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Published ahead of print on June 15, 2007, doi:10.1164/rccm.200611-1712OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 473-482, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200611-1712OC

Original Article

Sexual Dimorphism in Superantigen Shock Involves Elevated TNF-{alpha} and TNF-{alpha}–induced Hepatic Apoptosis

Lee Faulkner1, Daniel M. Altmann1, Stephan Ellmerich1, Ilpo Huhtaniemi2, Gordon Stamp3 and Shiranee Sriskandan1

Departments of 1 Infectious Diseases and Immunity, 2 Reproductive Biology, and 3 Histopathology, Imperial College, London, United Kingdom

Correspondence and requests for reprints should be addressed to Shiranee Sriskandan, F.R.C.P., Ph.D., Department of Infectious Diseases and Immunity, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: s.sriskandan{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: There is conflicting evidence regarding sex differences in the outcome from severe sepsis and toxic shock. Superantigen-mediated toxic shock affects a higher proportion of female patients.

Objectives: The objective of the current study was to investigate sexual dimorphism in superantigen-associated sepsis and in superantigen-mediated shock and to identify the key mechanisms responsible for this sex difference.

Methods: We measured mortality and serum cytokines after induction of sepsis with isogenic superantigen-positive and superantigen-negative Streptococcus pyogenes in HLA class II transgenics. During superantigen-mediated toxic shock, we measured mortality, T-cell responses, systemic tumor necrosis factor (TNF)-{alpha} and TNF receptors, TNF-{alpha}–induced hepatocyte apoptosis, and conditioning of these responses by tamoxifen treatment.

Measurements and Main Results: In both superantigen-associated sepsis and in superantigen-mediated shock, serum TNF-{alpha} was increased in females compared with males. This was not attributable to a detectable difference in splenic TNF-{alpha} transcription; rather, serum soluble TNF receptors were higher in males. Pretreatment of females with the estrogen receptor modulator tamoxifen increased serum soluble TNF receptors, reduced the early serum TNF-{alpha} response, and improved mortality in females challenged with staphylococcal enterotoxin B. Lethal superantigen shock was characterized by hepatocyte apoptosis, and was reproduced by injection of TNF-{alpha}. Females had enhanced susceptibility to TNF-{alpha}–mediated lethality. TNF-{alpha}–induced hepatocyte apoptosis was greater in females, and was reduced by tamoxifen pretreatment.

Conclusions: Sexual dimorphism in experimental superantigen toxic shock results from increased systemic TNF-{alpha} in females, coupled with an increased susceptibility to TNF-{alpha}–induced hepatocyte apoptosis. Both processes are abrogated by estrogen receptor modulators.

Key Words: superantigen • sepsis • tumor necrosis factor-{alpha} • apoptosis



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Females appear to be more susceptible to shock or death in the specific setting of severe sepsis and superantigen-mediated toxic shock.

What This Study Adds to the Field
Sexual dimorphism in toxic shock results from an early and enhanced presence of TNF-{alpha} in females, coupled with an increased susceptibility to TNF-{alpha}–-induced hepatocyte apoptosis, both of which are dependent on estrogen receptor signaling.

 
Bacterial sepsis leading to shock is a major cause of morbidity and mortality. Although the underlying molecular mechanisms of severe sepsis remain poorly characterized, there is a clear immunopathological component (1). This is particularly the case with respect to T-cell activation by bacterial superantigens, which can lead to toxic shock (2). We recently reappraised the early events in experimental toxic shock, revealing an important role for the early burst of T-cell–derived tumor necrosis factor (TNF)-{alpha} (3).

Notwithstanding the linkage of toxic shock syndrome with menstruation, clinical studies have shown that superantigen toxic shock affects female patients disproportionately (4). Intriguingly, superantigen-sensitive HLA class II transgenic mice also demonstrate a similar sexual dimorphism; in our studies, we found that female mice are highly susceptible to superantigen shock, whereas males are almost entirely resistant. Females are known to develop stronger antibody and T-cell responses than males, and studies have shown that females are more effective than males at resolving infection by parasites, viruses, bacteria, and fungi (5). Although females show better recovery from surgery and are less likely to develop sepsis than males (6, 7), conflicting evidence exists regarding potential sex differences in outcome from severe sepsis (6, 8, 9). Enhanced immune effectiveness in females may also result in a propensity for developing autoimmune disease. Striking sex differences are observed in Sjögren's syndrome, systemic lupus erythematosus (SLE), autoimmune thyroid disease, and rheumatoid arthritis where the majority of patients are women (10).

Because the influence of sex on immune responsiveness usually becomes apparent after sexual maturity, a crucial role in this process has been attributed to sex hormones, such as estrogens and androgens (9). Baseline estrogen concentrations do not differ markedly between the sexes, but females have cyclically high concentrations during the ovulation stage of the estrous cycle. The peak immune responsiveness in female mice corresponds to the peak level of estrogen (11, 12). The influence of estrogens on the incidence and course of autoimmune diseases has been clearly established in mouse models of SLE (13), collagen-induced arthritis (14), and autoimmune encephalomyelitis (15). In murine models of sepsis and surgical recovery after shock, administration of estrogen or suppression of androgens was also beneficial (16, 17).

