NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance.

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant V alpha 14J alpha 281 TCR because J alpha 281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in J alpha 281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.

MIP-2 recruits NKT cells to the spleen during tolerance induction.

Peripheral tolerance occurs after intraocular administration of Ag and is dependent on an increase in splenic NKT cells. New data here show that macrophage inflammatory protein-2 (MIP-2) is selectively up-regulated in tolerance-conferring APCs and serves to recruit NKT cells to the splenic marginal zone, where they form clusters with APCs and T cells. In the absence of the high-affinity receptor for MIP-2 (as in CXCR2-deficient mice) or in the presence of a blocking Ab to MIP-2, peripheral tolerance is prevented, and Ag-specific T regulatory cells are not generated. Understanding the regulation of lymphocyte traffic during tolerance induction may lead to novel therapies for autoimmunity, graft acceptance, and tumor rejection.

Differential production of prostaglandin E(2) in male and female mice subjected to thermal injury contributes to the gender difference in immune function: possible role for 15-hydroxyprostaglandin dehydrogenase.

We have previously reported a macrophage-mediated gender difference in postburn immunosuppression, which was dependent upon elevated levels of circulating 17beta-estradiol (E(2)) and, in part, interleukin-6. Herein we examined the role of prostaglandin E(2) (PGE(2)), a potent suppressor of cell-mediated immunity. Circulating levels of PGE(2) were significantly elevated in females but not males at 10 days postburn (P < 0.01), and indomethacin treatment fully restored the delayed-type hypersensitivity and splenocyte proliferative responses of thermally injured females. While there was no difference in cyclooxygenase-2 protein expression in the lungs and liver of thermally injured male and female mice, there was a marked decrease in the protein expression of 15-hydroxyprostaglandin dehydrogenase in females. These data demonstrate that PGE(2) is a critical mediator of immunosuppression in thermally injured female mice and that the increase in circulating PGE(2) is derived, in part, from decreased degradation and clearance of PGE(2).

Anti-interleukin-6 antibody treatment restores cell-mediated immune function in mice with acute ethanol exposure before burn trauma.

BACKGROUND: Previous studies from this laboratory reported that suppression of cell-mediated immune function was coincident with elevated interleukin (IL)-6 production after acute ethanol exposure before burn trauma, compared with either insult alone. The goal of this study was to investigate whether treatment with an anti-IL-6 antibody could restore immunocompetence in mice subjected to burn trauma with previous exposure to alcohol, as assessed by delayed-type hypersensitivity (DTH) and mitogen-induced splenocyte proliferative responses. METHODS: Mice given an ethanol treatment designed to reach a blood alcohol level of 100 mg/dl before a 15% total body surface area burn injury were treated with an anti-IL-6 antibody at 30 min and 24 hr postinjury. RESULTS: Burn/ethanol mice exhibited a 91% suppression of the DTH response ( < 0.01) and a 76% suppression of mitogen-induced splenocyte proliferation (p < 0.01) at 48 hr postinjury, along with increased levels of circulating and splenic macrophage-derived IL-6, compared with all other treatment groups. After anti-IL-6 antibody administration to burn/ethanol mice, there was a 25% (p < 0.05) and 63% (p < 0.01) recovery of the DTH and splenocyte proliferative responses, respectively. Addition of exogenous IL-6 to splenocyte cultures isolated from anti-IL-6 antibody-treated burn/ethanol mice resulted in a 70% inhibition of mitogen-induced proliferative responses (p < 0.03). CONCLUSIONS: These data confirm previous findings that burn in combination with acute ethanol exposure suppresses cell-mediated immune function compared with either insult alone. Furthermore, the ability of the anti-IL-6 antibody treatment to improve cellular immune responses in the burn/ethanol group suggests that blocking this cytokine may be beneficial for the ethanol-exposed, thermally injured individual.

Gender difference in cell-mediated immunity after thermal injury is mediated, in part, by elevated levels of interleukin-6.

The gender difference in normal immune function has been well documented, however, there is only limited information regarding whether such a difference occurs after injury. To investigate this, we examined cell-mediated immune responses in male and female mice given a 15% total body surface area dorsal scald or sham injury. Both delayed-type hypersensitivity (DTH) and splenocyte proliferative responses were significantly suppressed in males at 1 day and in females at 7 and 10 days post burn (P < 0.01). The decreased splenocyte proliferation was found to be macrophage-dependent and suppression of both immune parameters corresponded with elevated interleukin-6 (IL-6) levels. Furthermore, post-burn treatment with an anti-IL-6 antibody partially restored the DTH response in males at 1 day and females at 10 days post injury and completely restored splenocyte proliferation. These data demonstrate a possible mechanism for the gender difference in cell-mediated immune responses after thermal injury.

