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Role of lipid rafts in the regulation of LFA-1 function

University of British Columbia

Leukocyte function-associated molecule-1 (LFA-1) plays an essential role in cell-cell interactions in the immune system. LFA-1 may be activated by conformational changes and by clustering. In this study, the role of lipid rafts in the regulation of LFA-1 was investigated. Lipid rafts are plasma membrane microdomains enriched in cholesterol and ganglioside GM1, and they are thought to regulate functions of various cell surface receptors by mediating their clustering. Disruption of rafts on T cells by cholesterol depletion inhibited phorbol-ester-induced LFA-1 binding to its ligand ICAM-1, suggesting that rafts regulate LFA-1 functions. Isolation of rafts by non-ionic detergents followed by sucrose gradient centrifugation as well as sensitivity to cholesterol depletion and co-capping experiments suggested that the rafts of primary resting T cells are heterogeneous and that LFA-1 associates with a subset of lipid rafts that are rich in cholesterol but poor in GM1. To further elucidate the role of lipid rafts in LFA-1 function, the formation of the immunological synapse on cytotoxic T lymphocytes (CTL) was examined. Disruption of rafts by cholesterol depletion inhibited CTL cytotoxicity. However, the LFA-1 on CTL was constitutively active and was not inhibited by cholesterol depletion. Instead, rafts seemed to mediate the association between LFA-1 and the CD3/T cell receptor (TCR) complex on CTL, but not on resting T cells. Deconvolution and confocal microscopy as well as 3-D image reconstruction showed that LFA-1, CD3/TCR and GM1 co-distribute in the periphery of the CTL immunological synapse in an antigen-independent manner while cholesterol was distributed more widely. These results suggest that the binding of LFA-1 on CTL to ICAM-1 on target cells initiates the formation of the synapse on CTL and recruits the CD3/TCR complex to the synapse through their co-distribution in rafts. The role of LFA-1 in the reorganization of the cytoskeleton following formation of the immunological synapse was also analyzed. LFA-1 ligation induced antigen-independent F-actin accumulation in the synapse, whereas tyrosine phosphorylation in the synapse and translocation of the microtubule organizing center required recognition of specific antigen by the TCR. Thus, LFA-1-mediated signalling is important for the activation of the actin cytoskeleton, but not sufficient for microtubules reorganization in CTL.

Thesis/Dissertation

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2009-12-01

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http://hdl.handle.net/2429/16062

Experimental Medicine

University of British Columbia

UBCV

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