The present study set out to dissect the nature of observed sex differences in susceptibility to both superantigen-associated sepsis and superantigen-induced toxic shock using established HLA class II transgenic models. Sexual dimorphism in susceptibility to superantigen shock was found to be related to increased serum TNF-{alpha}, lower soluble TNF receptor (sTNF-R), and enhanced TNF-{alpha}–induced hepatic cell death in females. Both increased serum TNF-{alpha} and enhanced TNF-{alpha}–induced apoptosis were reversed by the estrogen receptor modulator tamoxifen.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
HLA-DQ8 mice (18) were backcrossed for several generations with C57BL/6 Abeta0 mice (19). HLA-DQ8 mice and HLA-DR1 mice (20) were bred on site and BALB/c mice purchased from Harlan UK Ltd. (Bicester, UK). All mice were maintained in accordance with UK government Home Office guidelines. Unless otherwise stated, male and female mice were weight-matched and aged between 6 and 16 weeks, with an age difference of no more than 8 weeks. Females were studied at random phases of the estrous cycle as demonstrated by the distribution of serum luteinizing hormone (LH) levels, measured as described previously (21) (data not shown).

Bacteria
Streptococcus pyogenes strain H305 (speA+smeZ+) was used in initial studies. Isogenic S. pyogenes strains H377 (speA–smeZ–) (22) and H432 (speA+smeZ+) were used subsequently; both are derivatives of H293, a clinical necrotizing fasciitis isolate. H432 was derived from H293 (speA–smeZ+) by transformation with pDL278speA (23). Strains were grown overnight at 37°C in Todd Hewitt broth. Inocula in sterile saline were quantified by plating and were 6 x 108 cfu for H305, 9 x 107 cfu for H432, and 4 x 107 cfu for H377.

Experimental Groups
For infection, S. pyogenes was administered into the right leg muscle of HLA-DQ8 mice. For toxic shock, 2 to 200 µg staphylococcal enterotoxin B (SEB), streptococcal pyrogenic exotoxin A (SPEA) (Toxin Technology, Sarasota, FL), streptococcal mitogenic exotoxin Z (SMEZ), or TNF-{alpha} (200 ng or 1 µg) (R&D Systems, Abingdon, UK) in 0.2 ml saline was administered intraperitoneally to HLA-DR1 or HLA-DQ8 mice. D-galactosamine (Dgal) (20 mg) in 0.2 ml saline was also administered immediately prior to toxin injection. Survival was monitored for 74 hours (sepsis) and for 24 hours (Dgal) using defined humane endpoints. Preliminary studies (3) established that Dgal-sensitized HLA DR1 mice receiving superantigen reached defined endpoints within 8 hours of challenge; any mice surviving to 24 hours were found to be long-term survivors. Hence, studies using Dgal-sensitized mice were monitored for 24 hours only.

For tamoxifen pretreatment, 4-week-old female HLA-DR1 mice were injected intraperitoneally with 50 µg/10g tamoxifen daily for 15 days (50 µl/mouse). Tamoxifen was dissolved in ethanol and administered in 2-hydroxypropyl beta-cyclodextrin or olive oil; control animals received the same volume of ethanol in vehicle (< 2 vol/50 vol). On Day 16, mice were challenged intraperitoneally with Dgal and 20 µg SEB or 200 ng TNF-{alpha} in 0.2 ml saline. Blood was taken by tail bleed or cardiac puncture. Dgal, 2-hydroxypropyl beta-cyclodextrin, and tamoxifen were from Sigma-Aldrich (Poole, UK). SMEZ was a gift of Thomas Proft (University of Auckland, Auckland, NZ).

Spleen Cell Analysis
Measurement of proliferation and cytokine release by superantigen-stimulated spleen cells was carried out as described by Faulkner and colleagues (3). Flow cytometry of spleen cells was also carried out as described in Faulkner and colleagues' report (3). Directly labeled antibodies to HLA-DR, TNF-{alpha} converting enzyme (TACE) (R&D Systems), and to murine CD3, CD4, CD8, CD14, Mac-1, B220, T-cell receptor (TCR) Vbeta (Becton Dickinson, Oxford, UK), CD95, CD95L, FoxP3 Fix/Perm, and Perm solution (eBioscience, London, UK) were used.

Reverse Transcriptase–Polymerase Chain Reaction Analysis
The presence of TNF-{alpha} mRNA and 18S ribosomal RNA in the spleen was assessed by reverse transcriptase–polymerase chain reaction (see the online supplement). Data were analyzed using the 2-{Delta}{Delta}Ct method (24); samples were normalized using 18S, and female HLA-DR1 mice provided the baseline value at 0 minutes. Statistical analysis was performed using the REST (Relative Expression Software Tool) analysis (25).

Cytokine Detection
Soluble TNF-RI and TNF-RII were detected using Quantikine kits; IL-6 and TNF-{alpha} were detected by ELISA using matched antibody pairs and according to the manufacturer's instructions (R&D Systems). Sham-treated mice did not demonstrate significant increases in cytokine levels.

