The CD45 antigen family is a group of high molecular weight glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. We have previously shown that epitopes of CD45 are involved in neutrophil chemotaxis to the chemoattractants, leukotriene B4 and recombinant human C5a (rHuC5a). The purpose of this project is to generate and characterize stable CD45 negative myeloid cell clones and determine the role of CD45 in phagocyte function. The Abelson-murine leukemia virus transformed macrophage cell line, RAW 264, was selected for this project because RAW 264 macrophages exhibit dose-dependent chemotaxis to Rhuc5a. RAW 264 cells were mutagenized with the alkylating agent, ethyl methanesulfonate, for 24 hours in tissue culture flasks. Mutagenized cells were scraped off the flask, replated into several flasks, and allowed to grow in Minimal Essential Medium containing 10% fetal calf serum. Cells were harvested from the flasks when they reached confluent growth and were treated with a biotinylated-monoclonal antibody (mAb) to murine CD45. Streptavidin- conjugated magnetic beads were incubated with the CD45 Mab-treated cells and the mixture was run over a magnetized column to enrich for the CD45 negative cells. After multiple rounds of CD45 negative selection and expansion of the enriched population in culture, 75 CD45 negative clones were identified by flow cytometric analysis. Twenty clones were screened for chemotactic responsiveness to Rhuc5a and >90% of the clones had defective chemotactic responses. Current studies are in progress to transfect the CD45 negative mutants with CD45 cDNA and determine whether normal chemotactic function returns upon expression of CD45. These results indicate that CD45 expression on macrophage plasma membranes is important for normal chemotactic responsiveness to Rhuc5a. The CD45 negative RAW 264 mutant clones provide a unique cellular system to probe the functional activity of CD45 in myeloid cells.
