Apoptosis-inducing Membrane Vesicles
2001; Elsevier BV; Volume: 276; Issue: 43 Linguagem: Inglês
10.1074/jbc.m107005200
ISSN1083-351X
AutoresSatoshi Jodo, Sheng Xiao, Andreas M. Hohlbaum, David Strehlow, Ann Marshak‐Rothstein, Shyr‐Te Ju,
Tópico(s)RNA Interference and Gene Delivery
ResumoThe CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novoprotein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL. The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novoprotein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL. CD95 Fas ligand soluble FasL recombinant soluble FasL ionomycin cycloheximide matrix metalloproteinase phorbol 12-myristate 13-acetate peripheral blood T cells vesicle preparation kilobase(s) interleukin-2 PMA plus ionomycin enzyme-linked immunosorbent assay monoclonal antibody lymphocyte function-associated antigen-1 intercellular adhesion molecules CD95 (Fas)1 is a type I transmembrane protein expressed by a variety of nucleated cells (1Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2701) Google Scholar). 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 (2Takahashi T. Tanaka M. Brannan C.I. Jenkins N.A. Copeland N.G. Suda T. Nagata S. Cell. 1994; 76: 969-976Abstract Full Text PDF PubMed Scopus (1464) Google Scholar, 3Lynch D.H. Watson M.L. Alderson M.L. Baum P.R. Miller R.E. Tough T. Gibson M. Davis-Smith T. 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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 (2Takahashi T. Tanaka M. Brannan C.I. Jenkins N.A. Copeland N.G. Suda T. Nagata S. Cell. 1994; 76: 969-976Abstract Full Text PDF PubMed Scopus (1464) Google Scholar, 3Lynch D.H. Watson M.L. Alderson M.L. Baum P.R. Miller R.E. Tough T. Gibson M. Davis-Smith T. Smith C.A. Hunter K. Bhat D. Din W. Goodwin R.G. Seldin M.F. Immunity. 1994; 1: 131-136Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 4Dhein J. Walxzak H. Baumler C. Debatin K.-M. Krammer P.H. Nature. 1995; 373: 438-441Crossref PubMed Scopus (1605) Google Scholar, 5Brunner T. Mogil R.J. LaFace D. Yoo N.J. Mahoubi A. Wcheverri F. Martin S.J. Force W.R. Lynch D.H. Ware C.F. Green D.R. Nature. 1995; 373: 441-444Crossref PubMed Scopus (1269) Google Scholar, 6Ju S.-T. Panka D.J. Cui H. Ettinger R. El-Khatib M. Sherr D.H. Stanger B.Z. 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Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar, 32Schneider P. Holler N. Bodmer J.-L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (696) Google Scholar). 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 (29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar, 30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar, 32Schneider P. Holler N. Bodmer J.-L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (696) Google Scholar). Compared with cell-associated FasL, sFasL is a relatively poor mediator of cytotoxicity. Indeed, under certain conditions, sFasL can actually inhibit the cytotoxicity of FasL-expressing cells (30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar). Thus, sFasL release effectively down-regulates 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 (33Albanese J. Meterissian S. Kontogiannea M. Dubreuil C. Hand A. Sorba S. Dainiak N. Blood. 1998; 91: 3862-3874Crossref PubMed Google Scholar, 34Martinez-Lorenzo M.J. Anel A. Gamen S. Monleon I. Lasierra P. Larrad L. Pineiro A. Alava M.A. Naval J. J. Immunol. 1999; 163: 1274-1281PubMed Google Scholar), 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 (35Jodo S. Hohlbaum H. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 36Strehlow D. Jodo S. Ju S.-T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4209-4214Crossref PubMed Scopus (16) Google Scholar, 37Jodo S. Strehlow D. Ju S.-T. J. Immunol. 2000; 164: 5062-5069Crossref PubMed Scopus (16) Google Scholar).