Tissue Histology and Apoptosis
Tissues were fixed in formalin, embedded in paraffin wax, and stained with hematoxylin–eosin. Sections were coded and reviewed by an experienced histopathologist. Liver samples were snap frozen and stored at –80°C, until homogenized in 200 µl of lysis buffer per 50 mg wet weight. Oligosomal DNA was detected by the Cell Death Detection ELISA kit, according to the manufacturer's instructions (Roche Diagnostics, Burgess Hill, UK). Data were normalized to the positive control and the background subtracted (liver samples from untreated mice).

Statistics
Survival data were analyzed by log-rank analysis. Other data were analyzed using an unpaired t test. Box plots show the median and range from the 25th to the 75th percentile.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate potential sex differences in experimental superantigen shock, we used two types of HLA class II transgenic model. First, we used an HLA-DQ8 model of S. pyogenes sepsis in which superantigens are known to influence inflammation during intramuscular infection and disease severity can be monitored by serum IL-6 and TNF-{alpha} (22, 26). Second, we used an HLA-DR1 model of lethal toxic shock in which mice receive an injection of Dgal with purified superantigen, and susceptibility is dependent on an early burst of splenic T-cell–derived TNF-{alpha} (3). This model results in fulminant hepatocyte death without evidence of other organ failure, reminiscent of other Dgal-sensitized models of endotoxic shock (27).

Female Mice Are More Sensitive to Superantigen-associated Sepsis
HLA-DQ8 mice infected with an M1 S. pyogenes strain H305 (known to produce superantigens SPEA and SMEZ) developed prominent swelling and necrotizing lesions at the site of infection, conjunctivitis, and lethal bacteremia (26). When weight-matched HLA-DQ8 mice were infected with 6 x 108 cfu S. pyogenes, it was noted that female mice were more rapidly susceptible to sepsis than male mice (log-rank test p < 0.01; Figure 1A). Serum cytokines measured at 24 hours also showed a sex difference, in that female HLA-DQ8 mice had higher levels of IL-6 and TNF-{alpha} than male HLA-DQ8 mice (Figure 1B). Blood bacterial counts did not significantly differ between male and female mice (data not shown). The same result was obtained when age-matched HLA-DQ8 mice were used (data not shown). "


Figure 1
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  		Figure 1. Female mice are more sensitive to streptococcal sepsis. HLA-DQ8 mice were injected intramuscularly with 6 x 108 cfu Streptococcus pyogenes H305 in the right leg muscle. (A) Survival was monitored for 74 hours. Log-rank test, P < 0.01. (B) Blood samples were taken after 24 hours and serum cytokines measured by ELISA. The data shown are means and SD of values from 10 individual mice, *P < 0.001 by unpaired t test.

 
To further assess the role of superantigen responses in the sex difference observed during streptococcal sepsis, we performed two further studies using isogenic M89 S. pyogenes strains that differed only in the production of superantigens SPEA and SMEZ. The M89 strains used do not result in lethal infection or elevated serum TNF-{alpha}, necessitating measurement of serum IL-6 as an indicator of disease severity. As observed for the M1 strain, a sexually dimorphic response in IL-6 production was seen when female and male HLA-DQ8 mice were infected with an smeZ+speA+ strain (H432). Females produced significantly more IL-6 (7,415.8 ± 1,375 pg/ml) than males (2,717.9 ± 1,325 pg/ml, p = 0.0003, t test) at 24 hours. However, when mice were infected with an smeZ–speA– isogenic strain (H377) that has no superantigenic activity in HLA-DQ8 mice, no sex difference in IL-6 levels was observed between female and male mice (females, 6,726.8 ± 1,533 pg/m;; males, 6,138 ± 1,452 pg/ml; p = 0.275). Taken together, the data strongly suggested that the sex difference in susceptibility to S. pyogenes sepsis arose as a consequence of sexual dimorphism in the response to superantigens.

Female Mice Are More Sensitive to Purified Superantigen than Male Mice
Next, we assessed the response of mice to lethal toxic shock triggered by Dgal and purified superantigens. Female HLA-DQ8 mice were more susceptible to toxic shock induced by SPEA or SMEZ and female HLA-DR1 mice were more susceptible to toxic shock induced by SEB than male mice (Table 1). As little as 2 µg SEB was lethal for female HLA-DR1 mice, whereas male HLA-DR1 mice were capable of surviving a 100-fold higher dose (Table 1). The observed sexual dimorphism was not restricted to HLA transgenic mice because even BALB/c mice demonstrated greater female sensitivity to Dgal and SEB (Table 1). Weight-matched mice were used for these experiments, although age-matched mice also gave the same result (data not shown). Thus, the sex difference observed in streptococcal shock was reproduced in response to purified superantigen, and the sex difference was not restricted to a specific superantigen or to a specific strain of HLA transgenic or nontransgenic mouse. For subsequent in vivo experiments, we focused on the SEB model of toxic shock.


