The Rac GTPase-controlled NADPH oxidase of human phagocytes is responsible for the formation of superoxide anion and other oxidants used by these cells for bacterial killing, and is also an important contributor to the inflammatory response. NADPH oxidase is directly stimulated by LPS in monocytes, and LPS serves to """"""""prime"""""""" oxidant production in other leukocytes, thereby enhancing the inflammatory damage occurring during septic shock. We will investigate the connections between Rac GTPase-regulated responses of leukocytes and the signaling mechanisms utilized by LPS. Novel lipid mediators formed in response to LPS will be tested for the ability to disrupt regulatory complexes of Rho family GTPases and RhoGDI. We will use both direct binding measurements and activity assays to evaluate possible effects on Rac signing by this mechanism. The contribution of Rac to the phagocyte oxidative responses induced by 125 will be tested using genetic approaches. Signaling via Rac to p38 MAPK will be examined as well, and we will evaluate the hypothesis that Rac- regulated kinases such as p38 control the coordinated assembly of the NADPH oxidase. Sites on NADPH oxidase components that are phosphorylated by p38 will be identified, and the contribution of this enzyme to oxidase assembly assessed. Finally, the possible connection of Rac GTPase to activation of the LPS- and cytokine-regulated transcription factor NF-kB will be evaluated. These studies should clarify the mechanisms by which LPS mediates inflammatory tissue damage during septic shock syndromes.
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