REGULATION OF CELL-SURFACE APO-1 FAS (CD95) LIGAND EXPRESSION BY METALLOPROTEASES/

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca2+-independent cytotoxic ity in perforin knockout mice are mediated by APO-1L. To define whethe r APO-1L is expressed on the surface of activated T cells and to inves tigate the mechanisms leading to the release of a soluble form, we dev eloped rabbit anti-APO-IL antibodies (Ab). The purified rabbit Ab dete cted the mature forms of the human and mouse APO-1L of approximately 4 2 and 40 kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a moleculas mass of 26 kDa . Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ion omycin induced a significant increase in cell surface APO-1L only in t he presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevent ed the increase in surface APO-1L observed in the presence of 1,10-phe nanthroline. Blocking of other classes of proteases (serine- and acid- proteases, chymotrypsin) had no effect. Increased expression of surfac e APO-1L by metalloprotease inhibitors was not dependent on T cell act ivation, as the metalloprotease inhibitors also modulated the low leve l of constitutive APO-1L expression. These results suggest that cell s urface expression of human APO-1L is regulated by Zn2+-dependent metal loproteases. Cleavage of surface APO-1L may act as a regulatory mechan ism to prevent accumulation of the membrane-bound form and may cause s ystemic effects of the APO-1L.