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  		TABLE 1. FEMALE MICE ARE MORE SENSITIVE TO TOXIC SHOCK

 
Female Mice Have Elevated Serum TNF-{alpha} in Response to SEB
Systemic release of TNF-{alpha} occurs 0.5 to 2 hours after treatment with Dgal and SEB, and this early burst of TNF-{alpha} is known to determine lethality (3). Female HLA-DR1 mice treated with Dgal and SEB had significantly elevated serum TNF-{alpha} compared with male mice (Figure 2A). This suggested that the sex difference in response to superantigen shock might be explained by sexual dimorphism in serum TNF-{alpha}. Although there was a significant increase in TNF-{alpha} mRNA in the spleens of both male and female HLA-DR1 mice 30 to 90 minutes after treatment with Dgal and SEB (unpaired t test, P < 0.01 for 30, 60 and 90 min; Figure 2C), significant differences in TNF-{alpha} mRNA levels were not detected between male and female HLA-DR1 mice at these time points.


Figure 2
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  		Figure 2. Female mice show rapid elevation of serum tumor necrosis factor (TNF)-{alpha} after staphylococcal enterotoxin B (SEB) administration. HLA-DR1 mice were injected with 20 mg D-galactosamine (Dgal) and 20 µg SEB (A, C), or 100 µg SEB alone (B, D). Serum TNF-{alpha} was measured by ELISA (A, B), and TNF-{alpha} mRNA levels in the spleen by reverse transcriptase–polymerase chain reaction (C, D). The data shown in (A, B) are means and SD from five individual mice. *P < 0.01 by unpaired t test. The data shown in (C, D) are from five individual mice.

 
In the absence of Dgal, a single dose of superantigen is not lethal in HLA-DR1 mice. To investigate whether the observed sex difference was maintained in the absence of Dgal, HLA-DR1 mice were treated with 100 µg SEB alone, and TNF-{alpha} was measured 2 hours later. Again, significantly more serum TNF-{alpha} was found in female mice compared with males (Figure 2B). TNF-{alpha} mRNA in the spleen rose after treatment in both male and female mice compared with untreated mice (unpaired t test, P < 0.01 for 30, 60 and 90 min; Figure 2D), although there was no consistent difference between males and females. Thus, transcriptional regulation of TNF-{alpha} from spleen cells was unlikely to fully explain the observed sexual dimorphism in TNF-{alpha} after SEB exposure.

Size of T-cell Subsets in Relation to Superantigen Responses in Males and Females
Because splenic T cells were identified as the major source of systemic TNF-{alpha} in the SEB shock model (3), we investigated whether differences in T-cell subsets or T-cell activity could explain the sexual dimorphism in serum TNF-{alpha} levels.

Although there was no difference in the total number of spleen cells between male and female HLA-DR1 mice (data not shown), flow cytometry of spleen cells showed a sex difference in the proportion of T-cell subsets in the spleen. Female HLA-DR1 mice had a higher proportion of CD4 and CD8 cells than male HLA-DR1 mice (Table 2), although the CD4/CD8 ratio was similar (female, 2.63; male, 2.77). There was, however, no difference in the proportion of CD3 cells expressing CD95L or in the proportion of CD4 cells expressing intracellular FoxP3 between male and female mice. Neither TACE nor CD95 was detected on CD3-positive cells. Differences were not seen in the proportion of CD14, B220, HLA-DR, and Mac-1–expressing cells in the spleen (data not shown).


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  		TABLE 2. SPLENIC T-CELL SUBSET RATIOS

 
Superantigen activation of T cells results in the expansion of specific TCR Vbeta families (2). SEB is reported to expand murine T cells bearing Vbeta3, Vbeta7, and Vbeta8 (28), although, in HLA-DR1 mice, SEB preferentially expands T cells expressing Vbeta3, Vbeta5.1, Vbeta6, and Vbeta7 in vitro (unpublished observations). Importantly, a proportionate size difference in these SEB-responsive subsets was not detected between males and females (Table 2).

Sexually Dimorphic Responses to Superantigen Are Not Reproduced in Ex Vivo Culture of Spleen Cells
Exposure of spleen cells from HLA-DR1 mice to SEB in vitro resulted in T-cell activation as shown by a clear dose response in proliferation and TNF-{alpha} release. Although there was a tendency for higher proliferation and TNF-{alpha} release (Figures 3A and 3C) in spleen cells from female HLA-DR1 mice, the response was not statistically different from male mice. Similarly, there was no significant sex difference in proliferation or TNF-{alpha} release when spleen cells from HLA-DQ8 mice were exposed to SPEA in vitro (Figures 3B and 3D). Spleen cell cultures also released IL-6 and IFN-{gamma} in response to superantigens and, again, no sex difference was detected (data not shown). Overall, we were unable to reproduce the in vivo findings of raised serum TNF-{alpha} in females using cell culture of spleen cells alone.