Estrogen mediates the sex difference in post-burn immunosuppression.

Previous studies in our laboratory have demonstrated that cell-mediated immune function was suppressed in female, but not male, mice at 10 days after burn injury and was mediated, in part, by increased production of interleukin-6 (IL-6). Because 17beta-estradiol (E(2)) influences immune function after trauma and the hormone is known to regulate IL-6 production, the effect of E(2) on immune function after thermal injury was examined. Increased circulating concentrations of E(2) corresponded with suppressed delayed-type hypersensitivity (DTH) and splenocyte-proliferative responses, and increased circulating concentrations of IL-6 in female mice after burn. Ovariectomy restored the suppressed DTH response and decreased IL-6 concentrations, and administration of exogenous E(2) to both ovariectomized females and intact male mice resulted in a suppressed DTH response. In addition, in vitro treatment with E(2) suppressed splenocyte proliferation in a macrophage-dependent manner and enhanced macrophage production of IL-6. These results strongly suggest that the sex difference in cell-mediated immunity 10 days after burn injury is mediated by altered concentrations of E(2), which in turn modulate key macrophage-derived immunoregulatory cytokines.

Neutrophil chemokine production in the skin following scald injury.

The present study was conducted to determine whether local production of neutrophil chemoattractant cytokines preceded the influx of neutrophils following dermal scald injury. To accomplish this, dermal tissue was examined for inflammatory infiltrate and the level of KC, a murine homolog of human interleukin-8, at various time points after scald injury. The studies reveal that there was a largely neutrophilic infiltrate at 1 day post-injury which persisted for 4 days. Dermal KC levels increased significantly at 4 h, returned to baseline at 8 h and were elevated again from 1 to 3 days post-burn (P < 0.01). At 3 days post-burn, KC was elevated 15-fold above the level in sham treated mice (P < 0.01). These observations demonstrate that the influx of neutrophils into the skin follows the expression of KC in the skin. This suggests that it should be possible to alter neutrophil accumulation at the wound site by manipulating the local chemokine signal.

Elevation in pulmonary neutrophils and prolonged production of pulmonary macrophage inflammatory protein-2 after burn injury with prior alcohol exposure.

Various studies have shown that alcohol exposure before thermal injury leads to increased morbidity and mortality. Pulmonary failure is a major complication seen in these patients. This study examines the effects of prior alcohol exposure on lung pathology after burn injury. There is a marked increase in neutrophil recruitment in the lung after thermal injury, and herein we show that this appears to be significantly elevated in animals given alcohol before burn injury. Consequently, we chose to determine whether there is a difference in pulmonary production of macrophage inflammatory protein (MIP)-2, a potent neutrophil chemoattractant, in mice subjected to a 15% total body surface area scald (or sham) injury with or without prior ethanol treatment. Lung tissue was obtained at various time points after injury and homogenates were assayed for MIP-2 by enzyme-linked immunosorbent assay. At 2 h after injury, peak levels of the chemokine were produced in both burn and burn + alcohol-treated mice. This represents a 7-fold increase above baseline. In mice exposed to burn injury alone, the level of MIP-2 returned to baseline within 8 h. In contrast, mice given alcohol before burn injury continued to show elevated levels of the chemokine at 8 h, after which MIP-2 decreased. This study may provide a basis for understanding the mechanism responsible for the increased neutrophil presence in the lung after thermal injury in individuals who have consumed alcohol. Subsequently, this may lead to the enhanced neutrophil-mediated pulmonary damage observed in these patients.

Glucocorticoids protect against suppression of T cell responses in a murine model of acute ethanol exposure and thermal injury by regulating IL-6.