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.DISCUSSIONPrevious studies have demonstrated that a major pathway of FasL turnover is the release of sFasL resulting from MMP digestion. Here, we showed that FasL-expressing cells also release cell membrane FasL as vesicles capable of inducing apoptosis of target cells. The biological significance of releasing cell membrane FasL as vesicles is unclear. This process could down-regulate membrane expression of FasL. On the other hand, FasL presented by vesicles could be functional and could have an important biological function (33Albanese J. Meterissian S. Kontogiannea M. Dubreuil C. Hand A. Sorba S. Dainiak N. Blood. 1998; 91: 3862-3874Crossref PubMed Google Scholar, 34Martinez-Lorenzo M.J. Anel A. Gamen S. Monleon I. Lasierra P. Larrad L. Pineiro A. Alava M.A. Naval J. J. Immunol. 1999; 163: 1274-1281PubMed Google Scholar, 35Jodo S. Hohlbaum H. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 36Strehlow D. Jodo S. Ju S.-T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4209-4214Crossref PubMed Scopus (16) Google Scholar, 37Jodo S. Strehlow D. Ju S.-T. J. Immunol. 2000; 164: 5062-5069Crossref PubMed Scopus (16) Google Scholar). Using gene-transferred hFasL-PA317 and hFasL-3T3 cell lines and conducting quantitative analyses of FasL protein expression levels, we have shown that FasL is released from cells in the form of vesicles, and this process is as effective as the MMP-mediated release of sFasL. In contrast, FasL-expressing T cells release lower levels of FasL-containing vesicles and thus contribute modestly for T cell FasL turnover. Unlike sFasL, which is unable to kill many of the targets sensitive to FasL-expressing cells, vesicle-associated FasL retained the ability to kill these targets. These observations suggest that the rapid release of FasL-bearing vesicles may serve a more complex role than just simply down regulating the cell-associated FasL as is the case of sFasL release.Our analyses based on quantitative hFasL-specific ELISA demonstrated that release of FasL-bearing vesicles is as effective a turnover mechanism as sFasL release, because large amounts of FasL accumulated as vesicles in the supernatants of hFasL-PA317 and hFasL-3T3 cells. The FasL accumulated in vesicles was comparable to or slightly more than the amounts of sFasL released. In contrast to hFasL-PA317 and hFasL-3T3 cell lines, the P/I-activated Jurkat, PBT, and pIL-2-hFasL-EL-4 T cells expressed variable levels of FasL from very low to moderate. Taking this factor into consideration, the FasL levels of vesicles generated by these activated T cells ranged from 23% to 71% of the cell-associated FasL (Table I), whereas the sFasL levels were significantly higher. This indicates that the release of FasL-bearing vesicles contributes modestly to the FasL turnover in activated T cells. The modest accumulation of FasL-bearing vesicles is not likely due to an overactive MMP that degrades the full-length FasL of vesicles, because the same high level of vesicle-associated FasL was obtained when hFasL-3T3 cells were cultured with activated pIL-2-hFasL-EL-4 T cells (data not shown). In an earlier study, sFasL was shown to be the major species detected in culture supernatants of T lymphoma cells that overexpress hFasL (29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar). Our results obtained from three additional and different FasL-expressing T cell populations not only support the previous observation but also indicate that the release of vesicles contributes modestly to T cell FasL turnover. Whether different types of cells utilize different mechanisms,i.e. membrane shedding or secretion of exosomes (50Raposo G. Nijman H.W. Stoorvogel W. Leijendekker R. Harding C.V. Melief C.J.M. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2383) Google Scholar), for the release of FasL-containing vesicles remains to be established.T cell-mediated cytotoxicity depends on cell interaction forces. Antibodies against LFA-1 and ICAM are effective inhibitors of FasL-mediated cytotoxicity of T cells (45Wang J. Lenardo M.J. J. Exp. Med. 1997; 186: 1171-1176Crossref PubMed Scopus (43) Google Scholar, 46Kojima H. Eshima K. Takayama H. Sitkovsky M.V. J. Immunol. 