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.

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.