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Abstract

Intercellular adhesion molecule-1 (ICAM-1, CD54) and its interactions with LFA-1 and Mac-1 are critical for the proper functioning of our immune system. In addition to these associations, other molecules such as a-actinin of the actin cytoskeleton, and a sialoglycoprotein, CD43, have been suggested to bind human ICAM-1 but the precise nature of these interactions and their physiological significance are not known. This thesis has examined the possible associations of ICAM-1 with these later two molecules, in the murine system, to further elucidate the mechanisms responsible for the upregulation of ICAM-1 adhesion during inflammatory and immune responses. Punctate distribution of ICAM-1 on the surface of fibroblasts suggested the association of ICAM-1 with cytoskeletal components. However, detergent extraction of cell lines expressing endogenous ICAM-1, as well as those transfected with truncated ICAM-1 lacking the cytoplasmic domain or recombinant ICAM-1 anchored by glycdsyl-phosphatidyl inositol, suggested there is very little or no association of ICAM-1 with the cytoskeleton. In all cases, ICAM-1 was completely soluble. To determine if ICAM-1 associated with another molecule which in turn associated with the cytoskeleton, chemical cross-linking studies were performed. In Western blot analysis, a band of approximately 200 KD was observed which suggests ICAM-1 may self associate in a fibroblast and T cell hybridoma cell line. It was concluded that murine ICAM-1 does not associate with the cytoskeleton in the cell types tested and that there must be some other underlying mechanism responsible for the punctate distribution of ICAM-1 on fibroblasts. Whether this affects cell adhesion remains to be determined. The report demonstrating the binding of human ICAM-1 to a leukocyte sialoglycoprotein, CD43, lead to further investigation of this interaction in the murine system in this thesis. Fibroblasts transfected with murine CD43 were unable to adhere to soluble immobilized ICAM-1 in an adhesion assay. As well, soluble ICAM-1 coated microspheres were unable to bind to CD43+ fibroblasts. The results of these studies conclude murine CD43 interaction with ICAM-1 can not be detected by these conventional methods and thus bring into question the original reports on CD43/ICAM-1 interaction. A new model system was established in an attempt to detect a more sensitive interaction that may not withstand an adhesion assay. This system utilized cell death via the cross-linking of an ICAM-1/Fas chimeric molecule as a read out for an association. Although this system was unable to determine whether CD43 and ICAM-1 interact, it did suggest that LFA-1/ICAM-1 interaction may be one of monovalency. This however, requires further investigation.