1997; 159: 2728-2734PubMed Google Scholar). In the present study we showed that the FasL-mediated cytotoxicity of the P/I-activated pIL-2-hFasL-EL-4 cells but not hFasL-3T3 cells depended on LFA-1/ICAM interaction. Because of this cell interaction, the former cells are more potent than the latter cells even though their FasL is only one-sixth of the latter cells. In contrast to the P/I-activated pIL-2-hFasL-EL-4 cells, the cytotoxicity of vesicles prepared from the same cells was not inhibited by anti-LFA-1 mAb. Thus, release of FasL-containing vesicles could effectively down-regulate FasL function of T cells. Although we have clearly shown that vesicles contain FasL, whether or not LFA-1 could be released in the form of vesicles and whether a vesicle co-expresses both FasL and LFA-1 have not been firmly established.One reason that sFasL release has been considered a down-regulatory mechanism for cell-associated FasL is that sFasL is unable to kill many targets that are sensitive to the FasL-expressing cells. In addition, affinity-purified and concentrated sFasL was shown to inhibit killing mediated by cell-associated FasL (30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar). The sFasL prepared from hFasL-3T3 cells also inhibited killing mediated by FasL-bearing vesicles or FasL-expressing cells. This loss and gain of function was not observed for FasL-bearing vesicles, which do not inhibit the killing mediated by FasL-expressing cells (data not shown). Moreover, vesicle-associated FasL retained the ability to kill a panel of targets that are sensitive to the FasL-expressing cells from which the vesicles were derived. This suggests that release of FasL-bearing vesicles may have a different biological function than the MMP-mediated release of sFasL. For example, certain tumor cells (25Hahne M. Rimoldi D. Schroter M. Romero P. Schreiter M. French L.E. Schneider P. Bornand T. Fontana A. Lienard D. Cerottini J.-C. Tschopp J. Science. 1996; 274: 1363-1366Crossref PubMed Scopus (1191) Google Scholar, 26O'Connell J. O'Sullivan G.C. Collin J.K. Shanahan F. J. Exp. Med. 1996; 184: 1075-1082Crossref PubMed Scopus (867) Google Scholar, 27Bennett M.W. O'Connell J. O'Sullivan G.C. Chrady C. Roche D. Collins J.K. Shanahan F. J. Immunol. 1998; 160: 5669-5675PubMed Google Scholar), cells in the immune privileged sites (24Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1097) Google Scholar, 28Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar), and cells outside the immune system (51Galle P.R. Hofmann W.J. Walczak H. Schaller H. Otto G. Stremmel W. Krammer P.H. Runkel L. J. Exp. Med. 1995; 182: 1223-1230Crossref PubMed Scopus (675) Google Scholar, 52Strand S. Hofmann W.J. Grambihler A. Hug H. Volkmann M. Otto G. Wesch H. Mariani S.M. Hack V. Stremmel W. Krammer P.H. Galle P.E. Nat. Med. 1998; 4: 588-593Crossref PubMed Scopus (227) Google Scholar, 53Moller P. Walczak H. Reidl S. Strater J. Krammer P.H. Am. J. Pathol. 1996; 149: 9-13PubMed Google Scholar) constitutively express FasL under specific conditions. Their FasL is likely maintained at a steady level despite continuous release of vesicles that bear FasL. The accumulated FasL-bearing vesicles could provide additional killing power and could function beyond the local area controlled by FasL-expressing cells. Active release of FasL-bearing vesicles may help FasL-expressing cells mediate its functions rather than the inhibition as observed with sFasL. FasL-bearing vesicles may have long-lasting apoptosis-inducing power, because their expression is no longer dependent on de novoprotein synthesis. A recent study suggests sFasL bind to extracellular matrix and display an enhanced cytotoxicity against Jurkat target (54Aoki K. Kurooka M. Chen J.-J. Petryniak J. Nabel E.G. Nabel G.I. Nat. Immunol. 