Regulation of phagocyte function by Rac2

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The Rho family GTPase Rac plays a key role in regulating a variety of phagocyte functions, including NADPH oxidase-catalyzed superoxide generation, Fcgamma receptor-mediated phagocytosis, and cytoskeletal changes during membrane ruffling and cell movement. To examine the role of the hematopoietic specific Rac2 GTPase, which is approximately 90% identical to the more widely expressed Rac1 and Rac3 isoforms, mice with a targeted disruption of the Rac2 gene were generated. NADPH oxidase activation in Rac2-deficient neutrophils )which still express Rac1) was substantially reduced in response to fMLP, IgG- opsonized particles, and phorbol ester, but normal in response to complement-coated zymosan. Other neutrophil defects observed in initial studies include impaired actin polymerization, chemotaxis, and poor L-selectin-dependent adhesion. In addition, mice exhibited decreased exudate formulation in vivo and an increased susceptibility to invasive Aspergillosis. An important role for Rac in neutrophil function has been further established by the identification of a dominant-negative mutant of Rac2 in an infant who presented with recurrent pyogenic infections and functional neutrophil defects similar to those found in Rc2-/-. These data suggest that Rac2 regulates multiple cellular responses in phagocytes downstream of specific receptor-activated pathways, and has non-overlapping functions with Rac1 and Rac3 in the host infection and inflammation. Project 3 will focus on testing this hypothesis, particular as it relates to activation of the NADPH oxidase, and explore underlying biochemical mechanisms. There are four Specific Aims, which will take advantage of the Rac2-/- mouse. These propose to (1) further define defects in Rac2-/- phagocytes to examine macrophage superoxide production, phagocytosis, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes reflect selective activation, localization, or usage of Rac2, and if specific Rac2 effector sequences are required; (3) investigate upstream and downstream signaling events involved in Rac-dependent assembly of the NADPH oxidase complex and (4) determine the impact of Rac2 deficiency on host defense and inflammation. This work will provide further insight into how Rho-GTPases regulate phagocytic leukocyte functions and the biochemical mechanisms that mediate specific agonist-induced cellular responses. These studies may also result in new approaches to modulating phagocyte function in inflammation and host defense.