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Expression of FasL as a stable cell-surface molecule is important to its apoptotic function
Expression of FasL as a Stable Cell-Surface Molecule is Important to Its Apoptotic Function S. Lamhamedi Cherradi and H. Shirwan
A
POPTOSIS is induced by FasL in cells bearing the Fas receptor, and FasL has also been shown to be important in immune privilege and tolerance toward self-antigens.1 This aspect of FasL was exploited for immune regulation to alloantigens and autoantigens to prevent allograft rejection and abrogation of autoimmunity.2– 4 These studies, however, have generated mixed outcomes; whereas some reported success, others reported failure.2– 4 We think this may occur because different functions are performed by different forms of FasL. FasL is expressed as a type II cell-surface protein. This molecule, however, is cleaved from the cell surface by matrix metalloproteinases within minutes of expression to produce a soluble form.5 The soluble and the membrane-bound molecules perform different functions. In soluble form FasL is less effective in inducing apoptosis and is chemotactic for neutrophils.6,7 In contrast, the membranous form does not have chemotactic functions and is apoptotic.7 We modified rat FasL to eliminate the putative metalloproteinase site to generate a stable cell-surface molecule. This molecule is used as a tolerogenic factor to induce apoptosis in alloreactive lymphocytes for the prevention of allograft rejection and the induction of transplantation tolerance.
expression level in cells transfected with the wild-type molecule. Most importantly, Cos 1 cells expressing the modified FasL induced eight times more apoptosis in the Fas⫹ human Jurkat cells. The apoptotic efficiency of the modified FasL was time-independent, suggesting that this molecule is not cleaved from the cell surface. Our data clearly demonstrate that removal of the putative metalloproteinase site in rat FasL results in stable cell-surface expression and improves in vitro apoptotic activity. This modified molecule may therefore serve as a better immunoregulatory agent to inhibit alloreactive responses. Particularly, the inability of the cell-surface molecule to serve as a chemotactic factor for neutrophils avoids complications arising from neutrophils and facilitates its use as a tolerogenic factor in preventing allograft rejection.7
REFERENCES 1. Griffith TS, Brunner T, Fletcher SM, et al: Science 270:1189, 1995 2. Kang SM, Schneider DB, Lin Z, et al: Nat Med 3:738, 1997 3. Lau HT, Yu M, Fontana A, et al: Science 273:109, 1996 4. Zhang HG, Su X, Liu D, et al: J Immunol 162:1423, 1999
MATERIALS AND METHODS A functional cDNA for FasL was cloned from activated rat splenocytes using FasL-specific primers in RT-PCR. The metalloproteinase cleavage site was deleted using primers containing XhoI and excluding 12 amino acid residues encoding the metalloproteinase site in PCR. The PCR product was cut with XhoI, ligated, and cloned into the pcDNA3 expression vector to obtain the modified FasL. cDNA clones for the modified FasL, wild-type, and antisense were transiently transfected into Cos 1 cells, and transfectants were analyzed for cell-surface expression of FasL and apoptosis. Apoptosis was performed by coincubating transfectants with human Fas⫹ Jurkat cells for various periods of time. Jurkat cells were then analyzed in flow cytometry using propidium iodide and Annexin V.
RESULTS AND DISCUSSION
We detected higher levels of expression of the modified FasL in Cos 1 cells transfectants as compared with the
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
5. Tanaka M, Itai T, Adachi M, et al: Nat Med 4:31, 1998 6. Ottonello L, Tortolina G, Amelotti M, et al: J Immunol 162:3601, 1999 7. Suda T, Hashimoto H, Tanaka M, et al: J Exp Med 186:2045, 1997
From the Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA. This work was supported by grants to H.S. from NIH (AI 33587) and AHA (9650229N and 9951499V). Address reprint requests to Dr H. Shirwan, Institute for Cellular Therapeutics, University of Louisville, 570 South Preston St, Suite 404, Louisville, KY 40202. E-mail: [email protected].
