puncture and serum was stored at –20°C until analysis. Only female mice were used in experiments. Mice were age and weight matched.

Proliferation assays

A single cell suspension of spleen cells in RPMI 1640 medium containing 10% FCS, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin was plated at 2 x 105 cells/well in triplicate in 96 round-bottom plates. The cells were cultured at 5% CO2, 37°C for 72 h in the presence of 0–1 µg/ml SEB or 2 µg/ml Con A (Sigma-Aldrich) in a final volume of 200 µl. The cells were pulsed with 1µCi/well [3H]thymidine (Amersham Biosciences) for the last 18 h of culture. The incorporated radioactivity was measured by liquid scintillation. Duplicate cultures were set up for cytokine analysis where 150 µl/well of medium was removed after 48 or 72 h of culture and stored at –20°C until analysis.

Tissue and intracellular TNF-{alpha}

HLA-DR1 mice were treated with 20 mg of D-galactosamine and 20 µg of SEB. For measurement of tissue TNF-{alpha}, tissue samples were taken after 0, 2, and 7 h. One-hundred milligram spleen and liver samples were homogenized in 500 µl of RPMI 1640 medium containing Protease Inhibitor Cocktail III (Calbiochem) and 1 ml of the same medium was used for peritoneal lavage. The samples were freeze-thawed three times and spun at 2000 x g for 20 min to remove tissue debris.

For measurement of intracellular TNF-{alpha}, tissue samples were taken after 0, 1.5, and 6.5 h. Peritoneal cells were removed by lavage using 5 ml of RPMI 1640, 10 U/ml heparin and the spleen was homogenized to a single cell suspension. Cells were incubated with 50% FCS in PBS for 15 min and washed in 2% FCS in PBS, incubated with anti-CD3-FITC, anti-B220-FITC, anti-Mac-1-FITC, and isotype-matched control Abs for 20 min and washed, fixed for 15 min in Cell Fix and washed, incubated in 0.2% saponin in PBS for 10 min and washed, incubated with anti-TNF-{alpha}-PE and isotype-matched control Ab for 30 min and washed in 0.2% saponin in PBS. All incubations were carried at 4°C and all reagents were supplied by BD Biosciences. Samples were analyzed on a FACSCalibur and 10,000 events were acquired for each sample. Data analysis was performed using CellQuest software.

Cytokine detection

IL-6, IL-12, TNF-{alpha}, and IFN-{gamma} were detected by ELISA using matched Ab pairs and according to the manufacturer’s instructions (R&D Systems).

Statistics

Data were analyzed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HLA-DR1 transgenic mice show enhanced sensitivity to SEB in vitro

To establish a model of toxic shock in HLA-DR1 mice we first investigated the sensitivity of spleen cells to SEB in vitro. The proliferative response of HLA-DR1 spleen cells was at least 100-fold greater than control mice when exposed to 1–100 ng/ml SEB (Fig. 1A). Over the same concentration range of SEB, HLA-DR1 spleen cells also released significantly higher amounts of TNF-{alpha}, IL-6, and IFN-{gamma} compared with control spleen cells (Fig. 1, B–D). Thus, HLA-DR1 mice show enhanced sensitivity to SEB in vitro as we and others have already shown for other HLA class II transgenic mice (16, 18, 19, 37).



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  		FIGURE 1. Spleen cells from HLA-DR1 mice are more responsive to SEB in vitro. Spleen cells from control FVB/N and HLA-DR1 mice were exposed to 0–10 µg/ml SEB for 3 days. A, Proliferation was measured by [3H]thymidine uptake after 72 h. Proliferation in the presence of 2 µg/ml Con A was 245,547 ± 9,764 for controls and 238,015 ± 20,716 for HLA-DR1. Cytokine release after 72 h: B, TNF-{alpha}; C, IL-6; D, IFN-{gamma}. Data shown are means from three individual mice ± SD. *, p < 0.005 by t test.

 
The presence of TCR{alpha}{beta} T cells is an absolute requirement for in vitro responses to SEB

IL-6 is an acute phase cytokine released by a wide range of cell types and not generally associated with the specific activation of Th1 cells. To investigate whether this response can be elicited in the absence of TCR{alpha}{beta} cells, we backcrossed HLA-DR1 transgenic mice onto a homozygous deletion for the TCR{alpha} locus. Use of TCR{alpha}–/– mice was also of particular interest given that TCR{gamma}{delta} T cells are reported to respond to bacterial superantigen (38, 39).

