REGULATION OF PHAGOCYTE FUNCTION

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Human phagocytic white blood cells serve as a major cellular defense against bacterial infection. These cells undergo a complex series of responses, including chemotactic and motile responses, uptake of microorganisms by phagocytotic processes, and bacterial killing by secretion of granule contents and the generation of toxic oxygen metabolites. This highly organized and regulated series of events triggered by infectious agents can be understood at its basic level utilizing many of the same underlying mechanisms as in normal cellular processes. This includes the widespread involvement of GTP-binding proteins in phagocytic white blood cell activities. It has been shown that the NADPH oxidase, the site of superoxide production in human neutrophils, is regulated by the ras-related GTP- binding protein Rac2. The activity of this, and related GTP-binding proteins is determined by factors which regulate the binding of GTP (active) and GDP (inactive). Thus GTPase activating proteins (GAPs) stimulate GTP hydrolysis, while GDP dissociation stimulators (GDS) and GDP dissociation inhibitors (GDI) regulate nucleotide exchange. l propose to identify and investigate regulatory components for Rac2 which would determine the activity of the NADPH oxidase in response to infectious agents. In related studies, l will determine the role of Rac protein in regulating the cell biology of motility phagocytosis or bacterial uptake, and granule secretion. Assays for regulatory GDS and GAP proteins will be developed using recombinant Rac2, and these proteins will be identified in the human neutrophil. The proteins will be purified to homogeneity, sequenced, and cloned. We will then utilize these proteins to investigate the regulation of the NADPH oxidase. This system will provide a model to understand Rac regulation that will then be extended to other cellular systems in which Rac and related proteins are required. Methodologies for the transfection and expression of Rac, Rac mutants and regulatory proteins into HL-60 promyelocytic cells will be developed. Using dominant negative or constitutively active forms of Rac protein, we will determine whether Rac, or the related Rho protein, play critical roles in the cell biology of neutrophils and/or monocytes. The mechanisms by which other systems, crucial for bacterial uptake and killing, are regulated by Rac and Rac regulatory proteins will be investigated using our findings in the oxidase system as a working model. Such studies will lead to increased knowledge of the cell biology of human phagocytes at the molecular level, and to novel approaches in anti-infective therapy.