Our studies will test the hypothesis that during receptor-mediated activation of adherent or adherent phagocytosing polymorphonuclear leukocytes (PMN), the NADPH oxidase can be assembled on specific granule (SG) membranes which contain lactoferrin (LF) and cytochrome b558. Our studies will test whether phosphatidic acid (PA) initiates the assembly of the NADPH oxidase onto SG membranes either prior to or during exocytosis. Our studies will be directed to assess the role of calcium-binding proteins and diacylglycerol (DAG) in initiating and/or sustaining exocytosis. We will focus on the metabolic pathways involved in the assembly of the multicomponent NADPH oxidase onto SG, the biochemical documentation of NADPH activity on SG, and the subcellular localization of phosphatidic acid phosphohydrolase. We will employ radioactive substrates, which will allow documentation of phospholipase C or phospholipase D activities, as well as measure mass production of PA and DAG. We will monitor assembly of the NADPH oxidase P47phox onto SG biochemically, immunologically and by employing transmission immune electron microscopy. We will document the interrelationship between release of SG bearing LF and the respiratory burst in adherent PMN undergoing phagocytosis. Immunoelectron microscopy will be used to morphologically assess the assembly of the NADPH oxidase by monitoring the association of the oxidase component p47Phox with LF bearing SG. We will employ immunofluorescence intensified-enhanced microscopy to document the entry of oxidants into phagosomes. To link delivery of LF into phagosomes with the respiratory burst we will test the ability of BAPTA or MAPTAM/ionomycin treatment to affect LF delivery into the phagosomes by employing resonance energy transfer. Finally, we will study the regulation of exocytosis of SG by focusing on cell-free systems which will allow us to evaluate the role of calcium binding proteins along with diradylglycerol as cofactors in mediating fusion of SG with the plasma membrane. We will purify novel fusogenic proteins by chromatographic techniques and assess their ability to promote fusion of SG with PMN plasma membranes using lipid mixing assays. To confirm delivery of SG contents into PMN membranes, we will employ immune electron microscopy. These studies will document the intracellular signalling pathways leading to the assembly of the NADPH oxidase on SG membranes. They will provide further biochemical understanding of the factors regulating exocytosis of SG. The knowledge of both oxidative metabolism and degranulation in PMN should lead to novel therapeutic approaches to chronic inflammation.
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