Spleen cells from HLA-DR1 TCR{alpha}+/– mice, which have a normal T cell repertoire, proliferated in response to SEB following a similar dose response to that seen for HLA- DR1 spleen cells (Figs. 1A and 2A). In contrast, spleen cells from HLA-DR1 TCR{alpha}–/– mice, which lack TCR{alpha}{beta} T cells, did not proliferate at any concentration of SEB, even at 10 µg/ml SEB which was capable of inducing a marked response in control mice. Spleen cells from HLA-DR1 TCR{alpha}+/– mice released significant amounts of IL-6, TNF-{alpha}, and IFN-{gamma} in response to SEB whereas spleen cells from HLA-DR1 TCR{alpha}–/– mice did not (Fig. 2, B–D). Therefore, the proliferative and cytokine responses of spleen cells to SEB are entirely dependent on the presence of TCR{alpha}{beta} T cells. TCR{gamma}{delta} T cells present in HLA-DR1 TCR{alpha}–/– spleen cells were unable to substitute for the lack of TCR{alpha}{beta} T cells.



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  		FIGURE 2. Response to SEB in vitro is T cell dependent. Spleen cells from HLA-DR1 TCR mice were exposed to 0–10 µg/ml SEB for 3 days. A, Proliferation measured by [3H]thymidine uptake. Proliferation in the presence of 2 µg/ml Con A was 124,221 ± 49,264 for HLA-DR1 TCR{alpha}+/– and 6,293 ± 6,421 for HLA-DR1 TCR{alpha}–/–. Cytokine release after 48 h: B, TNF-{alpha}; C, IL-6; D, IFN-{gamma}. Data shown are means of values from three individual mice ± SD. All values except medium alone were p < 0.001 by t test.

 
Sensitivity of HLA-DR1 mice to SEB in vivo is also T cell dependent

Next, we investigated whether the enhanced sensitivity to SEB we had seen in vitro was mirrored by a similar sensitivity in vivo. Although a single dose of superantigen is rarely fatal to unsensitized mice, treatment with two doses can cause mortality (11, 32). Control and HLA-DR1 mice were given two doses of SEB 48 h apart and their survival monitored for a further 24 h. All control mice survived (5 of 5) this treatment whereas 40% of DR1 mice died (2 of 5 mortality). Control mice gained weight over the course of the experiment (gain 1.0 ± 0.5 g from 0 to 72 h, p < 0.005 by t test), whereas HLA-DR1 mice lost weight (lost 1.2 ± 0.6 g from 0 to 72 h, p < 0.005 by t test).

The role of T cells in the response to SEB in vivo was investigated by giving two doses of SEB to mice without TCR{alpha}{beta} T cells. All HLA-DR1 TCR{alpha}–/– mice survived (5 of 5) whereas only 40% of HLA-DR1 TCR+/– mice survived (2 of 5) two doses of SEB. HLA-DR1 TCR{alpha}+/– mice lost more weight over the course of the experiment (2.7 ± 1.52 g) than HLA-DR1 TCR{alpha}–/– mice (1.9 ± 0.60 g) but this difference was not statistically significant. Thus HLA-DR1 mice show enhanced sensitivity to SEB in vivo compared with control mice and mortality to SEB appeared to be dependent on TCR{alpha}{beta} T cells.

Serum cytokines produced in response to SEB are T cell dependent

The role of HLA class II and TCR{alpha}{beta} T cells in cytokine release in vivo was investigated by measuring serum cytokines after two doses of SEB. HLA-DR1 mice had significantly higher levels of serum IL-6, TNF-{alpha}, and IFN-{gamma} 7 h after the second SEB dose compared with control mice (Fig. 3A).



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  		FIGURE 3. Increased serum cytokines in HLA-DR1 mice after two doses of SEB are T cell dependent. Mice received injections i.p. with 50–100 µg of SEB 48 h apart. Serum cytokines were analyzed 7 h after the second dose. A, Control FVB/N and HLA-DR1 mice. B, HLA-DR1 TCR mice. Data are means of values from five individual mice ± SD. *, p < 0.001; +, p < 0.01 by t test.

 
HLA-DR1 TCR{alpha}–/– mice had significantly lower levels of serum IL-6, TNF-{alpha}, IL-12, and IFN-{gamma} 7 h after the second SEB dose compared with HLA-DR1 TCR{alpha}+/– mice (Fig. 3B) demonstrating that production of these cytokines was largely, though not completely, T cell driven. To our knowledge this is the first demonstration that superantigen induced cytokine responses in vivo depend upon an intact TCR{alpha}{beta} T cell repertoire.