2001; 2: 333-337Crossref PubMed Scopus (80) Google Scholar). However, the loss of cytotoxicity against various targets associated with sFasL release would significantly reduce the role of sFasL as a general cytotoxic mediator in vivo. Given that multiple roles of FasL in the immune system have been demonstrated, the present study indicates that release of FasL-bearing vesicles, like sFasL release, is an important factor to consider because of its distinct influence on the expression and function of cell-associated FasL. In this respect, our ongoing study has shown that FasL-bearing vesicles display two bioactivities in vivo, i.e. they act as a chemotactic factor for neutrophils when injected intraperitoneally, and they induce lethal fulminant hepatitis when injected intravenously. 2S. Jodo, and S.-T. Ju, unpublished observations. CD95 (Fas)1 is a type I transmembrane protein expressed by a variety of nucleated cells (1Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2701) Google Scholar). 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 (2Takahashi T. Tanaka M. Brannan C.I. Jenkins N.A. Copeland N.G. Suda T. Nagata S. Cell. 1994; 76: 969-976Abstract Full Text PDF PubMed Scopus (1464) Google Scholar, 3Lynch D.H. Watson M.L. Alderson M.L. Baum P.R. Miller R.E. Tough T. Gibson M. Davis-Smith T. Smith C.A. Hunter K. Bhat D. Din W. Goodwin R.G. Seldin M.F. Immunity. 1994; 1: 131-136Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 4Dhein J. Walxzak H. Baumler C. Debatin K.-M. Krammer P.H. Nature. 1995; 373: 438-441Crossref PubMed Scopus (1605) Google Scholar, 5Brunner T. Mogil R.J. 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Nakajima H. Henkart P.A. Ferguson T.A. Green D.R. Immunity. 1998; 9: 711-720Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Cross-linking of Fas induces cells to undergo apoptosis (12Trauth B.C. Klas C. Peters A.M. Matzku S. Moller P. Falk W. Debatin K.-M. Krammer P.H. Science. 1989; 245: 301-305Crossref PubMed Scopus (1655) Google Scholar, 13Yonehara S. Ishii A. Yonehara M. J. Exp. Med. 1989; 169: 1747-1756Crossref PubMed Scopus (1422) Google Scholar, 14Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1804) Google Scholar). 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 (2Takahashi T. Tanaka M. Brannan C.I. Jenkins N.A. Copeland N.G. Suda T. Nagata S. 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Regulation of FasL expression has been demonstrated at the transcriptional and post-transcriptional levels (4Dhein J. Walxzak H. Baumler C. Debatin K.-M. Krammer P.H. Nature. 1995; 373: 438-441Crossref PubMed Scopus (1605) Google Scholar, 5Brunner T. Mogil R.J. LaFace D. Yoo N.J. Mahoubi A. Wcheverri F. Martin S.J. Force W.R. Lynch D.H. Ware C.F. Green D.R. Nature. 1995; 373: 441-444Crossref PubMed Scopus (1269) Google Scholar, 6Ju S.-T. Panka D.J. Cui H. Ettinger R. El-Khatib M. Sherr D.H. Stanger B.Z. Marshak-Rothstein A. Nature. 1995; 373: 444-448Crossref PubMed Scopus (1451) Google Scholar, 29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar, 30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar, 32Schneider P. Holler N. Bodmer J.-L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (696) Google Scholar). 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 (29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar, 30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar, 32Schneider P. Holler N. Bodmer J.-L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (696) Google Scholar). Compared with cell-associated FasL, sFasL is a relatively poor mediator of cytotoxicity. Indeed, under certain conditions, sFasL can actually inhibit the cytotoxicity of FasL-expressing cells (30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar). Thus, sFasL release effectively down-regulates 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 (33Albanese J. Meterissian S. Kontogiannea M. Dubreuil C. Hand A. Sorba S. Dainiak N. Blood. 1998; 91: 3862-3874Crossref PubMed Google Scholar, 34Martinez-Lorenzo M.J. Anel A. Gamen S. Monleon I. Lasierra P. Larrad L. Pineiro A. Alava M.A. Naval J. J. Immunol. 