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Published online before print March 19, 2007

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(Journal of Leukocyte Biology. 2007;81:1414-1421.)
© 2007 by Society for Leukocyte Biology

Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains

Saotomo Itoh^*, Chie Susuki^*, Kana Takeshita^*, Kisaburo Nagata and Tsutomu Tsuji^*,1

^* Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan; and
Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, Japan

¹ Correspondence: Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: tsuji{at}hoshi.ac.jp

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte interactions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid microdomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8-treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-perturbing agents such as methyl-ß-cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL-8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with antibodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin.

Key Words: adhesion molecule • platelet-neutrophil microaggregates • cell polarization • reactive oxygen species

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