Apoptosis-inducing Membrane Vesicles

Abstract

CD95 (Fas)1 is a type I transmembrane protein expressed by a variety of nucleated cells (1). The physiological ligand for Fas (FasL) is a type II transmembrane protein expressed by activated T cells and non-T cells under a variety of conditions (2-11). Cross-linking of Fas induces cells to undergo apoptosis (12)(13)(14). This apoptosis pathway has been implicated in immune response regulation, self-tolerance, graft rejection, tumor escape of immune surveillance, and maintenance of the immune privileged sites (2-6, 15-28).Regulation of FasL expression has been demonstrated at the transcriptional and post-transcriptional levels (4 -6, 29 -32).Recent studies have suggested that the level of cell surface FasL is regulated by a mechanism involving matrix metalloproteinase (MMP) cleavage that releases from cells soluble FasL (sFasL) lacking the transmembrane and cytoplasmic domains (29 -32). Compared with cell-associated FasL, sFasL is a relatively poor mediator of cytotoxicity. Indeed, under certain conditions, sFasL can actually inhibit the cytotoxicity of FasLexpressing cells (30, 31). Thus, sFasL release effectively downregulates the function of cell-associated FasL. Here, we demonstrate that there is a second mechanism responsible for FasL turnover. This mechanism involves the release of cell surface FasL in the form of vesicles, which contain full-length FasL and are bioactive. Although the presence of FasL-bearing vesicles was implicated in previous studies (33, 34), the FasL expression level was so low that a quantitative study to determine its contribution to cell membrane FasL turnover was difficult. We have generated retroviral packaging cell lines that produce large amounts of FasL-expressing vesicles; however, it is not clear whether the retroviral packaging process has influenced the production of FasL membrane vesicles (35-37).To determine whether normal FasL-expressing cells produce apoptosis-inducing vesicles, we generated FasL-expressing 3T3 cells that do not produce virus. We generated T cells that produce high levels of membrane FasL upon activation. In addition, we studied normal T cells for cell membrane FasL turnover upon activation. We demonstrated that these cells release of FasL-bearing vesicles capable of inducing apoptosis in target cells. Our quantitative analysis indicated that release of vesicles contributes to the turnover of cell-associated FasL, but the extent of contribution varies in different cell lines examined. Interestingly, the apoptosis-inducing vesicles display unique properties. In contrast to sFasL, FasL-bearing vesicles fully retained the target range of the FasL-expressing cells. However, there is a reduction of specific activity in comparison with cell-associated FasL. These observations suggest that release of vesicles is a physiologically significant process regarding both the turnover of cell-associated FasL and the impact on FasL function of the FasL-expressing cells. EXPERIMENTAL PROCEDURESProduction of FasL-expressing Cell Lines-We obtained an hfasl cDNA construct ...

Diacylglycerol Kinase α Regulates the Secretion of Lethal Exosomes Bearing Fas Ligand during Activation-induced Cell Death of T Lymphocytes

“…It is remarkable that high order multimerization of the Fas death receptor has been shown to be a crucial event in apoptosis induction (28). Microvesicle-bound mFasL would guarantee cytotoxic potential, because it retains its multimerization ability and cross-linking efficiency and fully preserves the target range of the mFasL expressed on the cell surface (43). Therefore, mFasL on microvesicles provides evident advantages for apoptosis induction over cell surface mFasL and the secretion of the soluble protein, given the higher efficiency in triggering cell death of mFasL when compared with soluble FasL, and the fact that processing of mFasL to its soluble form leads to down-regulation of the apoptotic activity (31).…”

Anti-CD95-induced Lethality Requires Radioresistant FcγRII+ Cells

“…Target cells (2 ϫ 10 4 ), labeled with Na 2 51 CrO 4 (PerkinElmer Life Sciences), were cultured with various doses of Jo2 (BD Biosciences) or FasL vesicle preparation (FasL VP) (13,14) in 0.2 ml in individual wells of a 96-well plate. Supernatants were removed at 5 h after culture, and radioactivity (counts/min) was determined.…”

A Novel Signaling Mechanism for Soluble CD95 Ligand

“…We have also generated a tumor T cell line (pIL-2-hFasL-EL-4) in which the hfasl gene is put under the control of a 1.9-kb IL-2 promoter (28). The production and characterization of these cell lines have been described (28,34).…”

“…Western blot analysis demonstrated that FasL VP is composed of full-length FasL of 40,000 daltons and sFasL contains only sFasL of 26,000 -27,000 daltons (35). FasL concentrations in various preparations were determined using a capture ELISA kit (Oncogene, Boston) as described previously (28).…”

“…We have shown recently (28) that cells transfected with human FasL gene produce not only the expected transmembrane FasL and sFasL but also microvesicles (FasL VP) that express the transmembrane protein. Cleavage of the membrane FasL at position 130 yields a 152-amino acid polypeptide with a molecular mass of 26,000 -27,000 daltons (24,30,31).…”