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The Journal of Immunology, 2001, 167: 5814-5823.
Copyright © 2001 by The American Association of Immunologists

Association of Fc{gamma}RII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation1

Osamu Katsumata*, Miki Hara-Yokoyama2,{dagger}, Catherine Sautès-Fridman{ddagger}, Yasuko Nagatsuka§, Toshiaki Katada, Yoshio Hirabayashi||, Kazufumi Shimizu§, Junko Fujita-Yoshigaki*, Hiroshi Sugiya* and Shunsuke Furuyama*

* Department of Physiology, Nihon University School of Dentistry, Matsudo, Japan; {dagger} Department of Hard Tissue Engineering, Biochemistry, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; {ddagger} Centre de Recherches Biomédicals des Cordeliers, Paris, France; § Department of Microbiology, Nihon University School of Medicine, Tokyo, Japan; Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and || Brain Science Institute, Institute of Physical and Chemical Research, Wako, Japan

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{gamma}R-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, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, Fc{gamma}Rs 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 Fc{gamma}Rs as well as p120c-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 p120c-cbl with DRMs. In addition, although the physical association between Fc{gamma}RIIa 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-{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.




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