GENERATION OF MUTANT-CELLS WITH CONSTITUTIVELY ACTIVE BETA(2)-INTEGRIN LFA-1

To elucidate the molecular mechanisms behind regulation of integrin ad hesiveness, we have developed a system for the generation of mutant ce ll lines where the adhesive functions of specific molecules were modul ated. The human lymphatic leukemia cell line HPB-ALL was chemically mu tagenized and rare cells expressing constitutively active beta(2)-inte grin LFA-1 (alpha(1) beta(2), CD11a/CD18) were selected from the pool of mutagenized cells, by repeated cycles of binding to immobilized ICA M-1. The selected population showed 20-fold more binding to ICAM-1 com pared to the starting population. A sub-clone of the selected populati on, termed HAP4, was further analyzed. In contrast to the original cel l line, HAP4 had constitutively active LFA-1 and adherence to ICAM-1 w as insensitive to inhibition by okadaic acid and cytochalasin B or to inhibition of ATP synthesis. The activation of LFA-1 appeared specific since the cells did not show a general increase in adhesiveness. No m ajor aberration in cell surface carbohydrate expression was found and the expression of cell surface molecules including CD43, CD4, CD8, and MHC class I and II was normal. However, apparent cell surface express ion of integrin beta(1) chain was lost and alpha(4) chain was greatly diminished. Our study demonstrates the feasibility of generating cells with mutated adhesive functions and the characterized cells will serv e as tools to further dissect the mechanisms underlying integrin regul ation.