Injection Of Staphylococcal Enterotoxin B Research Articles

Berberine attenuate staphylococcal enterotoxin B-mediated acute liver injury via regulating HDAC expression

Staphylococcal enterotoxin B (SEB) has been documented to be implicated in the pathogenesis of liver injury in the experimental models of hepatitis. However, the underlying mechanism of SEB-induced acute liver injury (ALI) remains to be further explored. In our study, we explored the therapeutic effectiveness of berberine (BBR), a natural isoquinoline alkaloid, in the SEB-induced ALI. In our study, we found that injection of SEB into d-galactosamine (d-gal)-sensitized mice induced ALI, as demonstrated by an increase of levels of alanine aminotransferase and aspartate aminotransferase, massive infiltration of immune cells into the liver, and pro-inflammatory cytokine release. However, intragastric administration of BBR attenuated SEB-induced ALI in mice. Meanwhile, we discovered that BBR treatment suppressed activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Moreover, mechanistic analyses demonstrated that BBR was effective at inhibiting the expression of class I HDAC, but not class II, in SEB-stimulated splenocytes. Furthermore, trichostatin A, a standard HDAC inhibitor, alleviated activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Taken together, we inferred from these results that BBR attenuated SEB-mediated ALI through repressing the class I HDAC enzyme, suggesting that BBR may constitute a novel therapeutic modality to prevent SEB-mediated inflammation and ALI.

Natural indoles, indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells.

Staphylococcal enterotoxin B is involved in aggravation and recurrence of murine experimental autoimmune uveoretinitis via Vβ8+CD4+ T cells

Endogenous uveitis is a common cause of visual disability and blindness. The etiology of uveitis remains largely unknown but reasonable etiologic factors include infections. Superantigens are regarded as one of the leading causes of infectious etiology in autoimmune disease. However, the role of superantigens in uveitis remains unclear. In the present study, we investigated the effect of Staphylococcal enterotoxin B (SEB), a member of the superantigens, using an experimental model of autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein (IRBP) peptide, and the severity of EAU disease was scored. Vehicle (PBS) alone or SEB dissolved in PBS was administered by intravenous injection on post-immunization day 10 or on post-immunization day 24. In addition, a systemic immune response study was performed to address the effects of SEB on systemic immunity. EAU was aggravated significantly by the injection of SEB at post-immunization day 10. Furthermore, relapse was induced by the injection of SEB at day 24. On the other hand, SEB injection without IRBP peptide immunization elicited no inflammatory changes in the uvea or retina. Furthermore, SEB enhanced not only the IRBP-specific T-cell proliferative responses but also IFN-γ and IL-17 production. Moreover, the intraocular expression levels of these cytokines was also enhanced by SEB injection. Both anti-CD4 and -Vβ8 Ab administration suppressed disease aggravation and the enhancement of IRBP-specific T-cell responses caused by SEB. These results suggest that SEB is involved significantly in the aggravation or recurrence of endogenous uveitis through activation of autoreactive uveitogenic T cells.

Plasminogen Activator Inhibitor Type-1-Deficient Mice Have an Enhanced IFN-γ Response to Lipopolysaccharide and Staphylococcal Enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.

Role of CD44 and Its v7 Isoform in Staphylococcal Enterotoxin B-Induced Toxic Shock: CD44 Deficiency on Hepatic Mononuclear Cells Leads to Reduced Activation-Induced Apoptosis That Results in Increased Liver Damage

Exposure to bacterial superantigens such as staphylococcal enterotoxin B (SEB) leads to the induction of toxic shock syndrome which results in multiorgan failure, including liver damage. In the present study, we investigated the role of CD44 in SEB-induced liver injury. Injection of SEB into d-galactosamine-sensitized CD44 wild-type (WT) mice led to a significant increase in CD44 expression on liver T cells, NK cells, and NKT cells. Administration of SEB to CD44 knockout (KO) mice caused significantly enhanced liver damage which correlated with elevated numbers of T cells, NK cells, NKT cells, and macrophages in the liver and increased production of tumor necrosis factor alpha and gamma interferon compared to CD44 WT mice. Furthermore, liver mononuclear cells from CD44 KO mice were resistant to SEB-induced apoptosis, and cDNA microarray analysis revealed that SEB activation of such cells led to the induction of several antiapoptotic genes and repression of proapoptotic genes. Examination of CD44 isoforms revealed that SEB exposure altered CD44 variant 7 (v7) isoform expression. Interestingly, mice bearing a specific deletion of the CD44v7 exon exhibited increased susceptibility to SEB-induced hepatitis. Finally, treatment of CD44 WT mice with anti-CD44 monoclonal antibodies reduced expression of CD44 in liver mononuclear cells and caused increased susceptibility to SEB-induced liver injury. Together, these data demonstrate that the expression of CD44 and/or CD44v7 on SEB-activated liver mononuclear cells facilitates their rapid apoptosis, thereby preventing severe liver injury in wild-type mice, and suggest that CD44 plays an important role in the regulation and elimination of immune cells in the liver.