Previous reports by this laboratory demonstrated that acute alcohol exposure combined with a 15% body surface area dorsal scald injury results in significant reductions in delayed-type hypersensitivity (DTH) and splenocyte proliferative responses compared to either insult alone. Previous studies by this lab have also shown that these defects are mediated, in part, by increased production of interleukin-6 (IL-6). Because both alcohol exposure and thermal injury are known to modulate glucocorticoid (CORT) levels, and CORT regulates IL-6 gene expression, the relationship between circulating CORT and IL-6 production in burn + ethanol mice was examined. At 24 and 48 h post-burn, a positive correlation existed between circulating CORT levels and measurements of cellular immune function. Administration of exogenous CORT to burn + ethanol-treated mice resulted in significant restoration (to 60% of control) of DTH and splenocyte proliferative responses. This restoration was concomitant with a down-regulation of circulating and macrophage-derived IL-6. The specificity of CORT in modulating these responses was tested by assessing cellular immune function and IL-6 levels after glucocorticoid receptor blockade with RU486. Taken together, these data strongly suggest that under normal circumstances CORT protects burned mice from severe immune dysfunction, a protection that is not afforded to burn + ethanol-treated mice. Furthermore, the immune dysfunction observed in burn + ethanol mice may be due to a lack of glucocorticoid attenuation of IL-6.

Acute ethanol exposure prior to thermal injury results in decreased T-cell responses mediated in part by increased production of IL-6.

Previous studies by our laboratory have demonstrated that acute ethanol exposure prior to thermal injury results in suppression of cellular immune responses when compared with thermal injury alone. Ethanol exposure and burn injury are independently known to result in elevated IL-6, a cytokine with potent immunosuppressive properties. Therefore, we examined the role of IL-6 in the immune dysfunction in mice following a 15% body surface area scald (or sham) injury combined with acute ethanol (or vehicle) treatment. At 24 h post-injury, we observed slightly suppressed splenocyte proliferative responses and elevated circulating IL-6 (149+/-15 pg/mL) in mice receiving burn alone compared with those receiving sham injury (31+/-7 pg/mL). In contrast, burn + ethanol treated mice showed a profound suppression of splenocyte proliferation (20% of control) and significantly elevated circulating IL-6 levels (738+/-218 pg/mL). The suppressed splenocyte proliferative response was found to be macrophage dependent. Furthermore, IL-6 production was significantly elevated (p < .05) in splenic macrophage cultures from burn + ethanol mice (159+/-6 pg/mL) when compared with burn alone (109+/-10 pg/mL). Treatment of the splenocyte cultures from burn + ethanol mice with an anti-IL6 monoclonal antibody resulted in partial restoration of splenocyte proliferation. Taken together, these data strongly suggest that the immune dysfunction observed in ethanol-exposed, thermally injured mice is mediated in part by elevated levels of IL-6.

Effects of acute ethanol exposure on cellular immune responses in a murine model of thermal injury.

To test the effects of acute ethanol exposure on immune function after thermal injury, mice with blood alcohol levels of 100 mg/dL were given a 15% total body surface area dorsal scald or sham injury. Bacterial challenge resulted in 100 and 40% mortality in burn + ethanol- and burn + vehicle-treated mice, respectively. Delayed-type hypersensitivity responses were also significantly suppressed in burn + ethanol-treated mice. At 1 and 4 days post-burn, concanavalin A (ConA) -induced total splenocyte proliferation in burn + ethanol-treated groups was significantly decreased (P < 0.01) compared with burn + vehicle- or sham-treated animals. This decrease was not observed in total splenocytes cultured with anti-CD3epsilon or among adherence-depleted splenocytes given ConA or anti-CD3epsilon. FACS analyses revealed no changes in splenocyte sub-type ratios in burn + ethanol mice. The data herein demonstrate that acute ethanol exposure before thermal injury results in enhanced susceptibility to bacterial infection and markedly suppressed cellular immunity, which appears to be macrophage dependent.

Estrogen regulation of JE/MCP-1 mRNA expression in fibroblasts.

We have recently demonstrated that 17 beta-estradiol (E2) inhibits peritoneal adhesion formation. Because macrophages play a central role in inflammation and wound healing, we chose to investigate whether the E2 could inhibit the expression of JE, the murine monocyte chemoattractant protein-1 (MCP-1). To accomplish this, murine fibroblasts were cultured with physiological concentrations of E2 (3-300 pg/ml) with or without inducers of JE/MCP-1 mRNA expression. Untreated cells failed to express the message, but, following stimulation, a marked increase in JE/MCP-1 mRNA expression was observed. At 10-30 pg/ml, E2 had no effect on JE/MCP-1 mRNA expression in stimulated fibroblasts. In contrast, lower and higher doses of E2 inhibited the expression of JE/MCP-1 mRNA in stimulated fibroblasts. Treatment with tamoxifen reversed the E2-inhibition of expression of the message. These data demonstrate that JE/MCP-1 mRNA expression is controlled, in part, by estrogen and suggest that macrophage recruitment may be affected by circulating levels of E2.