Figure 3
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  		Figure 3. In vitro spleen cell proliferation and tumor necrosis factor (TNF)-{alpha} release are similar in male and female mice. Spleen cells from HLA-DR1 mice (A, C) and HLA-DQ8 mice (B, D) were incubated with staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPEA), respectively, for 72 hours. Proliferation was measured by [3H]-thymidine uptake for the last 18 hours of culture (A, B). Culture supernatants were analyzed for TNF-{alpha} after 48 hours (C, D). Data shown are means and SD from three individual mice.

 
sTNF-Rs in the Serum Are Lower in Female HLA-DR1 Mice
Circulating sTNF-Rs are an important natural mechanism for the clearance of TNF-{alpha} during disease (29, 30). Surprisingly, baseline levels of sTNF-RI (p55) and sTNF-RII (p75) in serum were significantly lower in female mice compared with males. Levels of sTNF-R increased after SEB treatment in both male and female mice (Figure 4). However, even after SEB administration, female mice had significantly lower levels of sTNF-RI and sTNF-RII in serum than male mice, raising the possibility that clearance of endogenous TNF-{alpha} may be less effective in females than males.


Figure 4
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  		Figure 4. Circulating soluble tumor necrosis factor (TNF) receptors are lower in female mice. HLA-DR1 mice were injected with 20 mg D-galactosamine (Dgal) and 20 µg staphylococcal enterotoxin B (SEB) or 100 µg SEB alone. Serum samples were taken at 0 minutes and after 90 minutes and the presence of soluble TNF-RI (A) and soluble TNF-RII (B) measured by ELISA. The data shown are means and SD from five individual mice. *P < 0.001 by unpaired t test.

 
Female Mice Exhibit Greater Liver Damage during Toxic Shock
Lethality in the toxic shock model occurs 7 to 8 hours after treatment and is triggered by TNF-{alpha}. To characterize the effects of toxic shock on different organs, tissue samples were obtained for histologic examination from HLA-DR1 mice 7 hours after treatment with Dgal and 20 µg SEB. Inspection of tissue sections from spleen, liver, kidney, heart, lung, thymus, colon, and lymph nodes from female and male HLA-DR1 mice failed to identify any gross histologic differences in any tissues except the liver. Female mice demonstrated more liver damage than male mice, as shown by greater numbers of apoptotic hepatocytes and a greater influx of blood into necrotic areas (Figures 5A and 5B). The difference in levels of hepatocyte apoptosis was confirmed by measurement of oligosomal DNA in liver samples taken from Dgal- and SEB-treated mice. Female HLA-DR1 mice had significantly higher levels of oligosomal DNA in the liver than male mice (Figure 5C). Another method for assessing liver integrity is the measurement of metabolites, such as alanine aminotransferase (ALT), which are released from damaged hepatocytes into the blood (31). Female HLA-DR1 mice treated with Dgal and 20 µg SEB for 7 hours had significantly higher levels of serum ALT than male HLA-DR1 mice (Figure 5D). Thus, in addition to raised levels of serum TNF-{alpha}, the increased susceptibility of female mice to lethal toxic shock was also associated with clear sexual dimorphism in hepatic cell injury and death.


Figure 5
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  		Figure 5. Female mice show greater liver damage after toxic shock. HLA-DR1 mice were injected with 20 mg D-galactosamine (Dgal) and 20 µg staphylococcal enterotoxin B (SEB) or 20 mg Dgal alone. Blood and liver samples taken after 7 hours. Liver samples were fixed in formalin, stained with hematoxylin and eosin, and viewed at a magnification of x40. (A) Male HLA-DR1 mouse after Dgal and SEB; (B) female HLA-DR1 mouse after Dgal and SEB. (C) Oligosomal DNA in liver samples was measured by ELISA. (D) Serum alanine aminotransferase was measured by an automated process. The data shown in (C, D) are means and SD of values from five individual mice. *P < 0.01 by unpaired t test.

 
Female Mice Show Enhanced Sensitivity to the Effects of TNF-{alpha}
Next, we investigated the possibility that female HLA-DR1 mice might be more sensitive to the effects of TNF-{alpha} compared with male mice. When HLA-DR1 mice were treated with Dgal and 200 ng TNF-{alpha}, female mice had significantly greater mortality than male mice (log-rank analysis, P < 0.01; Figure 6A). Hence, female mice not only produced more circulating TNF-{alpha} in response to SEB but were also more sensitive to the lethal effects of TNF-{alpha} than male mice. TNF-{alpha} can induce expression of IL-6 in a number of different tissues (32, 33). Early serum IL-6 levels in HLA-DR1 mice treated with Dgal and 200 ng TNF-{alpha} did not differ between male and female mice (Figure 6B). Furthermore, serum IL-6 did not differ between male and female HLA-DR1 mice treated with 200 ng TNF-{alpha} alone (Figure 6B).


Figure 6
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  		Figure 6. Female mice show enhanced sensitivity to tumor necrosis factor (TNF)-{alpha} in vivo. (A) HLA-DR1 mice were injected with 20 mg D-galactosamine (Dgal) and 200 ng TNF-{alpha}. Survival was monitored over 24 hours. Log-rank test, P < 0.01. (B) HLA-DR1 mice were injected with 20 mg Dgal and 200 ng TNF-{alpha} (n = 5) or 1 µg TNF-{alpha} alone (n = 4). Liver samples were taken after 5 hours and oligosomal DNA was measured by ELISA. (C) HLA-DR1 mice were injected with 20 mg Dgal and 200 ng TNF-{alpha} or 200 ng TNF-{alpha} alone. Blood was taken after 2 hours and serum IL-6 was measured by ELISA (n = 5). Data shown in (B, C) are means and SD of values. *P < 0.05 by unpaired t test.