1999; 163: 1274-1281PubMed Google Scholar), 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 (35Jodo S. Hohlbaum H. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 36Strehlow D. Jodo S. Ju S.-T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4209-4214Crossref PubMed Scopus (16) Google Scholar, 37Jodo S. Strehlow D. Ju S.-T. J. Immunol. 2000; 164: 5062-5069Crossref PubMed Scopus (16) Google Scholar). 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. DISCUSSIONPrevious studies have demonstrated that a major pathway of FasL turnover is the release of sFasL resulting from MMP digestion. Here, we showed that FasL-expressing cells also release cell membrane FasL as vesicles capable of inducing apoptosis of target cells. The biological significance of releasing cell membrane FasL as vesicles is unclear. This process could down-regulate membrane expression of FasL. On the other hand, FasL presented by vesicles could be functional and could have an important biological function (33Albanese J. Meterissian S. Kontogiannea M. Dubreuil C. Hand A. Sorba S. Dainiak N. Blood. 1998; 91: 3862-3874Crossref PubMed Google Scholar, 34Martinez-Lorenzo M.J. Anel A. Gamen S. Monleon I. Lasierra P. Larrad L. Pineiro A. Alava M.A. Naval J. J. Immunol. 1999; 163: 1274-1281PubMed Google Scholar, 35Jodo S. Hohlbaum H. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 36Strehlow D. Jodo S. Ju S.-T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4209-4214Crossref PubMed Scopus (16) Google Scholar, 37Jodo S. Strehlow D. Ju S.-T. J. Immunol. 2000; 164: 5062-5069Crossref PubMed Scopus (16) Google Scholar). Using gene-transferred hFasL-PA317 and hFasL-3T3 cell lines and conducting quantitative analyses of FasL protein expression levels, we have shown that FasL is released from cells in the form of vesicles, and this process is as effective as the MMP-mediated release of sFasL. In contrast, FasL-expressing T cells release lower levels of FasL-containing vesicles and thus contribute modestly for T cell FasL turnover. Unlike sFasL, which is unable to kill many of the targets sensitive to FasL-expressing cells, vesicle-associated FasL retained the ability to kill these targets. These observations suggest that the rapid release of FasL-bearing vesicles may serve a more complex role than just simply down regulating the cell-associated FasL as is the case of sFasL release.Our analyses based on quantitative hFasL-specific ELISA demonstrated that release of FasL-bearing vesicles is as effective a turnover mechanism as sFasL release, because large amounts of FasL accumulated as vesicles in the supernatants of hFasL-PA317 and hFasL-3T3 cells. The FasL accumulated in vesicles was comparable to or slightly more than the amounts of sFasL released. In contrast to hFasL-PA317 and hFasL-3T3 cell lines, the P/I-activated Jurkat, PBT, and pIL-2-hFasL-EL-4 T cells expressed variable levels of FasL from very low to moderate. Taking this factor into consideration, the FasL levels of vesicles generated by these activated T cells ranged from 23% to 71% of the cell-associated FasL (Table I), whereas the sFasL levels were significantly higher. This indicates that the release of FasL-bearing vesicles contributes modestly to the FasL turnover in activated T cells. The modest accumulation of FasL-bearing vesicles is not likely due to an overactive MMP that degrades the full-length FasL of vesicles, because the same high level of vesicle-associated FasL was obtained when hFasL-3T3 cells were cultured with activated pIL-2-hFasL-EL-4 T cells (data not shown). In an earlier study, sFasL was shown to be the major species detected in culture supernatants of T lymphoma cells that overexpress hFasL (29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar). Our results obtained from three additional and different FasL-expressing T cell populations not only support the previous observation but also indicate that the release of vesicles contributes modestly to T cell FasL turnover. Whether different types of cells utilize different mechanisms,i.e. membrane shedding or secretion of exosomes (50Raposo G. Nijman H.W. Stoorvogel W. Leijendekker R. Harding C.V. Melief C.J.M. