The oxidative responses of leukocytes crawling through an extracellular matrix under physiological conditions differ dramatically from those of isolated leukocytes. In nonadherent cells, an oxidative response of modest extent is rapidly stimulated by chemoattractants and/or cytokines. In contrast, the response of adherent leukocytes exhibits a prolonged delay, followed by a dramatically enhanced and prolonged generation of oxygen metabolites. The basis for this crosstalk between adhesion receptors (integrins) and chemoattractant receptors that initiate the oxidative response has not been determined. Substantial data from our group suggests the hypothesis that this crosstalk occurs at the level of Rho GTPases which control both integrin signaling to the cytoskeleton and chemoattractant- induced activation of the phagocyte NADPH oxidase. In order to systematically investigate the adhesion-regulated oxidative response, we will determine the signaling pathways controlling Rho GTPase function and NADPH oxidase activation in adherent leukocytes. We will determine whether NADPH oxidase assembly and/or activation is modified in adherent cells. The function of individual oxidase regulatory proteins will be examined using intact cell and broken cell systems. The activation of Rho family GTPases will be measured during the adhesion-regulated oxidative response using novel biochemical assays. Signal transduction events initiated by adhesion will be quantified and correlated pharmacologically with changes in GTPase activity and NADPH oxidase activation. The role of specific signaling pathways will be examined using biochemical means and genetic approaches in which dominant active or dominant negative mutants will be introduced into cells using a viral expression system. Finally, transgenic mice lacking specific signaling proteins will be used to confirm the roles of these pathways in regulating components of the adhesion-regulated oxidative response.
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