 
Female HLA-DR1 mice treated with Dgal and TNF-{alpha} had significantly greater levels of liver apoptosis (Figure 6C) and significantly higher levels of serum ALT than male HLA-DR1 mice (females: 1,627 units/ml; SD, 1,119; males: 374 units/ml; SD, 185; unpaired t test, P < 0.05). When HLA-DR1 mice were treated with 1 µg TNF-{alpha}, which is not lethal in the absence of Dgal, the sex difference in liver apoptosis was still maintained, albeit to a lesser degree (Figure 6C). Thus, there was a fundamental sex difference in TNF-{alpha}–induced hepatocyte apoptotic responses, which did not extend to all TNF-{alpha}–mediated pathways and which contributed to the sexual dimorphism in lethal responses to superantigen.

Tamoxifen Modulation of TNF-{alpha}, TNF-{alpha}–induced Hepatocyte Apoptosis and sTNF-R
To investigate whether tamoxifen would alter the sensitivity of female HLA-DR1 mice to toxic shock, 4-week-old female HLA-DR1 mice were treated daily for 2 weeks with tamoxifen and then challenged with Dgal and 20 µg SEB. Tamoxifen treatment had a partially protective effect, in that more tamoxifen-treated mice survived toxic shock than control mice (tamoxifen treatment, 8/9 survived; control treatment, 3/9 survived). Measurement of serum TNF-{alpha} after Dgal and SEB treatment clearly showed that tamoxifen reduced the early burst of superantigen-induced TNF-{alpha} at 2 hours, in addition to reducing the later rise of TNF-{alpha} after 7 hours, before death (Figure 7A).


Figure 7
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  		Figure 7. Tamoxifen treatment reduced circulating tumor necrosis factor (TNF)-{alpha} after toxic shock. (A) Four-week-old female HLA-DR1 mice were treated for 2 weeks with tamoxifen or vehicle before induction of toxic shock with 25 mg D-galactosamine (Dgal) and 20 µg staphylococcal enterotoxin B. Blood samples were taken after 2 and 7 hours and TNF-{alpha} measured by ELISA. The data shown are means and SD of values from nine individual mice. *P < 0.01 by unpaired t test. (B) Four-week-old female HLA-DR1 mice were treated for 2 weeks with tamoxifen or vehicle before intraperitoneal injection with 25 mg Dgal and 200 ng TNF-{alpha}. Liver samples were taken after 5 hours and analyzed for oligosomal DNA by ELISA. The data shown are means and SD of values from five individual mice. *P < 0.001 by unpaired t test. Con = control vehicle; Tam = tamoxifen.

 
Intriguingly, tamoxifen had an additional effect on TNF-{alpha}–induced hepatocyte apoptosis. Tamoxifen-treated female HLA-DR1 mice challenged with Dgal and 200 ng TNF-{alpha} had significantly lower levels of liver apoptosis than control-treated mice (Figure 7B; unpaired t test, P < 0.001). Thus, tamoxifen modulated SEB-induced TNF-{alpha} production and modulated TNF-{alpha}–induced hepatocyte apoptosis.

Serum levels of sTNF-RI after challenge with Dgal and SEB were significantly elevated in female mice that had received pretreatment with tamoxifen compared with control mice (Figure 8A). Tamoxifen had a similar effect in mice challenged with Dgal and TNF-{alpha} (Figure 8B). Hence, the protection against elevated serum TNF-{alpha} and TNF-{alpha}–induced hepatocyte apoptosis afforded by tamoxifen was associated with increased production or release of sTNF-RI.


Figure 8
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  		Figure 8. Tamoxifen treatment and increased soluble tumor necrosis factor receptor (sTNF-R) levels. (A) sTNF-RI levels in serum from tamoxifen-treated or control mice challenged with D-galactosamine (Dgal) and staphylococcal enterotoxin B (n = 9 per group). (B) sTNF-RI levels in serum from tamoxifen-treated or control mice challenged with Dgal and TNF-{alpha} (n = 5 per group). Differences between tamoxifen-treated and control groups were significant (P < 0.01 by unpaired t test). The data shown are means and SD of values from individual mice.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Over the past 2 decades there has been a major shift in the etiology of bacterial sepsis, with an increasing prevalence of sepsis caused by gram-positive bacteria (5). Although females are generally less susceptible to infection (5–7), there may be an increased female propensity for severe shock once sepsis is established (8, 9). Intriguingly, there is an unexplained female preponderance of cases of superantigen-associated staphylococcal toxic shock, which persists even after cases of menstruation-associated toxic shock syndrome are accounted for (4). In this article, we demonstrate a previously unreported sexual dimorphism in susceptibility of mice to superantigen-associated sepsis and lethal superantigen shock. Enhanced female susceptibility was associated with significantly increased circulating TNF-{alpha} in response to superantigen, combined with a clearly enhanced hepatic cell death response to TNF-{alpha}. We speculate that enhanced serum TNF-{alpha} and TNF-{alpha}–mediated cell death observed in females during experimental superantigen-induced disease might account for some aspects of the sexual dimorphism seen between male and female patients.