Behavioural endocrine immune-conditioned response is induced by taste and superantigen pairing

Administration of bacterial superantigen, such as staphylococcal enterotoxin B (SEB), induces in vivo stimulation of T cell proliferation and cytokine production such as interleukin-2 (IL-2). It has been previously reported that SEB administration induces fever, c-Fos expression in the brain, and hypothalamus–pituitary–adrenal axis activation, demonstrating that the brain is able to sense and respond to SEB. Previously it had been shown that immune functions can be behaviourally conditioned pairing a novel gustatory stimulus together with an immunomodulatory drug or an antigen. We designed an experimental protocol using Dark Agouti rats in which saccharin taste, as conditioned stimulus, was paired with an i.p. injection of SEB (2 mg/kg), as unconditioned stimulus. Six days later, when conditioned animals were re-exposed to the conditioned stimulus they displayed strong conditioned taste avoidance to the saccharin. More importantly, re-exposure to the conditioned stimulus significantly increased IL-2, interferon-γ and corticosterone plasma levels, in comparison with conditioned animals which had not been re-exposed to saccharin taste. These results demonstrate a behavioural-immune-endocrine conditioned response using a superantigen as unconditioned stimulus. In addition, they illustrate the brain abilities to mimic the unconditioned effects of a superantigen by yet unknown mechanisms.

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance.

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.

Mycobacterium bovis BCG-infected mice are more susceptible to staphylococcal enterotoxin B-mediated toxic shock than uninfected mice despite reduced in vitro splenocyte responses to superantigens.

Type 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice with Mycobacterium bovis BCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-gamma) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-gamma levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-gamma. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-gamma from purified T cells. The diminished IFN-gamma levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.

Suppression of superantigen-induced lung injury and vasculitis by preadministration of human urinary trypsin inhibitor.

We examined whether the lung injury produced in rats by intraperitoneal injection of the superantigen, staphylococcal enterotoxin B (SEB), could be inhibited by intravenous preadministration of human urinary trypsin inhibitor (UTI), which exhibits multipotent inhibitory effects on serine proteinases such as plasmin, chymotrypsin, or human leukocyte elastase or cathepsin G, since preliminary experiments showed the ability of UTI to bind lipopolysaccharides and bacterial toxins. For ligand blotting analysis, four kinds of toxins were run on a slab gel and the binding of UTI to the toxins was visualized by immunoblotting. Lung tissue from 26 rats was used for immunohistochemistry using a mouse antirat CD 45 mAb and an antirat macrophage mAb. Lung tissue from 31 rats was used for measurement of myeloperoxidase activity before and after intraperitoneal injection of SEB, after infusion of PBS, UTI, PBS-SEB or UTI-SEB combination. Ten of the 26 rats described above were used for electron microscopy. Rat sera were used for measurement of TNF-alpha. Statistical analysis was performed using the Mann-Whitney U-test. Intraperitoneal injection of SEB caused an increase in the number of punctate areas of haemorrhage on the surface of the lung with time, and histological examination revealed lung injuries with different extents, vasculitis where inflammatory cells were concentrated, and infiltration of numbers of eosinophils into the alveolar septa. However, preadministration of UTI for rats markedly attenuated lung injury and vasculitis induced by intraperitoneal injection of SEB. This revealed, from a marked reduction in the number of inflammatory cells and the extent of injury, a marked inhibition of serum TNF-alpha production and reduction of myeloperoxidase content of rat lungs compared to controls. UTI may have defensive effects to infection by suppressing the early responses of stimulated cells to activated stimulus such as SEB as well as the release of stimulant-mediated cytokines via trapping of bacterial toxins.

A unique response to staphylococcal enterotoxin B by intrahepatic lymphocytes and its relevance to the induction of tolerance in the liver.