Lethal effects of D-galactosamine and SEB in HLA-DR1 mice are T cell-dependent

The sensitivity of mice to SEB can be further enhanced by coadministration of D-galactosamine such that lethal toxic shock is induced within 8 h. HLA-DR1 mice were highly susceptible to toxic shock induced by D-galactosamine and SEB. In contrast, control mice showed no adverse effects to this treatment and survived even the highest dose of SEB given. Neither D-galactosamine alone nor 200 µg of SEB alone was lethal to HLA-DR1 mice (Table I).


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  		Table I. Mortality of mice from toxic shock induced by D-galactosamine and SEBa

 
Next we investigated whether TCR{alpha}{beta} T cells were responsible for lethality in this sensitized model of toxic shock. HLA-DR1 TCR{alpha}+/– mice showed a similar sensitivity to the lethal effects of 20 µg of SEB in the presence of D-galactosamine, as HLA-DR1 mice (Table I). In contrast, all HLA-DR1 TCR{alpha}–/– mice survived without ill effects, clearly demonstrating for the first time that TCR{alpha}{beta} T cells are critical to the lethal effects of toxic shock in this model.

Both groups of mice lost the same amount of weight over the 8 h of the experiment (HLA-DR1 TCR{alpha}+/– lost 1.3 ± 0.4 g, HLA-DR1 TCR{alpha}–/– lost 1.3 ± 0.5 g; both p < 0.001). Previously, Marrack et al. (25) found that weight loss correlated with lethality from a single superantigen dose and that nude mice or mice treated with cyclosporine were protected from weight loss. In this instance, mice were sensitized with D-galactosamine which may account for the difference in results. However, the weight loss in HLA-DR1 TCR{alpha}–/– mice was transient and mice returned to their starting weights overnight (data not shown).

HLA-DR1 mice show an early and late burst of serum cytokines after D-galactosamine and SEB

Next we investigated the systemic release of cytokines in the D-galactosamine-sensitized model. HLA-DR1 transgenic mice showed a much stronger serum cytokine response to SEB than control mice (Fig. 4, A–D). This difference was not due to differing sensitivity to D-galactosamine, because D-galactosamine alone produced little or no cytokines. HLA-DR1 mice showed an early burst of TNF-{alpha}, IL-2, and IL-6 release 2 h after treatment followed by a drop in levels and a further rise in TNF-{alpha} and IL-6 just before death from toxic shock (Fig. 4, E and F). In contrast, serum IL-12 and IFN-{gamma} levels in HLA-DR1 mice showed a gradual increase over 7 h coinciding with the second phase of TNF-{alpha}. IL-12 levels peaked at 6 h and preceded the rise in serum IFN-{gamma} (Fig. 4G). Thus, the response to SEB in this model can be broadly considered to encompass a rapid, early IL-2, IL-6, and TNF-{alpha} response, followed some hours later by build up of a more classically Th1, IL-12-driven IFN-{gamma} response.



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  		FIGURE 4. Serum cytokines are higher in HLA-DR1 mice after treatment with D-galactosamine and SEB. Mice received injections i.p. with 20 mg of D-galactosamine with and without 20 µg of SEB. Serum cytokines at 7 h for control FVB/N and HLA-DR1 mice: A, TNF-{alpha}; B, IL-6; C, IFN-{gamma}; D, IL-12. Serum cytokine time course for HLA-DR1 mice: E, IL-2; F, TNF-{alpha} and IL-6; G, IL-12 and IFN-{gamma}. Data are means of values from three individual mice for D-galactosamine treatment and five individual mice for D-galactosamine and SEB treatment ± SD. *, p < 0.01; +, p < 0.05 by t test.

 
Early but not late release of TNF-{alpha} is a critical mediator in lethality due to SEB

To determine which of these cytokines might be key mediators of lethality in the D-galactosamine model, HLA-DR1 mice were pretreated with Abs to remove systemic TNF-{alpha} or IFN-{gamma} before challenge with D-galactosamine and SEB.

