Association of FcγRII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation

Abstract

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcγRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcγR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcγRs resulted in a marked increase in the tyrosine phosphorylation of FcγRIIa, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcγRs and tyrosine-phosphorylated proteins including p120c-cbl were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcγRs as well as p120c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-β-cyclodextrin, the cross-linking did not induce the association of FcγRs as well as p120c-cbl with DRMs. In addition, although the physical association between FcγRIIa and p58lyn 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-β-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcγRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcγRII to lipid rafts is important for the activation of FcγRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

“…Disruption of the integrity of the DRMs by cholesterol-affecting agents, such as M␤CD, is known to alter intracellular signaling events and functional responses in neutrophils. M␤CD interferes with the membrane-dependent initiation of tyrosine phosphorylation (25), inhibits the phosphorylation of ERK1/2 induced by IL-8 (52), increases the phosphorylation of p38 MAPK, and inhibits NADPH oxidase activity. These findings thus suggest that lipid microdomains may mediate the efficiency of the oxidase by coupling NADPH oxidase components to certain intracellular signaling pathways in neutrophils.…”

“…The involvement of lipid rafts in regulating neutrophil functions is suggested by the fact that the neutrophil oxidative burst can be severely curtailed by depleting the level of cholesterol in the plasma membranes using methyl-␤-cyclodextrin (M␤CD) (23,25). It remains to be determined whether lipid raft microdomains play a regulatory role in the IL-8-induced priming of the oxidative burst in human neutrophils.…”

Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells

“…DIM fractions were prepared as described (26,27) with slight modifications. Untreated and rGL-7-treated HL60 cells (10 8 cells) were lysed with 1 ml of lysis buffer consisting of 1% Triton X-100 and TN buffer (25 mM Tris-HCl, pH 7.5͞150 mM NaCl͞0.1 mM phenylmethylsulfonyl fluoride͞1 mM NaHSO 3 ͞1 mM benzamidene͞2 mM CaCl 2 ͞5 mM MgCl 2 ͞ 0.15 mM spelmin͞0.3 mM spelmidine).…”

Cholesterol-Binding Peptides and Phagocytosis

“…Syk activation in turn initiates a signaling cascade, including activation of phosphatidylinositol 3-kinase (PI 3-kinase) and of the small GTPases Rac and Cdc42, which coordinate actin remodeling (Henry et al, 2004). As was reported (Kwiatkowska & Sobota, 2001;Katsumata et al, 2001;Kono et al, 2002;Kwiatkowska et al, 2003), one of the earliest signal events after the cross-binding of Fcγ receptors is lateral raft assembly that occurs before the activation of kinases of the Src family and independent of their activity. To trigger the lateral assembly of rafts, sufficient was the expression of ligand-binding monomer FcγR, without signal subunits carrying the activating fragment with tyrosine.…”