CELL-SURFACE DISTRIBUTION OF HIGH-AVIDITY LFA-1 DETECTED BY SOLUBLE ICAM-1-COATED MICROSPHERES

Murine recombinant soluble ICAM-1 (sICAM-1) was immobilized on polysty rene microspheres. The binding of sICAM-1-coated microspheres to splen ic T cells required previous activation of the cells and was inhibited by antibody to LFA-1 (CD11a/CD18), indicating that sICAM-1-coated mic rospheres bind exclusively to high-avidity LFA-1. The cytoskeleton inh ibitor cytochalasin B did not inhibit the binding oi sICAM-1-coated mi crospheres to PMA-activated splenic T cells, whereas their adhesion to sICAM-1 immobilized on microtiter wells was almost completely inhibit ed. The murine T hybridoma T28 cells on activation with PMA also bound sICAM-1-coated microspheres, and the binding sites on the cell surfac e 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 e ven distribution of sICAM-1 binding sites. To further investigate the distribution of high-avidity LFA-1, murine fibroblast L cells expressi ng LFA-1 were generated by gene transfer. The transfected L cells cons titutively expressed high-avidity LFA-1 and bound sICAM-1-coated micro spheres without previous activation. Interestingly, the binding sites seemed highly localized on most cells. In contrast, the binding sites for anti-LFA-l 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 cel ls. These results show that 1) sICAM-1-coated microspheres represent a useful tool in identifying high-avidity LFA-1, 2) the binding of sICA M-1-coated microspheres to high-avidity LFA-1 does not require an inta ct 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 s eems highly localized on some cells,