Circulatory shock and critical organ injury during gram-negative bacteremia (GNB) result from inappropriate regulation of a cytokine network involving tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-1beta, IL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to cardiopulmonary dysfunction and ischemic- hypoxic cellular injury. Although GNB-induced cytokine production by the liver and lungs can initiate the gram-negative septic shock syndrome, the mechanisms by which cytokine expression in these organs escapes host regulation remain unclear. Ischemia-reperfusion (I/R) and hypoxia- reoxygenation (H/R) upregulate cytokine biosynthesis owing to reactive O2 species, activated complement fragments (C'), and alpha-adrenoceptor stimulation. The central premise of this proposal is that selective I/R or H/R of the liver and lungs following GNB downregulates cytokine gene expression independent of these mechanisms, thereby modifying organ injury. As a corollary, we propose that autoregulatory cyclooxygenase- derived eicosanoids in ischemic tissues downregulate GNB-induced cytokine production during subsequent I/R, but not during equivalent reductions in O2 delivery during H/R. The objective of the proposed research is to test the HYPOTHESIS that changes in oxidative metabolism from low-flow I/R vs. constant-flow H/R of the liver and lungs following GNB modulate post- transcriptional control of cytokine expression. We further propose that such interactions modify tissue repair by altering nitric oxide synthase (NOS) and collagen gene expression. We plan to vary the timing of E. coli GNB in ex situ perfused rat liver and lungs with respect to graded I/R or H/R. Serial changes in cytokine, NOS and collagen isoform transcripts will be characterized by Northern analyses, as will the kinetics of the co- induced anti-inflammatory genes for IL-4, IL-10, and TGF-beta. Transcriptional vs. translational effects will be distinguished by nuclear run-on assays. Corresponding protein and prostaglandin E2 levels will be analyzed in perfusates with and without prior eicosanoid and xanthine oxidase inhibition. In parallel studies, the effects of C' and alpha- adrenoceptor stimulation will be determined. These events will be correlated with changes in tissue microbial burden and organ performance. Parallel studies will be performed in cultured Kupffer cells and alveolar macrophages. In conscious animals, cytokine-mediated changes in lung inflammation during GNB will be analyzed during focal hepatic ischemia and biliary cirrhosis. Results from these vertically-oriented studies will enable a new synthesis integrating the determinants of GNB-induced cytokine production and liver lung interactions-during ischemic-hypoxic stress. This should provide novel insights into the regulation of cytokine expression during gram negative sepsis while identifying mechanistic therapies to ameliorate acute lung injury and multiple organ failure.
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