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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Hinkovska-Galcheva, Vania; Clark, Andrea; VanWay, Susan et al. (2008) Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells. J Lipid Res 49:531-42
Mansfield, Pamela J; Hinkovska-Galcheva, Vania; Borofsky, Michael S et al. (2005) Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA. Biochem Biophys Res Commun 331:132-8
Hinkovska-Galcheva, Vania; Boxer, Laurence A; Kindzelskii, Andrei et al. (2005) Ceramide 1-phosphate, a mediator of phagocytosis. J Biol Chem 280:26612-21
Mansfield, Pamela J; Carey, Shannon S; Hinkovska-Galcheva, Vania et al. (2004) Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes. Blood 103:2363-8
Malawista, Stephen E; de Boisfleury Chevance, Anne; Brown, Eric J et al. (2003) Chemotaxis of non-compressed blood polymorphonuclear leukocytes from an adolescent with severe leukocyte adhesion deficiency. Am J Hematol 73:115-20
Hinkovska-Galcheva, Vania; Boxer, Laurence; Mansfield, Pamela J et al. (2003) Enhanced phagocytosis through inhibition of de novo ceramide synthesis. J Biol Chem 278:974-82
Mansfield, Pamela J; Hinkovska-Galcheva, Vania; Carey, Shannon S et al. (2002) Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D. Blood 99:1434-41
Mansfield, Pamela J; Hinkovska-Galcheva, Vania; Shayman, James A et al. (2002) Granulocyte colony-stimulating factor primes NADPH oxidase in neutrophils through translocation of cytochrome b(558) by gelatinase-granule release. J Lab Clin Med 140:9-16
Mansfield, P J; Shayman, J A; Boxer, L A (2000) Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase. Blood 95:2407-12
Rengan, R; Ochs, H D; Sweet, L I et al. (2000) Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells. Blood 95:1283-92

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