Monoclonal antibodies that interact with functional antigens on phagocytes are particularly useful as specific probes to study the role of surface structures in cell activation. By fusing spleen cells from mice immunized with human peripheral blood granulocytes with mouse myeloma cells, we have developed a highly specific monoclonal antibody, DL1.2, that induces chemotaxis in neutrophilic granulocytes (PMN). DL1.2 also stimulates f-met-leu-phe (fMLP)-induced chemotaxis. By means of flow cytometry and immunofluorescence, DL1.2 was found to react predominantly (greater than 85%) with mature PMN and not myeloid or monocytic precursor cells. DL1.2 binds to a 120 kD protein on PMN that we have purified by affinity chromatography. The overall objective of this proposal is to further characterize the chemotactic antigen on PMN that binds DL1.2 and to elucidate the mechanism by which it stimulates chemotaxis. Our hypothesis is that DL1.2 directly activates a tyrosine specific protein kinase resulting in phosphorylation of critical membrane proteins and cytoskeletal elements which subsequently leads to directed migration. Preliminary studies indicate that DL1.2 induces tyrosine specific protein phosphorylation in PMN, but has no effect on mobilization of calcium from intracellular stores. Furthermore, the effects of DL1.2 on PMN are not inhibited by the guanine nucleotide binding protein inhibitor, pertussis toxin. Thus, DL1.2, like phorbol myristate acetate, may bypass the calcium requiring segment of the activation sequence in PMN by acting directly on a protein kinase. To test this hypothesis, we plan to examine the effects of DL1.2 alone and in combination with fMLP on tyrosine kinase activity and protein phosphorylation in PMN. We will also study the effects of the antibody on intracellular calcium mobilization and on phosphatidylinositol turnover since these processes have been linked to chemoattractant induced PMN activation. Studies on DL1.2 and its relationship to other antibody binding sites may help to elucidate the transduction signals that regulate PMN migration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI020183-04A1
Application #
3129678
Study Section
Experimental Immunology Study Section (EI)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
Schools of Pharmacy
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901