While the molecular basis of granulocyte function is largely unknown, glycoproteins of the plasma membrane are believed to play a prominent role in many of the cell's functions. The study of granulocyte membrane structure and function has been hindered by difficulty in obtaining membrane components of high purity. Our long-term goal is to identify, purify, and characterize a variety of granulocyte membrane proteins and to examine their roles in granulocyte structure and function. This goal will be approached by developing a variety of monoclonal antibodies directed against specific granulocyte membrane antigens. We have already developed several such antibodies and begun characterizing their respective antigens. One of these antibodies, AHN-1, selectively inhibits phagocytosis by neutrophils and reacts with two distinct membrane proteins. We plan to purify these proteins, produce new monoclonal antibodies which react with unique epitopes on these proteins, and use these antibodies to further characterize their role in cell function. We have also found that the vast majority of monoclonal antibodies produced from mice immunized with human granulocytes are directed against the same highly antigenic carbohydrate recognized by AHN-1. Therefore granulocyte membranes will be solubilized and passed over an affinity column containing immobilized AHN-1; the column effluent containing other antigens of interest will be used as the immunogen for the production of more monoclonal antibodies. We will characterize these antibodies as we have AHN-1. Proteins bearing antigens recognized by these antibodies will be identified by polyacrylamide gel electrophoresis of proteins immunoprecipitated from detergent extracts of radiolabeled cells. To define the role of these antigens in granulocyte development and function, we will study antigen expression on hemopoietic progenitor cells and test the effects of the antibodies on measurable neutrophil functions. Proteins of interest will be purified by monoclonal antibody affinity chromatography, and, if necessary, further chromatographic techniques. These purified proteins will be used for further biochemical characterization as well as immunogens for the production of polyclonal antisera, which will recognize a variety of different antigens present on the molecule. Antibodies produced in this project should provide useful tools to study the molecular nature of normal and abnormal granulocyte surfaces and to probe the role of these antigens in cell development and function. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA036248-04
Application #
3173775
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1990-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Skubitz, K M; Ducker, T P; Goueli, S A (1992) CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils. J Immunol 148:852-60
Skubitz, K M; Ehresmann, D D (1992) The angiogenesis inhibitor beta-cyclodextrin tetradecasulfate inhibits ecto-protein kinase activity. Cell Mol Biol 38:543-60
Ducker, T P; Skubitz, K M (1992) Subcellular localization of CD66, CD67, and NCA in human neutrophils. J Leukoc Biol 52:11-6
Ward, J C; Gitlin, J B; Garry, D J et al. (1992) Epidermolysis bullosa acquisita induced by GM-CSF: a role for eosinophils in treatment-related toxicity. Br J Haematol 81:27-32
Skubitz, K M; Stroncek, D F; Sun, B (1991) Neutrophil-specific antigen NB1 is anchored via a glycosyl-phosphatidylinositol linkage. J Leukoc Biol 49:163-71
Wasiluk, K R; Skubitz, K M; Gray, B H (1991) Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa. Infect Immun 59:4193-200
Stroncek, D F; Skubitz, K M; Plachta, L B et al. (1991) Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc-gamma receptor III with maternal deficiency of CD16 antigen. Blood 77:1572-80
Harvath, L; Balke, J A; Christiansen, N P et al. (1991) Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis. J Immunol 146:949-57
Skubitz, K M; Goueli, S A (1991) Basic fibroblast growth factor is a substrate for phosphorylation by human neutrophil ecto-protein kinase activity. Biochem Biophys Res Commun 174:49-55

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