It has been reported that the intrahepatic lymphocyte (IHL) population is somewhat differently constituted from lymphocytes in other lymphoid tissues. Staphylococcal enterotoxin B (SEB) is a superantigen which can induce T-cell tolerance in mice. The authors investigated the in vitro and in vivo responses of mouse IHL to SEB. An intravenous injection of SEB did not result in the augmentation of the proliferative response of IHL, while mesenteric and splenic lymphocytes (mLNC and SPC, respectively) had augmented responses. Interleukin-2 (IL-2) mRNA was clearly detected in mLNC and SPC by reverse transcriptase-polymerase chain reaction (RT-PCR) shortly after the administration of SEB, but it was scarcely expressed in IHL. The expression of CD25 (IL-2 receptor) was also augmented in mLNC and SPC in the early period, while it was not changed in IHL. These findings suggested that the time required for tolerance induction is different locally and that the loss of augmentation of IL-2 and IL-2 receptor production by IHL may be relevant to the rapid induction of T-cell tolerance in the liver.

Superantigen and endotoxin synergize in the induction of lethal shock.

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-gamma (IFN-gamma) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D-galactosamine (D-GalN). Opposed to D-GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-gamma monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-gamma is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.

Persistent production of TH2-type cytokines and polyclonal B cell activation after chronic administration of staphylococcal enterotoxin B in mice.

In order to study the immunopathological consequences of repeated exposure to bacterial superantigens, we evaluated the production of cytokines, the profile of serum immunoglobulins and the tissue damage in BALB/c mice injected twice a week for 3 weeks with 50 micrograms of staphylococcal enterotoxin B (SEB). First, we found that neither interleukin 2 (IL-2) nor interferon gamma (IFN-gamma) were detectable in serum after the 6th injection of SEB whereas both cytokines were released in the circulation after the first injection. In contrast, interleukin 4 (IL-4) and interleukin 10 (IL-10) serum levels were similar after the first and the sixth injection of SEB. Likewise, spleen cells from mice injected for 3 weeks with SEB produced much lower levels of IL-2 and IFN-gamma than spleen cells from control mice in response to SEB in vitro whereas their production of IL-10 was not significantly altered. Both IL-4 and IL-10 were found to be secreted by purified T cells from SEB-treated mice when rechallenged in vitro with SEB in presence of human accessory cells. This TH2-type cytokine profile was associated with a marked hyperimmunoglobulinemia and the appearance of anti-mouse immunoglobulin autoantibodies. By light microscopy, no tissue lesions were observed in mice chronically injected with SEB but mesangial deposits of IgG were found in their kidneys by immunofluorescence. We conclude that T cell anergy induced by repeated injections of SEB in BALB/c mice is associated with a persistent production of TH2-type cytokines and a polyclonal B cell activation.

Cutaneous Exposure to the Superantigen Staphylococcal Enterotoxin B Elicits a T-Cell-Dependent Inflammatory Response

We analyzed the impact of superantigens secreted by skin-colonizing Staphylococci on the skin and the associated lymphoid tissue following epicutaneous application and intracutaneous injection of small amounts of staphylococcal enterotoxin B (SEB). A single intracutaneous injection of 50 ng of SEB elicited a strong inflammatory response in the skin of BALB/c mice. Three to 6 h later, we observed langerhans cell activation, mast cell degranulation, vasodilation, upregulation of ICAM-1, and induction of VCAM-1 on dermal blood vessels, with vascular adhesion of granulocytes. by 12 to 24 h, cell infiltration of the dermis increased, reaching the epidermis. Among the infiltrating leukocytes, a substantial number of eosinophils was found. After 48 h, the infiltrate was dominated by mononuclear cells. The response to SEB was dose-dependent, and signs of inflammation slowly disappeared over 5 to 7 days. Although the induction of VCAM-1 on dermal blood vessels suggested a role for interleukin-1/tumor necrosis factor-alpha in this reaction, the activation of monocytes/macrophages was not able to substitute for lymphocytes, as severe combined immunodeficiency (SCID) mice (which are lymphocyte-deficient) did not mount an inflammatory skin response to intradermal injection of SEB. The fact that nude mice (T-cell-deficient) also did not mount an inflammatory response to SEB indicated the T-cell dependency of the response. The V beta specificity of the SEB effect was demonstrated by the fact that SJL/J mice, which lack V beta 8+ T cells (the major SEB-reactive T cell population in mice), exhibited much weaker responses. Deletion or tolerization of SEB-reactive V beta T cells was not observed after a single intradermal injection of such minute amounts of SEB.

