UBC Theses and Dissertations
Functional analysis of ICAM-1 : LFA-1 interaction in cell adhesion Welder, Clayton Anthony
This thesis presents the results of a project aimed firstly at exploring the use of a soluble form of ICAM-1 to inhibit cellular immune responses, which in general rely heavily on the interaction between the counter receptors ICAM-1 and LFA-1, and aimed secondly at studying the mechanisms regulating ICAM-1:LFA-1 mediated cell adhesion. Functional characterization of the purified sICAM-1 demonstrated that radio-iodinated sICAM-1 could bind to LFA-1 positive cells, albeit with an apparently low affinity. However, when immobilized on plastic, sICAM-1 was fully functional, efficiently facilitating LFA-1:ICAM-1 mediated cell adhesion. Two mechanisms of inducing LFA-1:ICAM-1 mediated cell adhesion were characterized: stimulation by the phorbol ester PMA and stimulation by the divalent cation Mn++. PMA induced adhesion was dependant on a functional actin cytoskeleton as judged by the inhibitory effect of cytochalasin B. PMA induced adhesion could not be inhibited by sICAM-1, suggesting that this cell adhesion was mediated by low affinity ICAM-1:LFA-1interaction. In contrast, Mn++ induced adhesion seemed to be mediated by an increase in the affinity of the ICAM-1:LFA-1 interaction; Mn++ induced homotypic aggregation could be specifically inhibited by sICAM-1. While monovalent sICAM-1 could not efficiently inhibit PMA induced adhesion, a multivalent form of sICAM-1 could inhibit PMA induced ICAM-1:LFA-1 mediated cell adhesion, demonstrating the importance of multivalent interaction in LFA-1:ICAM-1 mediated cell adhesion. Fluorescence microscopic studies aimed at determining the cell surface distribution of ICAM-1 and LFA-1 clearly showed that the distribution of ICAM-1 could be differentially regulated. With LFA-1, the regulation of it’s cell surface distribution could not be demonstrated using immunofluorescence microscopy. Studies were conducted to define a role for the cytoplasmic domain of ICAM-1 by transfectingICAM-1 with or without its cytoplasmic domain into ICAM-1 negative cells. These studies demonstrated that removal of the cytoplasmic domain of ICAM-1 resulted in a decreased but not abolished aggregative phenotype. However, a precise role for the cytoplasmic domain of ICAM-1 in determining the cell surface distribution of ICAM-1 could not be defined. These results are discussed in relation to the literature concerning soluble adhesion molecules and the regulatory mechanisms governing cell adhesion. It seems likely that sICAM-1, at least in its monovalent form, has little potential as an inhibitor of in vivo immuneresponses. It is hypothesized that there are three primary mechanisms regulating cell adhesion mediated by ICAM-1 and LFA-1. The first mechanism is simply the amount of LFA-1 or ICAM-1 a given cell expresses on its surface, the second mechanism is the modulation of the affinity of the interaction between ICAM-1 and LFA-1, and the third mechanism is the regulation of the distribution of adhesion molecules on the cell surface. Each of these regulatory mechanisms is discussed with reference to the results presented in this thesis as well as the results in the literature.
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