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2383) Google Scholar), for the release of FasL-containing vesicles remains to be established.T cell-mediated cytotoxicity depends on cell interaction forces. Antibodies against LFA-1 and ICAM are effective inhibitors of FasL-mediated cytotoxicity of T cells (45Wang J. Lenardo M.J. J. Exp. Med. 1997; 186: 1171-1176Crossref PubMed Scopus (43) Google Scholar, 46Kojima H. Eshima K. Takayama H. Sitkovsky M.V. J. Immunol. 1997; 159: 2728-2734PubMed Google Scholar). In the present study we showed that the FasL-mediated cytotoxicity of the P/I-activated pIL-2-hFasL-EL-4 cells but not hFasL-3T3 cells depended on LFA-1/ICAM interaction. Because of this cell interaction, the former cells are more potent than the latter cells even though their FasL is only one-sixth of the latter cells. In contrast to the P/I-activated pIL-2-hFasL-EL-4 cells, the cytotoxicity of vesicles prepared from the same cells was not inhibited by anti-LFA-1 mAb. Thus, release of FasL-containing vesicles could effectively down-regulate FasL function of T cells. Although we have clearly shown that vesicles contain FasL, whether or not LFA-1 could be released in the form of vesicles and whether a vesicle co-expresses both FasL and LFA-1 have not been firmly established.One reason that sFasL release has been considered a down-regulatory mechanism for cell-associated FasL is that sFasL is unable to kill many targets that are sensitive to the FasL-expressing cells. In addition, affinity-purified and concentrated sFasL was shown to inhibit killing mediated by cell-associated FasL (30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar). The sFasL prepared from hFasL-3T3 cells also inhibited killing mediated by FasL-bearing vesicles or FasL-expressing cells. This loss and gain of function was not observed for FasL-bearing vesicles, which do not inhibit the killing mediated by FasL-expressing cells (data not shown). Moreover, vesicle-associated FasL retained the ability to kill a panel of targets that are sensitive to the FasL-expressing cells from which the vesicles were derived. This suggests that release of FasL-bearing vesicles may have a different biological function than the MMP-mediated release of sFasL. For example, certain tumor cells (25Hahne M. Rimoldi D. Schroter M. Romero P. Schreiter M. French L.E. Schneider P. Bornand T. Fontana A. Lienard D. Cerottini J.-C. Tschopp J. Science. 1996; 274: 1363-1366Crossref PubMed Scopus (1191) Google Scholar, 26O'Connell J. O'Sullivan G.C. Collin J.K. Shanahan F. J. Exp. Med. 1996; 184: 1075-1082Crossref PubMed Scopus (867) Google Scholar, 27Bennett M.W. O'Connell J. O'Sullivan G.C. Chrady C. Roche D. Collins J.K. Shanahan F. J. Immunol. 1998; 160: 5669-5675PubMed Google Scholar), cells in the immune privileged sites (24Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1097) Google Scholar, 28Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar), and cells outside the immune system (51Galle P.R. Hofmann W.J. Walczak H. Schaller H. Otto G. Stremmel W. Krammer P.H. Runkel L. J. Exp. Med. 1995; 182: 1223-1230Crossref PubMed Scopus (675) Google Scholar, 52Strand S. Hofmann W.J. Grambihler A. Hug H. Volkmann M. Otto G. Wesch H. Mariani S.M. Hack V. Stremmel W. Krammer P.H. Galle P.E. Nat. Med. 1998; 4: 588-593Crossref PubMed Scopus (227) Google Scholar, 53Moller P. Walczak H. Reidl S. Strater J. Krammer P.H. Am. J. Pathol. 