Regulation of cell surface APO‐1/Fas (CD95) ligand expression by metalloproteases

Abstract

APO‐1/Fas (CD95) ligand (APO‐1L) induces apoptosis in sensitive target cells. Activation‐induced T cell death and Ca 2+‐independent cytotoxicity in perforin knockout mice are mediated by APO‐1L. To define whether APO‐1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti‐APO‐1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO‐1L of approximately 42 and 40kDa. 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/ionomycin induced a significant increase in cell surface APO‐1L only in the presence of metalloprotease inhibitors. Zn 2+, but not Ca 2+, prevented the increase in surface APO‐1L observed in the presence of 1,10‐phenanthroline. Blocking of other classes of proteases (serine‐ and acid‐proteases, chymotrypsin) had no effect. Increased expression of surface APO‐1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO‐1L expression. These results suggest that cell surface expression of human APO‐1L is regulated by Zn 2+‐dependent metalloproteases. Cleavage of surface APO‐1L may act as a regulatory mechanism to prevent accumulation of the membrane‐bound form and may cause systemic effects of the APO‐1L.