Pretreatment with anti-TNF-{alpha} Ab was protective (Table II) and intriguingly led to a marked reduction in the early but not later phase of cytokine response. Both IL-6 and TNF-{alpha} levels were reduced 1 h after D-galactosamine and SEB in HLA-DR1 mice treated with anti-TNF-{alpha} Ab compared with mice treated with control Ab. However, 7 h after challenge both control and treatment groups had similar levels of IL-6 and TNF-{alpha} (Fig. 5, A and B). Furthermore, the later surge in IFN-{gamma} levels was unaffected by treatment with anti-TNF-{alpha} Ab (Fig. 5C). This suggested that the early cytokine response is largely responsible for lethality in the D-galactosamine model as opposed to the later, conventional Th1 response involving IFN-{gamma}.


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  		Table II. Mortality of mice treated with anti-cytokine Abs, D-galactosamine, and SEBa

 


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  		FIGURE 5. Early acute phase cytokine response is inhibited by anti-TNF-{alpha} Ab. HLA-DR1 mice received injections i.p. with 500 µg of anti-TNF-{alpha} Ab or an isotype-matched control 1 h before challenge with 20 mg of D-galactosamine and 20 µg of SEB. Serum cytokines were measured at 1 and 7 h: A, TNF-{alpha}; B, IL-6; C, IFN-{gamma}. Data are means of values from five individual mice ± SD. *, p < 0.001 by t test.

 
To test this hypothesis, administration of anti-TNF-{alpha} Ab was delayed until after the early cytokine response but before the later rise in TNF-{alpha} (administration 4 h after D-galactosamine and SEB). As expected, anti-TNF-{alpha} Ab administered at 4 h reduced serum TNF-{alpha} and IL-6 just before death (7 h) although IFN-{gamma} and IL-12 levels were unaffected (data not shown). Delaying the anti-TNF-{alpha} Ab until after early cytokine response proved critical as HLA-DR1 mice were not protected from toxic shock and mortality was the same in both control and treatment groups (Table II).

Abs to IFN-{gamma} were not protective against toxic shock and had no effect on TNF-{alpha} production (Table II). Serum TNF-{alpha} levels were not significantly different between anti-IFN-{gamma}-treated mice (2940.5 ng/ml, SD 928.7) and control mice (3824.2 ng/ml, SD 902.4) at 7 h.

TCR{alpha}{beta} T cells facilitate both the early acute phase and later Th1 cytokine response to SEB in D-galactosamine-sensitized mice

We have shown that SEB-induced lethality is dependent on the acute phase TNF-{alpha} response in D-galactosamine-sensitized mice, and that T cells are critical to this lethal effect. To assess the contribution of TCR{alpha}{beta} T cells to early (acute phase) and late (classical Th1) cytokine production in toxic shock, HLA-DR1 TCR{alpha}+/– and HLA-DR1 TCR{alpha}–/– mice were treated with SEB in the presence of D-galactosamine. Cytokines were measured after 1 h to assess the acute phase response and at 7 h to assess the later cytokine rise found before death. Although there was some evidence of cytokine response in mice lacking TCR{alpha}{beta} cells, IFN-{gamma}, TNF-{alpha}, and IL-6 were significantly reduced in HLA-DR1 TCR{alpha}–/– mice compared with HLA-DR1 TCR{alpha}+/– mice (Fig. 6, A–C) at both time points. IL-12 was only significantly reduced in HLA-DR1 TCR{alpha}–/– mice at 7 h (Fig. 6D). Thus the presence of TCR{alpha}{beta} T cells enhanced both the acute phase and late Th1 cytokine response to SEB in the presence of D-galactosamine.



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  		FIGURE 6. Both early acute phase and late cytokine responses after treatment with D-galactosamine and SEB are T cell dependent. HLA-DR1 TCR mice received injections i.p. with 20 mg of D-galactosamine and 20 µg of SEB. Serum cytokines were analyzed at 1 and 7 h: A, IL-6; B, TNF-{alpha}; C, IFN-{gamma}; D, IL-12. Data are means of values from five individual mice ± SD. *, p < 0.005; +, p < 0.05 by t test.

 
T cells from the spleen are a source of early TNF-{alpha} in response to SEB in D-galactosamine-sensitized mice

To identify the source of TNF-{alpha} in SEB-induced toxic shock, HLA-DR1 mice received injections with D-galactosamine and SEB and tissue homogenates of spleen, liver, and peritoneal cells were subjected to cytokine analysis. There was a significant increase in TNF-{alpha} in spleen tissue and to a lesser extent peritoneal cells at 2 and 7 h but no such increase in the liver (Table III). Next we analyzed individual spleen and peritoneal cells to identify the specific cell type responsible for TNF-{alpha} production in mice injected with D-galactosamine and SEB.


