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Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Nature volume 461, pages 659–663 (2009)Cite this article

Abstract

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses1,2,3 and prevention of autoimmunity4,5. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding10,11. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs ) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm ) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasLΔm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

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Figure 1: Generation of mutant mice that specifically lack either sFasL or mFasL.
Figure 2: mFasL but not sFasL is essential for target cell killing and AICD.
Figure 3: mFasL but not sFasL is essential to prevent lymphadenopathy, splenomegaly and hyper-gammaglobulinaemia with anti-nuclear autoantibodies.
Figure 4: FasL Δm/Δm mice die considerably earlier than FasL gld/gld mice owing to SLE-like fatal glomerulonephritis and histiocytic sarcoma.

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References

  1. Hughes, P. D. et al. Apoptosis regulators Fas and Bim cooperate in shutdown of chronic immune responses and prevention of autoimmunity. Immunity 28, 197–205 (2008)

    Article  CAS  Google Scholar 

  2. Hutcheson, J. et al. Combined deficiency of proapoptotic regulators Bim and Fas results in the early onset of systemic autoimmunity. Immunity 28, 206–217 (2008)

    Article  CAS  Google Scholar 

  3. Weant, A. E. et al. Apoptosis regulators Bim and Fas function concurrently to control autoimmunity and CD8+ T cell contraction. Immunity 28, 218–230 (2008)

    Article  CAS  Google Scholar 

  4. Krammer, P. H. CD95's deadly mission in the immune system. Nature 407, 789–795 (2000)

    Article  ADS  CAS  Google Scholar 

  5. Peter, M. E. et al. The CD95 receptor: apoptosis revisited. Cell 129, 447–450 (2007)

    Article  CAS  Google Scholar 

  6. Watanabe-Fukunaga, R. et al. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J. Immunol. 148, 1274–1279 (1992)

    CAS  PubMed  Google Scholar 

  7. Takahashi, T. et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76, 969–976 (1994)

    Article  CAS  Google Scholar 

  8. Rieux-Laucat, F. et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268, 1347–1349 (1995)

    Article  ADS  CAS  Google Scholar 

  9. Fisher, G. H. et al. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81, 935–946 (1995)

    Article  CAS  Google Scholar 

  10. Trambas, C. M. & Griffiths, G. M. Delivering the kiss of death. Nature Immunol. 4, 399–403 (2003)

    Article  CAS  Google Scholar 

  11. Schulte, M. et al. ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death. Cell Death Differ. 14, 1040–1049 (2007)

    Article  CAS  Google Scholar 

  12. Schneider, P. et al. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205–1213 (1998)

    Article  CAS  Google Scholar 

  13. Tanaka, M., Itai, T., Adachi, M. & Nagata, S. Downregulation of Fas ligand by shedding. Nature Med. 4, 31–36 (1998)

    Article  CAS  Google Scholar 

  14. Hohlbaum, A. M., Moe, S. & Marshak-Rothstein, A. Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival. J. Exp. Med. 191, 1209–1219 (2000)

    Article  CAS  Google Scholar 

  15. Siegel, R. M. et al. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288, 2354–2357 (2000)

    Article  ADS  CAS  Google Scholar 

  16. Chen, J.-J., Sun, Y. & Nabel, G. J. Regulation of the proinflammatory effects of Fas ligand (CD95L). Science 282, 1714–1717 (1998)

    Article  ADS  CAS  Google Scholar 

  17. Wilson, N. S., Dixit, V. & Ashkenazi, A. Death receptor signal transducers: nodes of coordination in immune signaling networks. Nature Immunol. 10, 348–355 (2009)

    Article  CAS  Google Scholar 

  18. Ahn, J. H. et al. Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activated protein kinases and NF-κB-dependent gene expression. J. Biol. Chem. 276, 47100–47106 (2001)

    Article  CAS  Google Scholar 

  19. Xiao, S., Jodo, S., Sung, S. S., Marshak-Rothstein, A. & Ju, S. T. A novel signaling mechanism for soluble CD95 ligand. Synergy with anti-CD95 monoclonal antibodies for apoptosis and NF-κB nuclear translocation. J. Biol. Chem. 277, 50907–50913 (2002)

    Article  CAS  Google Scholar 

  20. Oliveira, J. B. et al. NRAS mutation causes a human autoimmune lymphoproliferative syndrome. Proc. Natl Acad. Sci. USA 104, 8953–8958 (2007)

