Association of Fc gamma RII with low-density detergent-resistant membranesis important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation

IgG immune complexes trigger Immoral immune responses by cross-linking of F cRs for IgG (Fc gamma Rs). In the present study, we investigated role of li pid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the F c gammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, c ross-linking of Fc gamma Rs resulted in a marked increase in the tyrosine p hosphorylation of Fc gamma RIIa, p58(lyn), and p120(c-cbl). which was inhib ited by a specific inhibitor of Ste family protein tyrosine kinases. After crosslinking, Fc gamma Rs and tyrosine-phosphorylated proteins including p1 20(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The ass ociation 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 neu trophils, superoxide generation induced by opsonized zymosan or chemoattrac tant fMLP was not affected or increased, respectively, after the methyl-bet a -cyclodextrin treatment, but the superoxide generation induced by the ins oluble immune complex via Fc gamma RII was markedly reduced. Accordingly, w e 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.