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UBC Theses and Dissertations

Regulation of LFA-1 (CD11a/CD18) function Pyszniak, Andrew M.


In the immune system, the complex cellular interactions that are necessary for the effective surveillance of the body against pathogens, and for launching an appropriate immune response to eliminate these pathogens, are mediated by adhesion molecules. The proper function of the immune system relies on the strict regulation of adhesive interactions between immune cells. The leukocyte integrin LFA-1 and its major ligand ICAM-1 constitute one pair of adhesion molecules that play critical roles in immune responses. This thesis presents the results of experiments designed to elucidate the mechanisms regulating the binding of LFA-1 to ICAM- 1. Murine recombinant soluble ICAM-1 was immobilized on polystyrene microspheres with a view to using the beads to probe the distribution of high-avidity LFA-1 on various cell lines. These microspheres bound specifically to high-avidity LFA-1 in a cytoskeleton-independent manner as treatment with cytochalasin B had no effect. Furthermore, the beads displayed a highly localized distribution on some cell types, whereas fluorescence staining indicated that LFA-1 was uniformly distributed on the cell surface. Thus, the cell surface distribution of highavidity LFA-1 can be different from that of LFA-1 in general, and is potentially significant in regulating LFA-1-mediated adhesion. The role of LFA-1 cytoplasmic domains in binding to ICAM-1 and in post-adhesion events was also investigated. Various truncated forms of LFA-1 α (CD 11 a) and β (CD 18) chains were generated by PCR and co-transfected into murine fibroblast TNR-2 cells in various combinations. The transfected fibroblasts were tested for their ability to adhere to ICAM-1 immobilized on plastic, and to spread out following this adhesion. The results indicated that both LFA-1 cytoplasmic domains are important for adhesion to ICAM-1, but that they play different roles. Furthermore, both cytoplasmic domains are required for post-receptor spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. As a further probe into the roles of LFA-1 cytoplasmic domains in regulating adhesion, we overexpressed chimeras consisting of the LFA-1 subunit cytoplasmic domains attached to the extracellular portion of murine CD4 in a B cell line as well as in a T cell line, and examined the effect on adhesion to ICAM-1 and fibronectin. The CD4/18 chimera drastically inhibited adhesion of both lines to ICAM-1 and fibronectin, whereas the CD4/11 chimera and a truncated form of CD4 lacking most of the cytoplasmic domain had no effect. Cell spreading after binding to ICAM-1 was also inhibited by the CD4/18 chimera but not the CD4/11 chimera. These results suggest that the CD 18 cytoplasmic domain interacts with intracellular molecules that regulate not only LFA-1 but also other integrins.

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