Our studies are directed toward determining the biochemical mechanisms involved in degranulation (secretion) of granulocytes (PMN) which affect the adherent properties of PMN. We propose to focus on protein kinase C and its role in phosphorylation of proteins which might be associated with the secretory process. Protein kinase C will be purified from human PMN using high performance liquid chromatography (HPLC). Protein phosphorylation patterns in whole cells, subcellular organelles, and membrane fractions will be determined and correlated with the PMN secretroy behavior. We will purify an endogenous inhibitor of PKC localized in specific granules of PMN. The inhibitor will be characterized with respect to its specific intracellular localization, molecular weight, and activity as a regulator of PKC. To understand PMN secretion at a subcellular level we will employ a model system suitable for quantitative analysis which will allow us to examine in detail the interrelationship between PKC binding to PMN membranes and subsequent functional effects. We will examine in detail some of the components involved in promoting the adherence and fusion of granules with membranes. We will evaluate the specific interaction of actin with granule membranes. We will explore the role of lipids, calmodulin, protein kinase C in promoting fusion of granule membranes to phagosomes and to plasma membranes. We will categorize by 2-dimensional gel electrophoresis similarities and differences between the plasma membrane and specific and primary granules and secretory vesicles of PMN. We will identify intracellular organelle constituents such as lactoferrin and C3bi on the plasma membrane of cells previously activated by electroblotting. We will examine the biochemical mechanisms underlying the hyperresponsive behavior of specific granule deficient PMNs which are unable to adhere normally to surfaces. We will also examine by pulse-chase experiments the synthesis of adhesive glycoproteins and their accumulation on the plasma membrane of normal and deficient canine monocytes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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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
Dale, D C; Person, R E; Bolyard, A A et al. (2000) Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 96:2317-22
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

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