0041-1345/01/$–see front matter PII S0041-1345(00)02004-2 259
Transplantation Proceedings, 33, 259 (2001)
A
POPTOSIS is induced by FasL in cells bearing the Fas receptor, and FasL has also been shown to be important in immune privilege and tolerance toward self-antigens.1 This aspect of FasL was exploited for immune regulation to alloantigens and autoantigens to prevent allograft rejection and abrogation of autoimmunity.2– 4 These studies, however, have generated mixed outcomes; whereas some reported success, others reported failure.2– 4 We think this may occur because different functions are performed by different forms of FasL. FasL is expressed as a type II cell-surface protein. This molecule, however, is cleaved from the cell surface by matrix metalloproteinases within minutes of expression to produce a soluble form.5 The soluble and the membrane-bound molecules perform different functions. In soluble form FasL is less effective in inducing apoptosis and is chemotactic for neutrophils.6,7 In contrast, the membranous form does not have chemotactic functions and is apoptotic.7 We modified rat FasL to eliminate the putative metalloproteinase site to generate a stable cell-surface molecule. This molecule is used as a tolerogenic factor to induce apoptosis in alloreactive lymphocytes for the prevention of allograft rejection and the induction of transplantation tolerance.
expression level in cells transfected with the wild-type molecule. Most importantly, Cos 1 cells expressing the modified FasL induced eight times more apoptosis in the Fas⫹ human Jurkat cells. The apoptotic efficiency of the modified FasL was time-independent, suggesting that this molecule is not cleaved from the cell surface. Our data clearly demonstrate that removal of the putative metalloproteinase site in rat FasL results in stable cell-surface expression and improves in vitro apoptotic activity. This modified molecule may therefore serve as a better immunoregulatory agent to inhibit alloreactive responses. Particularly, the inability of the cell-surface molecule to serve as a chemotactic factor for neutrophils avoids complications arising from neutrophils and facilitates its use as a tolerogenic factor in preventing allograft rejection.7
REFERENCES 1. Griffith TS, Brunner T, Fletcher SM, et al: Science 270:1189, 1995 2. Kang SM, Schneider DB, Lin Z, et al: Nat Med 3:738, 1997 3. Lau HT, Yu M, Fontana A, et al: Science 273:109, 1996 4. Zhang HG, Su X, Liu D, et al: J Immunol 162:1423, 1999
MATERIALS AND METHODS A functional cDNA for FasL was cloned from activated rat splenocytes using FasL-specific primers in RT-PCR. The metalloproteinase cleavage site was deleted using primers containing XhoI and excluding 12 amino acid residues encoding the metalloproteinase site in PCR. The PCR product was cut with XhoI, ligated, and cloned into the pcDNA3 expression vector to obtain the modified FasL. cDNA clones for the modified FasL, wild-type, and antisense were transiently transfected into Cos 1 cells, and transfectants were analyzed for cell-surface expression of FasL and apoptosis. Apoptosis was performed by coincubating transfectants with human Fas⫹ Jurkat cells for various periods of time. Jurkat cells were then analyzed in flow cytometry using propidium iodide and Annexin V.
RESULTS AND DISCUSSION
We detected higher levels of expression of the modified FasL in Cos 1 cells transfectants as compared with the
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
5. Tanaka M, Itai T, Adachi M, et al: Nat Med 4:31, 1998 6. Ottonello L, Tortolina G, Amelotti M, et al: J Immunol 162:3601, 1999 7. Suda T, Hashimoto H, Tanaka M, et al: J Exp Med 186:2045, 1997
From the Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA. This work was supported by grants to H.S. from NIH (AI 33587) and AHA (9650229N and 9951499V). Address reprint requests to Dr H. Shirwan, Institute for Cellular Therapeutics, University of Louisville, 570 South Preston St, Suite 404, Louisville, KY 40202. E-mail: [email protected].
0041-1345/01/$–see front matter PII S0041-1345(00)02004-2 259
Transplantation Proceedings, 33, 259 (2001)