Association of FcγRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (Fc gamma Rs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the Fc gammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of Fc gamma Rs resulted in a marked increase in the tyrosine phosphorylation of Fc gamma RIIa, p58(lyn), and p120(c-cbl). which was inhibited by a specific inhibitor of Ste family protein tyrosine kinases. After crosslinking, Fc gamma Rs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of Fc gamma Rs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta -cyclodextrin, the cross-linking did not induce the association of Fc gamma Rs as well as p120(c-cbl) with DRMs. In addition, although the physical association between Fc gamma RIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta -cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via Fc gamma RII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of Fc gamma RII to lipid rafts is important for the activation of Fc gamma RII-associated Src. family protein tyrosine kinases to initiate the tyrosine phosphorylation. cascade leading to superoxide generation.