BIOLOGICALLY-ACTIVE FAS ANTIGEN AND ITS COGNATE LIGAND ARE EXPRESSED ON PLASMA MEMBRANE-DERIVED EXTRACELLULAR VESICLES

Exfoliation of plasma membrane components is a directed process that c onsumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are r eleased on exfoliated vesicles. We examine here whether the Fas recept or and its cognate ligand (FasL) are present on vesicles shed from hig h metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free me dium conditioned by CX-1 cells are increased by 1.8-fold and 1.8-fold, respectively, relative to that in medium conditioned by MIP-101 cells . Although vesicles shed from both cancer cell lines contain Fas antig en, the amount of Fas per vesicle and the percentage of vesicles conta ining Fas are increased for vesicles isolated from MIP-101 cells, rela tive to those from CX-1 cells, as determined by immunogold particle la beling and electron microscopy and by immunofluorescence microscopy an d flow cytometry. Results of metabolic labeling with S-35-methionine i ndicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 ce lls, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells . Although mRNA for soluble Fas receptor is detectable in both cell li nes, depletion of shed vesicles from serum-free medium by ultracentrif ugation removes all detectable biological activity. Fast is detected o n vesicles exfoliated from HUT 78 cells by immunoelectron microscopy a nd Western blot analysis. Fast-bearing vesicles induce apoptosis of Fa s expressing cancer cells at the same level as observed by treatment w ith monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellul ar vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from Fast-induced and anti fas-mediated apoptosis, indicating th at Fas present on shed vesicles is biologically active. We conclude th at the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mecha nism for long-range signal-directed apoptosis while maintaining Fas/Fa st on a membrane surface. (C) 1998 by The American Society of Hematolo gy.