Lipid rafts uncouple surface expression of transmembrane TNF-alpha from its cytotoxicity associated with ICAM-1 clustering in Raji cells.

Since transmembrane tumor necrosis factor-alpha (tmTNF-alpha) has been reported to have a palmitoylated site at Cys(-47), and therefore its functions may be linked to lipid raft membrane microdomains. The present study tested a hypothesis that lipid rafts may serve as a signaling platform to mediate the bioactivity of tmTNF-alpha. We found that destruction of lipid rafts with methyl-beta-cyclodextrin (MCD) in Raji cells almost completely blocked the cytotoxicity of tmTNF-alpha, as did an anti-TNF-alpha antibody. Although a proportion of tmTNF-alpha was colocated with lipid rafts, either the replacement of Cys at -47 by Ala, destructing its possible lipid rafts-attaching site or the displacement of its cytoplasmic domain by the C-terminal sequence (131-157) of caveolin-1, making all tmTNF-alpha target to lipid rafts, had no effect on tmTNF-alpha cytotoxicity. The data suggest that the cytotoxicity of tmTNF-alpha is not associated with its lipid rafts location. Unparallel to decreased cytotoxicity, moreover, MCD significantly increased tmTNF-alpha expression on the cell surface, and these increased tmTNF-alpha molecules were capable of binding to sTNFR1. To further explore the mechanism of lipid rafts-mediated cytotoxicity of tmTNF-alpha, we demonstrated that MCD led to a marked decrease in adhesion of Raji cells to T24 cells, which was due to dissociation of adhesion molecule ICAM-1 from lipid rafts. These results indicate that lipid rafts importantly participate in the cytotoxicity of tmTNF-alpha through ICAM-1 clustering and consequent enhancement of the cell-cell contact. The data suggest that lipid rafts are essential for the killing of tmTNF-alpha through the cell-cell contact mediated by ICAM-1. However, lipid rafts may limit exposure of tmTNF-alpha to the cell surface.