Suppressed T-cell growth lymphokine production and proliferation have been demonstrated in patients with severe trauma, burn, and sepsis, as well as in experimental animals with comparable injuries. Such a cell- mediated immunosuppression in conjunction with the hyperactivation of the macrophage/monocyte proinflammatory mediator response can evidently exacerbate the systemic inflammatory response syndrome in the injured hosts. The mechanisms of T-cell suppression are not well understood. We hypothesize that the inhibitory mediators such as TGF-beta, IL4, IL-10, PGE and glucocorticoid, that are collectively released by macrophage/monocyte, certain T-cells and adrenal cortex, interfere with the T-cell receptor (TCR)-associated early signal transduction pathways involving protein tyrosine kinases (PTK), protein kinase C (PKC), and intracellular Ca2+ (Ca2+i) mobilization, to cause T-cell deactivation. We will test this hypothesis in models of early and late stages of intraabdominal sepsis in rats and mice, and in rat or mouse splenic macrophage and T-cell co-cultures exposed to LPS. T-cell responses IL-2 secretion and IL-2 receptor expression, and cell signaling pathways will be assessed in primary T-lymphocyte cultures. The first specific aim is to determine the time course the release of the inhibitory mediators and changes in T-cell responses in septic rats and mice. Systemic release of the inhibitory mediators, and splenic tissue generation of inhibitory cytokines and their mRNAs will be quantified. The second specific aim is to define the changes in T-cell signaling pathways (PTK, PKC, Ca2+ mobilization) in septic animals. Ca2+ will be quantified in cell suspensions by means of Fura-2 photofluorometry, and in single cells using the Ca2+ imaging technique. The third specific aim is to determine through in vitro studies the direct effects of the inhibitory mediators, individually and in some combinations as warranted by previous studies, on the T-cells' signaling pathways, and to demonstrate that inhibitors/antibodies of the inhibitory mediators can abrogate the T-cell signaling alterations, and suppression of IL-2 production and proliferation in the LPS exposed co-cultures of splenic macrophages and T-lymphocytes from rats or mice. The fourth specific aim is to examine the putative effects of the blockers of PTK, PKC, and Ca2+i mobilization on T-cells' responses and to ascertain that agonist of PKC and Ca2+ mobilization can restore T-cell responses in rat splenic macrophage and T-lymphocyte co-cultures exposed to LPS. Through these in vivo and in vitro studies we will evaluate correlations and causal relationships between the inhibitory mediators and T-cell signaling and responses during the septic injury process. The proposed studies will allow for the development of rational immunomodulant therapies against suppressed cell- mediated immunity in sepsis.
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