Pyszniak A M, Welder C A, Takei F
Department of Microbiology, University of British Columbia, Vancouver, Canada.
J Immunol. 1994 Jun 1;152(11):5241-9.
Murine recombinant soluble ICAM-1 (sICAM-1) was immobilized on polystyrene microspheres. The binding of sICAM-1-coated microspheres to splenic T cells required previous activation of the cells and was inhibited by antibody to LFA-1 (CD11a/CD18), indicating that sICAM-1-coated microspheres bind exclusively to high-avidity LFA-1. The cytoskeleton inhibitor cytochalasin B did not inhibit the binding of sICAM-1-coated microspheres to PMA-activated splenic T cells, whereas their adhesion to sICAM-1 immobilized on microtiter wells was almost completely inhibited. The murine T hybridoma T28 cells on activation with PMA also bound sICAM-1-coated microspheres, and the binding sites on the cell surface seemed localized on some of the cells, whereas fluorescence staining showed an even distribution of LFA-1 on the cell surface. In contrast, the murine B cell line A20A8 and monocytic line P388 showed a more even distribution of sICAM-1 binding sites. To further investigate the distribution of high-avidity LFA-1, murine fibroblast L cells expressing LFA-1 were generated by gene transfer. The transfected L cells constitutively expressed high-avidity LFA-1 and bound sICAM-1-coated microspheres without previous activation. Interestingly, the binding sites seemed highly localized on most cells. In contrast, the binding sites for anti-LFA-1 Ab-coated microspheres were randomly distributed on the transfected L cells. Furthermore, fluorescence staining also revealed a uniform punctate distribution of LFA-1 on the surfaces of these cells. These results show that 1) sICAM-1-coated microspheres represent a useful tool in identifying high-avidity LFA-1, 2) the binding of sICAM-1-coated microspheres to high-avidity LFA-1 does not require an intact cytoskeleton, and 3) the cell surface distribution of high-avidity LFA-1 can be different from that of LFA-1 in general, and the former seems highly localized on some cells.
将小鼠重组可溶性细胞间黏附分子-1（sICAM-1）固定在聚苯乙烯微球上。sICAM-1包被的微球与脾T细胞的结合需要细胞预先激活，并且被抗淋巴细胞功能相关抗原-1（LFA-1，CD11a/CD18）抗体所抑制，这表明sICAM-1包被的微球仅与高亲和力的LFA-1结合。细胞骨架抑制剂细胞松弛素B并不抑制sICAM-1包被的微球与佛波酯（PMA）激活的脾T细胞的结合，然而它们对固定在微量滴定孔上的sICAM-1的黏附几乎被完全抑制。用PMA激活的小鼠T杂交瘤T28细胞也能结合sICAM-1包被的微球，并且细胞表面的结合位点似乎定位于部分细胞上，而荧光染色显示LFA-1在细胞表面呈均匀分布。相反，小鼠B细胞系A20A8和单核细胞系P388显示sICAM-1结合位点分布更为均匀。为了进一步研究高亲和力LFA-1的分布，通过基因转移构建了表达LFA-1的小鼠成纤维细胞L细胞。转染的L细胞组成性表达高亲和力LFA-1，并且无需预先激活就能结合sICAM-1包被的微球。有趣的是，结合位点似乎在大多数细胞上高度定位。相反，抗LFA-1抗体包被的微球的结合位点在转染的L细胞上随机分布。此外，荧光染色还显示LFA-1在这些细胞表面呈均匀的点状分布。这些结果表明：1）sICAM-1包被的微球是鉴定高亲和力LFA-1的有用工具；2）sICAM-1包被的微球与高亲和力LFA-1的结合不需要完整的细胞骨架；3）高亲和力LFA-1的细胞表面分布可能与一般的LFA-1不同，前者似乎在某些细胞上高度定位。
