Previous studies from this project have established a critical role for reactive oxygen metabolites (""""""""free radicals"""""""") generated from endothelial cell xanthine oxidase as triggers of neutrophil-mediated microvascular injury, and consequent organ dysfunction and failure following ischemia/reperfusion, or following shock, leading to the development of multiple organ failure. We now propose to evaluate the novel hypothesis that microvascular endothelial cell surface xanthine oxidoreductase functions physiologically as a molecular switch for the reticuloendothelial system by transducing circulating inflammatory signals (cytokines) via the limited proteolytic activation of xanthine oxidase (XO) from xanthine dehydrogenase (XD), generating hydrogen peroxide as a second messenger to trigger the integrin-mediated adhesion, trapping and activation of circulating neutrophils to effect the trapping, phagocytosis and killing of circulating microorganisms within the venules Of most organs (vestigially) and the sinusoids and central veins of the liver (functionally). In our model system of microvascular endothelial cell monolayers in vitro, gamma-interferon primes this signalling mechanism by upregulating the synthesis of xanthine dehydrogenase, while TNFalpha triggers neutrophil adhesion by mediating proteolytic XD to XO conversion.
Specific Aims : 1. Confirm the necessary role for endothelial cell surface XO for the transduction of cytokine (TNFalpha) signals in endothelial cells in vitro to upregulate the expression of surface integrins by blocking XO activity with custom-made m-anti-XO, and by transfecting XO- negative endothelial cells with chicken or mutant xanthine dehydrogenase which is incapable of proteolytic activation to XO, and evaluating these cells for signalling activity. 2. Define the relationship of this novel signal transduction pathway to other, established pathways using conventional techniques. 3. Evaluate each component of the proposed pathway, including the roles of specific TNFalpha receptors, the SEKk kinase pathway, sphingomyelin/ceramide, phospholipase C, inositol triphosphate, intracellular calcium, and mu-calpain in effecting proteolytic activation of XO using established, but state-of-the art techniques. 4. Confirm the integration of the component of this system to effect phagocytic killing of microorganisms by macrophages and Kupffer cells in vitro, and by the hepatic reticuloendothelial system of the intact rat in """"""""i""""""""o. This project should therefore establish and characterize a novel cell signalling mechanism that is important for immune defense against circulating microorganisms, and which incidentally causes organ injury following shock or ischemia.
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