Our studies are directed toward determining the biochemical mechanism involved in degranulation (secretion) of granulocytes (P M N) which affect the adherent properties of P M N. Recently the cell surface phorbol diester receptor has been suggested to be a protein kinase C. Since P M N activation is associated with the active metabolism of inositide lipids leading to the formation of the 1,2 diacyglycerol, phosphatidic acid, and arachidonic acid, we suggest that these biochemical changes might be related to fundamental mechanisms involved in P M N secretion. We propose to focus on the function of protein kinases, particularly protein kinase C and their role in phosphorylation of proteins which might be associated with the secretory process. Protein kinase C which is activated by diacyglycerol, will be purified from rabbit P M N using classical and affinity purification techniques. Protein phosphorylation patterns in whose cells, subcellular organelles, and membrane fractions will be determined and correlated with the P M N secretory behavior. In order to understand P M N secretion at a subcellular level we will employ a model system suitable for quantitative analysis which will allow us to examine in detail some of the components involved in promoting adherence and fusion of lysosomal granules with phagosomes. We will evaluate the specific interaction of actin with granule membranes. We will explore the role of lipids and calmodulin in promoting fusion of granule membranes to phagosomes. We will categorize by two-dimensional gel electrophoresis similarities and differences between the plasmalemma and specific and primary granule membranes of P M N. We will identify specific granule constituents (such as lactoferrin) on the plasma membrane of cells previously activated by electroblotting. Since P M N lactoferrin contributes to the adhesive properties of the PMN we plan to determine whether P M N lactoferrin can be reduced by rendering animals severely iron deficient which in turn could affect the functional behavior of these cells. Finally, we will examine both the functional properties and cellular distributions of proteins and enzymes that affect the functional properties of the P M N in a patient with specific granule deficiency and in another whole P M N lack a surface glycoprotein. These lines of investigation should prove fruitful in revealing the underlying mechanisms of stimulus-response coupling in P M N which lead to secretion. These studies will have relevance not only to the basic understanding of cellular responses to external stimuli but also to the mechanisms by which inappropriate responses are mainfested in abnormally functioning P M N.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020065-04
Application #
3129573
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1982-08-01
Project End
1987-07-01
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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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