Role of CD45 in Phagocyte Motility and Signal Transduction Liana Harvath The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout cell line. In a CD45 knockout, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, a CD45 targeting vector was constructed that utilized insertion of a neomycin gene driven by a heterologous promoter into exon 12 of CD45. Transfection experiments with the exon 12 CD45 targeting vector showed that random integration into the genome greatly exceeded targeted integration and indicated that it may be necessary to screen several thousand clones before a successful recombinant would be found. When this situation has arisen with other genes, a more favorable targeting to random integration ratio has been obtained with a promoterless targeting vector that utilizes the genomic promoter of the target gene. In an exon 12 knockout cell line, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Studies are underway to construct a promoterless CD45 targeting vector, to investigate the targeting to random integration ratio, and to isolate a CD45 knockout. The isolation of a CD45 deficient cell line will provide a valuable tool for establishing the functional role of the molecule in phagocyte chemotaxis and host defense.