1996; 149: 9-13PubMed Google Scholar) constitutively express FasL under specific conditions. Their FasL is likely maintained at a steady level despite continuous release of vesicles that bear FasL. The accumulated FasL-bearing vesicles could provide additional killing power and could function beyond the local area controlled by FasL-expressing cells. Active release of FasL-bearing vesicles may help FasL-expressing cells mediate its functions rather than the inhibition as observed with sFasL. FasL-bearing vesicles may have long-lasting apoptosis-inducing power, because their expression is no longer dependent on de novoprotein synthesis. A recent study suggests sFasL bind to extracellular matrix and display an enhanced cytotoxicity against Jurkat target (54Aoki K. Kurooka M. Chen J.-J. Petryniak J. Nabel E.G. Nabel G.I. Nat. Immunol. 2001; 2: 333-337Crossref PubMed Scopus (80) Google Scholar). However, the loss of cytotoxicity against various targets associated with sFasL release would significantly reduce the role of sFasL as a general cytotoxic mediator in vivo. Given that multiple roles of FasL in the immune system have been demonstrated, the present study indicates that release of FasL-bearing vesicles, like sFasL release, is an important factor to consider because of its distinct influence on the expression and function of cell-associated FasL. In this respect, our ongoing study has shown that FasL-bearing vesicles display two bioactivities in vivo, i.e. they act as a chemotactic factor for neutrophils when injected intraperitoneally, and they induce lethal fulminant hepatitis when injected intravenously. 2S. Jodo, and S.-T. Ju, unpublished observations. Previous studies have demonstrated that a major pathway of FasL turnover is the release of sFasL resulting from MMP digestion. Here, we showed that FasL-expressing cells also release cell membrane FasL as vesicles capable of inducing apoptosis of target cells. The biological significance of releasing cell membrane FasL as vesicles is unclear. This process could down-regulate membrane expression of FasL. On the other hand, FasL presented by vesicles could be functional and could have an important biological function (33Albanese J. Meterissian S. Kontogiannea M. Dubreuil C. Hand A. Sorba S. Dainiak N. Blood. 1998; 91: 3862-3874Crossref PubMed Google Scholar, 34Martinez-Lorenzo M.J. Anel A. Gamen S. Monleon I. Lasierra P. Larrad L. Pineiro A. Alava M.A. Naval J. J. Immunol. 1999; 163: 1274-1281PubMed Google Scholar, 35Jodo S. Hohlbaum H. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 36Strehlow D. Jodo S. Ju S.-T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4209-4214Crossref PubMed Scopus (16) Google Scholar, 37Jodo S. Strehlow D. Ju S.-T. J. Immunol. 2000; 164: 5062-5069Crossref PubMed Scopus (16) Google Scholar). Using gene-transferred hFasL-PA317 and hFasL-3T3 cell lines and conducting quantitative analyses of FasL protein expression levels, we have shown that FasL is released from cells in the form of vesicles, and this process is as effective as the MMP-mediated release of sFasL. In contrast, FasL-expressing T cells release lower levels of FasL-containing vesicles and thus contribute modestly for T cell FasL turnover. Unlike sFasL, which is unable to kill many of the targets sensitive to FasL-expressing cells, vesicle-associated FasL retained the ability to kill these targets. These observations suggest that the rapid release of FasL-bearing vesicles may serve a more complex role than just simply down regulating the cell-associated FasL as is the case of sFasL release. Our analyses based on quantitative hFasL-specific ELISA demonstrated that release of FasL-bearing vesicles is as effective a turnover mechanism as sFasL release, because large amounts of FasL accumulated as vesicles in the supernatants of hFasL-PA317 and hFasL-3T3 cells. The FasL accumulated in vesicles was comparable to or slightly more than the amounts of sFasL released. In contrast to hFasL-PA317 and hFasL-3T3 cell lines, the P/I-activated Jurkat, PBT, and pIL-2-hFasL-EL-4 T cells expressed variable levels of FasL from very low to moderate. Taking this factor into consideration, the FasL levels of vesicles generated by these activated T cells ranged from 23% to 71% of the cell-associated FasL (Table I), whereas the sFasL levels were significantly higher. This indicates that the release