Lipopolysaccharide is required for the lethal effects of enterotoxin B after D-galactosamine sensitization

We tested the D-galactosamine sensitization model with staphylococcal enterotoxin B (SEB). LPS was required for the lethal effects of SEB in D-galactosamine sensitized mice. Only two (2/62) among the C3HeB/FeJ (H-2k), Balb/c (H-2d) and C57BL/6J (H-2b) mice died in response to SEB in the absence of LPS whereas injection of SEB and minimally lethal concentrations of LPS became highly toxic. Similar to LPS, the lethal effect of SEB was dependent on the mouse strain used. Mouse strains more sensitive to the effects of LPS (Balb/c and C57BL/6J) were also more sensitive to the effects of SEB in comparison to C3H mice when equivalent doses of LPS and SEB were used. Among Balb/c and C3HeB/FeJ but not C57BL/6J mice, SEB (20 μg) potentiated the lethal effects of LPS at low doses (0.1 μg LPS), but had an apparent protective effect at high doses (1 μg LPS). Lastly, there was an inverse correlation between pathogenesis and serum IL-2 concentrations and splenic T cell activation in C3H mice. However, macrophage mobilization did correlate with lethality. Therefore, some questions remain about the mechanisms involved in the D-galactosamine/SEB pathogenesis model. We conclude that when sensitizing mice with D-galactosamine and assessing the lethal effects of SEB, endotoxin contamination must be assessed.

Influence of staphylococcal enterotoxin B (SEB) on the course of murine listeriosis

Confrontation of the immune system with bacterial superantigens leads to an initial activation of the immune system followed by a state of profound immunosuppression. To investigate the role of a superantigen in an acute infection with a facultatively intracellular bacterium, we have studied the effect of staphylococcal enterotoxin B on the course of murine listeriosis. Intraperitoneal injection of SEB led to a statistically significant growth restriction of Listeria monocytogenes in the organs of mice infected intravenously or intraperitoneally when treatment with SEB and infection with L. monocytogenes were given simultaneously or when the mice were treated two days before infection. No effect of SEB on murine listeriosis was found when SEB was given more than two days before infection or one or more days after infection. We conclude that initial immunostimulation by SEB which is indicated by a massive liberation of all interleukins measured (IL1α, IL6, TNFα, IL2, IFNγ, IL4) is responsible for the growth restriction of L. monocytogenes in the organs of treated mice. Apoptosis of Vβ8 positive T cells which was accompanied by a 30% reduction of these cells at day 7 after treatment seems to be totally compensated.

Influence of staphylococcal enterotoxin B (SEB) on the course of murine listeriosis.

Confrontation of the immune system with bacterial superantigens leads to an initial activation of the immune system followed by a state of profound immunosuppression. To investigate the role of a superantigen in an acute infection with a facultatively intracellular bacterium, we have studied the effect of staphylococcal enterotoxin B on the course of murine listeriosis. Intraperitoneal injection of SEB led to a statistically significant growth restriction of Listeria monocytogenes in the organs of mice infected intravenously or intraperitoneally when treatment with SEB and infection with L. monocytogenes were given simultaneously or when the mice were treated two days before infection. No effect of SEB on murine listeriosis was found when SEB was given more than two days before infection or one or more days after infection. We conclude that initial immunostimulation by SEB which is indicated by a massive liberation of all interleukins measured (IL1 alpha, IL6, TNF alpha, IL2, IFN gamma, IL4) is responsible for the growth restriction of L. monocytogenes in the organs of treated mice. Apoptosis of V beta 8 positive T cells which was accompanied by a 30% reduction of these cells at day 7 after treatment seems to be totally compensated.

Differential expression of mRNA encoding interleukin-12 p35 and p40 subunits in situ.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the regulation of the immune response. For biological activity the expression of both subunits of IL-12, p35 and p40, is required. Moreover, in the mouse the p40 chain of IL-12 specifically inhibits the effects of the IL-12 heterodimer. In the present study we have analyzed by in situ hybridization the expression of the p35 and p40 mRNA in the spleens of BALB/c and mutant (SCID, nude, beige) mice, unstimulated and after in vivo stimulation with lipopolysaccharide (LPS) and with staphylococcal enterotoxin B (SEB). In unstimulated spleens of BALB/c mice p35 and p40 mRNA were only detectable in a few strongly stained single cells, p35 mRNA was expressed in addition weakly in the B cell areas. After injection of LPS or SEB, p40 mRNA was strongly induced in the T cell areas all over the spleen, whereas expression of p35 mRNA and its distribution pattern did not change. Surprisingly, most of the mRNA for p35 and p40 was localized in different areas of the spleen and was apparently produced by different cells. In macrophage-depleted spleens the increased expression of p40 mRNA in response to LPS was reduced but still detectable, demonstrating that other cells besides macrophages can up-regulate IL-12 p40 mRNA. Nude mice showed a stronger expression of p35 mRNA, SCID mice lacked the weak p35 staining of the B cell areas but showed a strong basal expression of both p35 and p40 mRNA and a focal response to LPS. The pattern of IL-12 mRNA expression in beige mice was the same as in normal mice. These data demonstrate a spatial dissociation of expression of the two chains of IL-12 and are compatible with a regulatory role of the isolated IL-12 p40 chain in vivo. In addition, they indicate that the demonstration of mRNA for both chains of IL-12 in whole tissues or cell mixtures is not necessarily indicative of functional IL-12.

