Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions.

After scald burn-injury, the intestinal immune system responds to maintain immune balance. In this regard CD4+T cells in Gut-Associated Lymphoid Tissues (GALT), like mesenteric lymph nodes (MLN) and Peyer's patches (PP) respond to avoid immune suppression following major injury such as burn. Therefore, we hypothesized that the gut CD4+T cells become dysfunctional and turn the immune homeostasis towards depression of CD4+ T cell-mediated adaptive immune responses. In the current study we show down regulation of mucosal CD4+ T cell proliferation, IL-2 production and cell surface marker expression of mucosal CD4+ T cells moving towards suppressive-type. Acute burn-injury lead to up-regulation of regulatory marker (CD25+), down regulation of adhesion (CD62L, CD11a) and homing receptor (CD49d) expression, and up-regulation of negative co-stimulatory (CTLA-4) molecule. Moreover, CD4+CD25+ T cells of intestinal origin showed resistance to spontaneous as well as induced apoptosis that may contribute to suppression of effector CD4+ T cells. Furthermore, gut CD4+CD25+ T cells obtained from burn-injured animals were able to down-regulate naïve CD4+ T cell proliferation following adoptive transfer of burn-injured CD4+CD25+ T cells into sham control animals, without any significant effect on cell surface activation markers. Together, these data demonstrate that the intestinal CD4+ T cells evolve a strategy to promote suppressive CD4+ T cell effector responses, as evidenced by enhanced CD4+CD25+ T cells, up-regulated CTLA-4 expression, reduced IL-2 production, tendency towards diminished apoptosis of suppressive CD4+ T cells, and thus lose their natural ability to regulate immune homeostasis following acute burn-injury and prevent immune paralysis.

Dendritic cells (DCs) play a vital role in presenting antigen to CD4+ CD25+ (Reg) T cells. We hypothesize that burn plus sepsis injury adversely affects antigen presenting cells, in particular the DCs, such that these DC are unable to adequately prime Reg T cell; the inadequate stimulation of Reg T cell by DCs may contribute to burn-induced impairment in T cell IL-2 production and proliferation. In this study, we examined the effect of presence of anti-CD3, anti-CD28, and/or IL-2 in the T cell cultures with or without the added APC/DCs. The results show when sham rat Reg T cells were cocultured with DCs or APCs from burn septic rats, IL-2 production and proliferation was decreased, compared to that in sham T cells cocultured with sham DCs or APCs. When exogenous IL-2 was added to sham Reg T cell and burn and sepsis rat APC cocultures, there was an enhancement in Reg T cell proliferation to the level found in cocultures of sham rat T cells with sham rat APCs. Sham rat T cell proliferation was enhanced in the presence of anti-CD3 but not to the level in cocultures with burn plus sepsis rat APCs. Similarly, addition of anti-CD28 produced but a small increase (~20%) in proliferation of sham rat Reg T cell cocultured with rat APCs, compared to the effect of anti-CD28 in cocultures of sham rat T cell with sham rat APCs. These studies indicate a role of burn-derived APC and DCs in modulating a Reg T cell response in burn and sepsis injury.

Immunosuppression occurs after burn injury with or without septic complications. We hypothesize that CD4+CD25+ T cells (Treg) are responsible for transmitting inhibitory signals to the IL-2-producing T cells in burn related inflammatory/infectious conditions. Enriched Treg were obtained from day-3 burn rats by flow cytometry and magnetic microbead cell sorting and given intravenously to sham rats. The rats were then sacrificed day 1 and day 3 post-adoptive transfer and T cell proliferation responses were assessed by thymidine assay. The results indicated that when burn Treg were adoptively transferred to sham rats, T cell proliferation (CD4+, CD4+CD25+ and CD4+CD25-) of sham rats was significantly depressed. Furthermore, the results showed that adoptive transfer of Treg did not influence the phenotypic expression of CD4+ T cells of the recipient rats. The percentage expressions of CD25, CD11a (LFA), and CD62L of the recipient rats remained same as before adoptive transfer. Experiments are currently underway to expand these findings to burn plus septic rats. These preliminary findings however allow us to hypothesize that while burn injury alone can produce attenuated CD4+ T cell mediated responses as a result of actions of Treg; burn injury with septic complications may further cause an irreversible loss of T cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with burn plus a critical infection.