Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity

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Abstract
Many surface receptors and signaling molecules are thought to associate with unique membrane microdomains termed lipid rafts. We examined the involvement of lipid rafts in the activation of leukocyte function–associated antigen-1 (LFA-1). Depletion or sequestration of cholesterol with methyl-β-cyclodextrin (MCD) or filipin, respectively, strongly inhibited LFA-1–mediated adhesion of T-cell lines and primary T cells. This inhibition was reversed by cholesterol reconstitution. LFA-1 on T-cell lines was detected in cold Triton X-100–insoluble lipid rafts, which were disrupted by MCD or filipin treatment. However, no LFA-1 on primary T cells was detected in lipid rafts isolated by the same procedures, and these rafts were resistant to cholesterol depletion or sequestration. Association of LFA-1 with lipid rafts of primary T cells could be detected only when they were isolated with another nonionic detergent, Brij 35. Upon treatment with MCD, LFA-1 in Brij 35–insoluble lipid rafts partially shifted to nonraft fractions. T-cell lines were found to have a high level of cholesterol and a low level of ganglioside GM1, a common marker for lipid rafts, whereas primary T cells have a much lower level of cholesterol and a very high amount of GM1. Cross-linking of LFA-1 on primary T cells induced cocapping of cholesterol but not GM1. These results suggest that lipid rafts of T cells are heterogenous, and LFA-1 associates with a subset of lipid rafts containing a high level of cholesterol. This association seems to regulate LFA-1 functions, possibly by facilitating LFA-1 clustering. (Blood. 2003;102: 215-222)