Hepatic injury and lethal shock in galactosamine-sensitized mice induced by the superantigen staphylococcal enterotoxin B

Background/Aims: Staphylococcal enterotoxin B (SEB) acts as a superantigen binding to class II major histocompatibility complex proteins, and this complex stimulates T cells. The aim of this study was to investigate the pathogenic effects of SEB on hepatic injury and lethal shock in mice. Methods: SEB was administered to D-galactosamine (GalN)-sensitized mice, and the degree of liver injury and levels of circulating cytokines were determined. In vitro cytokine production in response to SEB was also investigated. Results: Intraperitoneal administration of SEB (50 μg) caused lethal shock (50% mortality) associated with massive hepatic necrosis in GaIN-sensitized mice, with no mortality on injection of up to 100 μg SEB alone. Within 2 hours after injection of SEB, serum tumor necrosis factor α (TNF-α) levels reached a peak, followed by high levels of serum interferon-γ (IFN-γ) up to 10 hours after injection. Passive immunization with anti-TNF-α/β-neutralizing monoclonal antibody (mAb) protected GaIN-sensitized mice from the lethal effects of SEB, with less protection with anti-IFN-γ-neutralizing mAb. SEB induced the production of TNF-α and IFN-γ in a dose-dependent manner from splenic mononuclear cells in vitro. Conclusions: The results show that SEB contributes to lethal shock associated with severe hepatic injury in GaIN-sensitized mice and suggest that TNF-α and IFN-γ produced in response to SEB may be mediators of the lethal toxicity and hepatotoxicity of SEB.

Rabbit Plasma Fibronectin Levels Associated withStaphylococcusaureusEnterotoxin B: An Acute-Phase Reaction

Variation in fibronectin (Fn) levels and white blood cell counts (WBC) following staphylococcal enterotoxin B (SEB) or SEB + cryoprecipitate containing Fn challenge was studied in New Zealand white rabbits. Increased plasma Fn levels were observed 2 h after the intravenous injection of SEB and peaked at 48-72 h (from a mean level 194.6 +/- 4.5 micrograms/ml prechallenge Fn level to a 72-hour postchallenge mean level of 407.9 +/- 25.4 micrograms/ml). Fn levels then decreased over the succeeding 5 days to approximately prechallenge levels. The total WBC count decreased by 88% within 2 h after the SEB injection. A slow increase in circulatory WBC was observed over the next 24 h. SEB caused an increase in plasma Fn levels and decreased WBC counts with lymphopenia that was followed by a normal lymphocyte count within 5 days. These data suggest that an acute-phase reaction was induced by interleukin-1. Fn prophylaxis provided no change in clinical signs when given at the time of SEB injection.

SUPPRESSION OF THE IN VIVO HUMORAL AND CELLULAR IMMUNE RESPONSE BY STAPHYLOCOCCAL ENTEROTOXIN B (SEB)

SUMMARY In vitro blast transformation of mouse spleen and lymph node cells occurs in response to staphylococcal enterotoxin B (SEB), as measured by 3H-thymidine incorporation. This response appears to be similar to the stimulation by phytohaemagglutinin. Treatment with SEB immediately after skin grafting suppressed a first, but not a second-set allograft rejection in mice. Syngeneic lymph node cells stimulated in vitro by SEB and adoptively transferred to normal mice failed to transfer suppression of graft rejection. Immediate injection of SEB following immunization with sheep red blood cells exhibited a markedly suppressed plaque-forming and rosette-forming cell response, whereas no suppression of the antibody response to a thymus-indendent antigen Escherichia coli lipopolysaccharide, was observed. Introduction of SEB after the second immunization with sheep red blood cells showed no significant suppression effect in plaque formation. Formalinized or heat-treated SEB destroyed the mitogenic and immunosuppressive capability as tested by 3H-thymidine incorporation, skin rejection, and plaque formation. It is suggested that the mechanism of this immunosuppression involved the interaction of SEB with T cells, which in some manner block T cell function in the early recognition stage, probably through nonspecific mitogenic activity.