of FasL-bearing vesicles contributes modestly to the FasL turnover in activated T cells. The modest accumulation of FasL-bearing vesicles is not likely due to an overactive MMP that degrades the full-length FasL of vesicles, because the same high level of vesicle-associated FasL was obtained when hFasL-3T3 cells were cultured with activated pIL-2-hFasL-EL-4 T cells (data not shown). In an earlier study, sFasL was shown to be the major species detected in culture supernatants of T lymphoma cells that overexpress hFasL (29Kayagaki N. Kawasaki A. Ebata H. Ohmoto S. Ikeda S. Inoue S. Yoshino K. Okumura K. Yagita H. J. Exp. Med. 1995; 182: 1777-1783Crossref PubMed Scopus (775) Google Scholar). Our results obtained from three additional and different FasL-expressing T cell populations not only support the previous observation but also indicate that the release of vesicles contributes modestly to T cell FasL turnover. Whether different types of cells utilize different mechanisms,i.e. membrane shedding or secretion of exosomes (50Raposo G. Nijman H.W. Stoorvogel W. Leijendekker R. Harding C.V. Melief C.J.M. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2383) Google Scholar), for the release of FasL-containing vesicles remains to be established. T cell-mediated cytotoxicity depends on cell interaction forces. Antibodies against LFA-1 and ICAM are effective inhibitors of FasL-mediated cytotoxicity of T cells (45Wang J. Lenardo M.J. J. Exp. Med. 1997; 186: 1171-1176Crossref PubMed Scopus (43) Google Scholar, 46Kojima H. Eshima K. Takayama H. Sitkovsky M.V. J. Immunol. 1997; 159: 2728-2734PubMed Google Scholar). In the present study we showed that the FasL-mediated cytotoxicity of the P/I-activated pIL-2-hFasL-EL-4 cells but not hFasL-3T3 cells depended on LFA-1/ICAM interaction. Because of this cell interaction, the former cells are more potent than the latter cells even though their FasL is only one-sixth of the latter cells. In contrast to the P/I-activated pIL-2-hFasL-EL-4 cells, the cytotoxicity of vesicles prepared from the same cells was not inhibited by anti-LFA-1 mAb. Thus, release of FasL-containing vesicles could effectively down-regulate FasL function of T cells. Although we have clearly shown that vesicles contain FasL, whether or not LFA-1 could be released in the form of vesicles and whether a vesicle co-expresses both FasL and LFA-1 have not been firmly established. One reason that sFasL release has been considered a down-regulatory mechanism for cell-associated FasL is that sFasL is unable to kill many targets that are sensitive to the FasL-expressing cells. In addition, affinity-purified and concentrated sFasL was shown to inhibit killing mediated by cell-associated FasL (30Tanaka M. Itai T. Adachi M. Nagata S. Nat. Med. 1998; 4: 31-36Crossref PubMed Scopus (606) Google Scholar, 31Suda T. Hashimoto H. Tanaka M. Ochi T. Nagata S. J. Exp. Med. 1997; 186: 2045-2050Crossref PubMed Scopus (439) Google Scholar). The sFasL prepared from hFasL-3T3 cells also inhibited killing mediated by FasL-bearing vesicles or FasL-expressing cells. This loss and gain of function was not observed for FasL-bearing vesicles, which do not inhibit the killing mediated by FasL-expressing cells (data not shown). Moreover, vesicle-associated FasL retained the ability to kill a panel of targets that are sensitive to the FasL-expressing cells from which the vesicles were derived. This suggests that release of FasL-bearing vesicles may have a different biological function than the MMP-mediated release of sFasL. For example, certain tumor cells (25Hahne M. Rimoldi D. Schroter M. Romero P. Schreiter M. French L.E. Schneider P. Bornand T. Fontana A. Lienard D. Cerottini J.-C. Tschopp J. 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Cruikshank (Pulmonary Center, Boston University Medical Campus) for providing the phytohemagglutinin-activated human T cells, and Dr. D. H. Sherr for critical comments.
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