In other animal models, females show enhanced survival and recovery after injury, hemorrhagic shock, and caecal ligation and puncture (34). In contrast, we found, in our model of superantigen-associated sepsis, that disease severity was greater in female HLA-DQ8 mice. Because disease progression is dependent on superantigen production in this model, and because sex differences were only seen when using superantigen-producing bacteria, the sexual dimorphism appeared likely to reside in dimorphic responses to superantigen.

We went on to demonstrate that female mice did indeed have greater mortality than male mice when challenged with purified superantigen. Sexual dimorphism in superantigen responsiveness was observed using three distinct superantigens in three different mouse strains. Intriguingly, the dimorphism was associated with significantly elevated serum TNF-{alpha} and reduced sTNF-R in females compared with males. We could not reproduce the observed sex difference in SEB-induced TNF-{alpha} release using spleen cells cultured in vitro, suggesting that the T cells from female mice are not intrinsically more responsive to superantigens, although they may be conditioned by factors operating in vivo that were not active in the cell culture conditions used in vitro. sTNF-Rs are important in the clearance of TNF-{alpha} from the circulation. An increase in sTNF-R has been detected in response to sepsis, LPS treatment, and anti–CD3-mediated T-cell activation (29, 35, 36). This is the first time that an increase in sTNF-R during superantigen-induced toxic shock has been demonstrated, and the first time that a sex difference in sTNF-R has been noted. Male HLA-DR1 mice had higher baseline levels of sTNF-RI and sTNF-RII and higher levels of these receptors after exposure to SEB. We postulate that elevated sTNF-R in males may enhance immediate clearance of TNF-{alpha} in vivo, although further studies will be required to confirm this observation. Importantly, pretreatment with the estrogen receptor modulator tamoxifen led to increased sTNF-RI in female mice, and this was associated with reduced circulating TNF-{alpha} and improved survival, consistent with a key role for sTNF-R in the model.

It is unclear whether the higher proportion of T cells in female mice may also contribute to the observed sex difference in serum TNF-{alpha}. Although TNF-{alpha} mRNA transcripts in the spleen clearly increased after SEB administration, no sex difference in TNF-{alpha} mRNA transcripts in either SEB- or Dgal- and SEB-treated mice could be detected. TNF-{alpha} production and release are further subject to post-transcriptional and post-translational regulation (37), and we cannot rule out the possibility of a sex difference in these processes. We did not identify an overt difference between males and females with regard to the proportion of FoxP3-positive CD4 cells in the spleen. Increased female susceptibility to a number of human autoimmune diseases, including SLE and rheumatoid arthritis, have prompted considerable efforts to elucidate the underlying mechanisms in mouse models, demonstrating a role for regulatory T cells in these sex differences (38, 39). Notwithstanding the similarity in size of the FoxP3-positive CD4 populations in males and females, functional assessment of regulatory T-cell activity will be required to exclude a role for regulatory T cells in the toxic shock model.

Intriguingly, we found that tamoxifen treatment of female HLA-DR1 mice before induction of toxic shock partially protected the mice against lethality and clearly reduced the early, lethal burst of serum TNF-{alpha} that originates from splenic T cells. Whether acting directly or indirectly via regulatory T cells, estrogen is known to affect activation of peripheral T cells via surface estrogen receptors although some studies suggest that estrogen may act to reduce T-cell–mediated effects (13, 40). Together with data showing the influence of tamoxifen on sTNF-R levels, the results herein suggest that the superantigen-induced proinflammatory cascade may be enhanced by estrogen receptor–mediated signals at multiple points.

Randomly cycling female mice were used in the studies reported. Because estrogen appears to be important in the models reported, we hypothesize that smaller or larger differences between the sexes may have been observed had experiments been performed at a specific stage of the cycle. The random phase of cycle thus mainly increases the variability in the findings reported, but should not have caused false-positive or false-negative findings. Interestingly, administration of estrogen to male mice did not enhance susceptibility to Dgal and SEB (not shown), although fundamental sex differences in responses to estrogen, including receptor distribution, or novel receptors may explain these findings. We cannot exclude a protective role for androgens, and further studies using gonadectomized mice may clarify this.