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  		Table III. Tissue source of TNF{alpha}a

 
Cells were labeled for surface CD3, B220, and Mac-1 expression, fixed, permeabilized, and then labeled for intracellular TNF-{alpha}. Intracellular TNF-{alpha} expression was not detected in peritoneal cells (data not shown) at either time point. However, a discrete but definite rise in TNF-{alpha}-positive cells was detected in the spleen at 1.5 h. Intracellular staining demonstrated that CD3-positive cells accounted for the majority of the rise in TNF-{alpha}-positive cells (Table IV).


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  		Table IV. Cellular source of TNF{alpha}a

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The aim of the current study was to investigate which elements of immune activation are critical for the lethal effects of staphylococcal superantigens. In doing this, we have brought together several features necessary for a relevant model but which have not necessarily been considered in previous studies: an optimum model needs to encompass lethality, high sensitivity, and the ability to precisely analyze the role of T cell responses. Many previous studies have investigated the effect of superantigens in vivo after a single dose or under conditions that are not harmful (32, 40, 41). We have concentrated specifically on protocols which induce lethal toxic shock either by giving two doses of SEB or by giving a single dose of SEB with the sensitizing agent D-galactosamine. We have established a model system using HLA-DR1 transgenic mice to enhance the sensitivity to superantigens based on the observation that HLA-DR1 has a higher affinity for SEB than murine MHC class II. We have also investigated the specific role of TCR{alpha}{beta} cells in SEB responses and toxic shock by comparing HLA-DR1 mice with and without an intact TCR{alpha}{beta} cell population. Previous studies conducted in nude, SCID and cyclosporine-treated mice (11, 25) are more difficult to interpret due to the broad immune defects found or induced in these mice. Finally, we have used flow cytometry to directly and positively identify the cells which produce the lethal burst of TNF-{alpha}.

We have shown that HLA-DR1 mice are more sensitive to SEB both in vitro and in vivo than control mice. This is consistent with previous reports on streptococcal superantigens and HLA-DQ8 mice (16, 17) and reports on staphylococcal superantigens and HLA-DR3, HLA-DQ6 and HLA-DQ8 mice (18, 19, 42). In our study, proliferative and cytokine responses to SEB in vitro were entirely dependent on the presence of TCR{alpha}{beta} cells. Although TCR{gamma}{delta} cells have been shown to respond to superantigens in vivo (38, 39) it seems likely that the number of these cells in the systemic circulation was too low to mount a detectable or biologically significant response.

Elevated levels of IL-10, IL-12, and IFN-{gamma} have previously been associated with lethality in the double dose model of toxic shock (32, 40). Florquin et al. (32) also found that anti-IFN-{gamma} Abs were protective, strongly implicating T cells as critical for lethality. In this study, we have conclusively shown that lethality was dependent on the presence of TCR{alpha}{beta} cells because HLA-DR1 TCR{alpha}–/– mice were protected from toxic shock when given two doses of SEB. This also suggests that TCR{gamma}{delta} cells do not play a significant role in the response to SEB in vivo. Cytokine production in vivo was clearly dependent on TCR{alpha}{beta} cells because HLA-DR1 TCR{alpha}–/– mice had significantly lower serum IL-6, TNF-{alpha}, and IFN-{gamma} than HLA-DR1 TCR{alpha}+/– mice.

When D-galactosamine is used as a sensitizing agent then superantigens can induce toxic shock within hours. This is characterized by elevated serum TNF-{alpha}, TNF{beta}, IFN-{gamma}, IL-2, IL-10, and IL-12 (10, 11). T cell involvement in this process has been demonstrated by investigating cytokine release from purified splenic T cells (43) and PBMCs (9, 24, 44), by measuring expansion of specific TCR V{beta} T cell subsets (11, 45, 46), by using nude or SCID mice (11, 25, 46), by disrupting T cell function (11, 25, 45, 46), and by disrupting the HLA class II-superantigen-T cell interaction (47, 48). From this work has emerged the hypothesis that toxic shock develops as a result of superantigen-induced T cell activation resulting in uncontrolled Th1 cytokine release culminating in fatal liver and tissue damage. In our model, we found cytokine release was biphasic with an early burst of IL-2, TNF-{alpha}, and IL-6 followed by a gradual increase in serum IFN-{gamma}, preceded by a rise in IL-12, consistent with an ongoing Th1 response and the timing of death is concomitant with high serum levels of IFN-{gamma}.

