Neutrophils respond to chemoattractants, such as the bacteria derived N-formyl peptide fMet-Leu-Phe (fMLF), with special bactericidal functions such as degranulation and superoxide (O2-) generation. While all chemoattractants can stimulate directed migration of leukocytes, only a few of them are able to induce O2- production. The mechanism underlying chemoattractant-induced O2- production is not well understood. In this competitive renewal application, we propose to investigate how fMLF stimulates neutrophil NADPH oxidase activation using a novel reconstitution system that we recently developed based on transfected COS-7 cells expressing the 4 essential NADPH oxidase components (COS-phox). There are 4 specific aims.
In Aim 1, we will determine the role of PKCdelta in formyl peptide receptor (FPR)-mediated O2- generation. We have recently found that this novel PKC is key to fMLF-induced O2- production in both COS-phox cells and neutrophils. Emphasis will be placed on PKCdelta-mediated p47phox phosphorylation and how PKCdelta is activated.
Aim 2 focuses on the mechanism of fMLF-induced Rac activation and its role in O2- production. We found that Rac2, the predominant Rac in neutrophils, cannot reconstitute NADPH oxidase in FPR-transfected COS-phox cells whereas Rac1 can. We will determine whether Rac2 is activated, where it is targeted, and what are the upstream molecules that activate Rac2, in transfected COS-phox cells.
In Aim 3, we will study FPR mutants for their effects on fMLF-induced O2- generation. Among these are point mutations associated with localized juvenile periodontitis and FPR mutations that disrupt receptor endocytosis and desensitization. This study will determine whether FPR desensitization is responsible for the characteristically transient O2- production following fMLF stimulation. For these studies, we will develop new O2- reconstitution system based on hematopoietic cell lines.
In Aim 4, we will investigate why some chemoattractants stimulate potent O2- generation in neutrophils but others such as IL-8 do not. Experiments are designed to determine whether these chemoattractants activate PKC and Rac, two critical events associated with NADPH oxidase activation. Taken together, these studies utilize our experience in FPR research and our newly developed reconstitution assay to investigate the mechanisms underlying an important neutrophil bactericidal function.
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