Importantly, we have also demonstrated an unexpected difference in the sensitivity of male and female HLA-DR1 mice to TNF-{alpha}. When given the same dose of TNF-{alpha} in the presence of Dgal, female mice showed greater mortality and greater liver damage than male mice. Numerous studies have shown that hepatocytes are particularly sensitive to TNF-{alpha} and that TNF-{alpha} is a critical mediator of liver damage due to sepsis, LPS, and superantigens, particularly when hepatic transcription is blocked by agents such as Dgal (41–43). TNF-{alpha} can signal via membrane-bound TNF-R to yield either proinflammatory sequelae or cell death. TNF-{alpha}–induced death signals are restricted to TNF-RI via the death domain that involves Fas-associated death domain protein (FADD), leading to activation of initiator caspase-8 that, in turn, activates the executioner caspase-3, leading to apoptosis and cell death (44). TNF-{alpha}–induced proinflammatory signals occur via activation of nuclear factor (NF)-{kappa}B, which induces expression of a number of genes. Importantly, TNF-{alpha}–induced apoptotic signals are impeded by the NF-{kappa}B–induced gene expression pathway at a number of checkpoints (45), including cellular FLICE-inhibitory protein (c-FLIP) expression and c-Jun N-terminal kinase (JNK) deactivation. Dgal acts in vivo to arrest transcription in the liver, effectively terminating NF-{kappa}B–mediated gene expression, and removes these checkpoints, resulting in unopposed susceptibility to TNF-{alpha}–mediated apoptosis, which does not require de novo gene transcription (42, 46). Hence, in the presence of Dgal, TNF-{alpha} will induce apoptosis of hepatocytes without regulation by NF-{kappa}B–mediated gene expression (27, 45, 47).

TNF-{alpha}–induced IL-6 expression results from p38 mitogen-activated protein kinase activation pathway and was unaffected by sex in our experiments. Because hepatic transcription was switched off by Dgal in our studies, the IL-6 measured is likely to originate from additional tissues, such as the spleen. Taken together, our data show that hypersensitivity to TNF-{alpha} in female mice is localized to the TNF-RI apoptosis signaling pathway in hepatocytes. Mignon and colleagues (27) have shown that TNF-{alpha}–triggered hepatocyte apoptosis in Dgal-sensitized mice occurs as a direct result of caspase-3 activation. The findings of our work suggest that hepatocyte caspase-3 activation must constitutively differ between males and females, related either to effects on TNF-{alpha}–induced caspase or TNF-{alpha}–induced JNK activation.

A number of studies have shown sex differences in the response of the liver to various types of insult (48). In the case of hepatic ischemia, hemorrhagic shock, hepatectomy, viral hepatic fibrosis, and sepsis, females suffered less and estrogen was found to be protective. Only in the case of alcohol-related liver injury was there clear evidence that female liver was more susceptible to injury than male liver. Expression of TNF-{alpha} and hepatocyte apoptosis were higher in female rats after chronic alcohol intake (49, 50), and estrogen was implicated as the primary factory underpinning the sex difference (51, 52). Sexual dimorphism in hepatic function is well recognized and several intracellular signaling pathways differ between the sexes, in particular pathways dependent on STAT5b (signal transducer and activator of transcription 5b) (53, 54). Sexual dimorphism in apoptotic responses has been reported in other cell types (55, 56). It is unclear at present whether these observations have direct bearing on female susceptibility to autoimmune disease, particularly in those disorders characterized by T-cell activation and target cell apoptosis. Importantly, the data highlight the importance of sex selection in any studies where TNF-{alpha}–mediated apoptosis is relevant.

We found that, in addition to effects on systemic TNF-{alpha} and sTNF-R, tamoxifen treatment of female HLA-DR1 mice protected hepatocytes against apoptosis induced by Dgal and TNF-{alpha}. Estrogen receptor binding can yield both genomic effects (binding to genes with estrogen response elements resulting in altered transcription) and nongenomic effects, (interaction of membrane receptors with components of transcription pathways resulting in downstream regulatory changes). Additional non–receptor-dependent effects of both estrogen and tamoxifen have been reported. In separate experiments, an alternative, highly specific estrogen receptor antagonist, ICI 182780 (Faslodex; AstraZeneca, Luton, UK), was also able to completely protect female HLA-DR1 mice from fulminant hepatocyte death after challenge with Dgal and SEB (not shown), indicating that estrogen receptor–dependent events protected against TNF-{alpha}–mediated hepatocyte apoptosis. Apoptosis pathway genes, such as caspase-7, that contain estrogen response elements and that can bind estrogen receptors in vitro (54) may well be estrogen responsive in vivo. However, because induction of TNF-{alpha}–mediated hepatocyte apoptosis in our model occurred without requirement for transcription, it is feasible that the effects of tamoxifen and Faslodex are related to a direct (nongenomic) interaction of the estrogen receptor complex with the apoptosis signaling pathway, rather than a classical antagonistic effect on gene transcription.

We have previously established that lethality in superantigen-induced toxic shock in HLA class II transgenic mice is dependent on the presence of HLA class II, TCR{alpha}beta T cells and TNF-{alpha}. TNF-{alpha} is central to disease pathogenesis, because antibodies to TNF-{alpha} are completely protective when administered before superantigen in a range of superantigen shock models (3, 41). The data presented herein demonstrate a clear sexual dimorphism in susceptibility to death induced by exposure to superantigen in the context of both sepsis and of toxin administration. Systemic TNF-{alpha} from T cells and sTNF-R are modulated both by sex and by estrogen receptor modulators. These same factors also modulate TNF-{alpha}–induced apoptosis of hepatocytes. Both events are crucial to explaining the sex differences observed in this model of toxic shock and may have important ramifications in a range of human inflammatory conditions.


   FOOTNOTES
 
Supported by DARPA (Defence Advanced Research Projects Agency) and the U.S. Army Research Office.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200611-1712OC on June 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 27, 2006; accepted in final form June 15, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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