To directly address whether the Th1 response was central to lethality in the D-galactosamine-sensitized model of toxic shock, we compared HLA-DR1 with and without an intact TCR{alpha}{beta} repertoire. Protection of HLA-DR1 TCR{alpha}–/– mice from toxic shock correlated with lower serum levels of IL-6, IL-12, TNF-{alpha}, and IFN-{gamma}. However, it was noteworthy that lack of functional {alpha}{beta} T cells markedly affected the early TNF-{alpha} and IL-6 response, in addition to the expected effects on the later Th1 cytokine surge. Previous reports have found that weight loss correlated with superantigen dose and lethality (10, 25) so that mice protected from toxic shock did not lose weight. In contrast, we found that weight loss was not entirely controlled by TCR{alpha}{beta} T cells. Both IL-6 and TNF-{alpha} have been implicated in weight loss (10) and it may be than the levels of these cytokines in HLA-DR1 TCR{alpha}–/– mice are sufficient to cause transient weight loss.

There are, however, several pieces of evidence which do not support the assumption that toxic shock in the D-galactosamine sensitized model is caused by accumulation of superantigen-induced Th1 cytokines. Administration of anti-cytokine Abs to remove IL-2, IL-12, and IFN-{gamma} has not proved effective in protecting mice in various models of toxic shock (10, 30, 31, 32, 49). We also found that Abs to IL-12 (data not shown) or IFN-{gamma} were not protective in our lethal model of toxic shock.

In contrast to classical Th1 cytokines, we and others have shown that Abs against TNF-{alpha} are protective when given just before SEB and D-galactosamine (11, 31, 49). Importantly, the protective effect of anti-TNF-{alpha} pretreatment was associated with inhibition of the early TNF-{alpha} and IL-6 cytokine response in HLA-DR1 mice but not the later burst of TNF-{alpha} which was associated with rising IL-12 and IFN-{gamma}. When administration of anti-TNF-{alpha} Abs was delayed until after the early cytokine response, no protective effect was found despite the fact that the late rises in TNF-{alpha} and IL-6 were markedly reduced. This strongly suggests that it is the early cytokine burst which is responsible for lethality in toxic shock rather than the later rise in Th1 cytokines. The key mechanism of lethality is probably TNF-{alpha}-induced liver damage (22, 49, 50).

Next, we addressed the tissue and cellular origin of the early serum TNF-{alpha} seen in our model. The predominant cell types first exposed to SEB in this model are monocytes and macrophages in the peritoneum. SEB is rapidly absorbed into the circulation where it can induce cytokine production in both lymphoid and nonlymphoid tissue such as the spleen and liver (49). In a nonlethal model of toxic shock, Bette et al. (26) identified early TNF-{alpha} mRNA production in the PALS area of the spleen, which is predominantly, although not exclusively a T cell area. We also found that the spleen is a major source of early TNF-{alpha} in our lethal model of toxic shock. Some TNF-{alpha} was also detected in the peritoneum but the cellular source could not be identified. Importantly, we showed that the increase in TNF-{alpha} in the spleen originated from CD3 positive cells, providing direct evidence for the first time that T cells are responsible for the very early burst of TNF-{alpha} seen during toxic shock. The rapidity of TNF-{alpha} release in vivo may result from the proximity between HLA class II-expressing and TCR-expressing cells in a solid organ such as the spleen which contrasts with the cumulative release of TNF-{alpha} and classical Th1 cytokines seen in vitro following superantigen stimulation.

In summary, using a novel transgenic model we have shown that mortality and serum cytokines in toxic shock are critically dependent on the presence of TCR{alpha}{beta} T cells. The accumulation of Th1 cytokines before death is not the decisive factor in lethality; lethality is crucially dependent on an early burst of TNF-{alpha} that originates from TCR{alpha}{beta} T cells in the spleen.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Defense Advanced Research Projects Agency/Army Reserve Office. Back

2 Address correspondence and reprint requests to Dr. Shiranee Sriskandan, Department of Infectious Diseases, Imperial College, Hammersmith Hospital, Du Cane Road, London, W12 0NN, U.K. E-mail address: s.sriskandan{at}imperial.ac.uk Back

3 Abbreviation used in this paper: SEB, staphylococcal enterotoxin B. Back

Received for publication April 28, 2005. Accepted for publication August 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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