    Article  ADS  CAS  Google Scholar 

  21. Kono, D. H. & Theofilopoulos, A. N. Genetic susceptibility to spontaneous lupus in mice. Curr. Dir. Autoimmun. 1, 72–98 (1999)

    Article  CAS  Google Scholar 

  22. Watanabe, D., Suda, T., Hashimoto, H. & Nagata, S. Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders. EMBO J. 14, 12–18 (1995)

    Article  CAS  Google Scholar 

  23. Ogasawara, J. et al. Lethal effect of the anti-Fas antibody in mice. Nature 364, 806–809 (1993)

    Article  ADS  CAS  Google Scholar 

  24. Huang, D. C. et al. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL . Proc. Natl Acad. Sci. USA 96, 14871–14876 (1999)

    Article  ADS  CAS  Google Scholar 

  25. Strand, S. et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells—a mechanism of immune evasion? Nature Med. 2, 1361–1366 (1996)

    Article  CAS  Google Scholar 

  26. Fredrickson, T. N. & Harris, A. W. Atlas of Mouse Hematopathology (Harwood Academic, 2000)

    Google Scholar 

  27. Frith, C. H. & Wiley, L. D. Morphologic classification and correlation of incidence of hyperplastic and neoplastic hematopoietic lesions in mice with age. J. Gerontol. 36, 534–545 (1981)

    Article  CAS  Google Scholar 

  28. Suzuki, I. & Fink, P. J. Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med. 187, 123–128 (1998)

    Article  CAS  Google Scholar 

  29. Sun, M. & Fink, P. J. A new class of reverse signaling costimulators belongs to the TNF family. J. Immunol. 179, 4307–4312 (2007)

    Article  CAS  Google Scholar 

  30. Karray, S. et al. Complete loss of Fas ligand gene causes massive lymphoproliferation and early death, indicating a residual activity of gld allele. J. Immunol. 172, 2118–2125 (2004)

    Article  CAS  Google Scholar 

  31. Schwenk, F., Baron, U. & Rajewsky, K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 5080–5081 (1995)

    Article  CAS  Google Scholar 

  32. Phillips, J. M., O'Reilly, L., Bland, C., Foulis, A. K. & Cooke, A. Patients with chronic pancreatitis have islet progenitor cells in their ducts, but reversal of overt diabetes in NOD mice by anti-CD3 shows no evidence for islet regeneration. Diabetes 56, 634–640 (2007)

    Article  CAS  Google Scholar 

  33. Pileri, S. A. et al. Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology 41, 1–29 (2002)

    Article  ADS  CAS  Google Scholar 

  34. Blott, E. J., Bossi, G., Clark, R., Zvelebil, M. & Griffiths, G. M. Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail. J. Cell Sci. 114, 2405–2416 (2001)

    CAS  PubMed  Google Scholar 

  35. Naik, S., Vremec, D., Wu, L., O'Keeffe, M. & Shortman, K. CD8α+ mouse spleen dendritic cells do not originate from the CD8 α-dendritic cell subset. Blood 102, 601–604 (2003)

    Article  CAS  Google Scholar 

  36. Hibbs, M. L. et al. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83, 301–311 (1995)

    Article  CAS  Google Scholar 

  37. Lyons, A. B. & Parish, C. R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994)

    Article  CAS  Google Scholar 

  38. Kilbourne, E. D. Future influenza vaccines and the use of genetic recombinants. Bull. WHO 41, 643–645 (1969)

    CAS  PubMed  Google Scholar 

  39. Flynn, K. J. et al. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8, 683–691 (1998)

    Article  CAS  Google Scholar 

  40. Belz, G. T., Xie, W., Altman, J. D. & Doherty, P. C. A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge. J. Virol. 74, 3486–3493 (2000)

    Article  CAS  Google Scholar 

  41. Belz, G. T., Xie, W. & Doherty, P. C. Diversity of epitope and cytokine profiles for primary and secondary influenza a virus-specific CD8+ T cell responses. J. Immunol. 166, 4627–4633 (2001)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Genentech, in particular A. Ashkenazi and S. Masters, for Fas-Fc fusion proteins. We also thank our colleagues at the Walter and Eliza Hall Institute of Medical Research (Melbourne), G. Siciliano, N. Iannarella, J. Coughlin for animal care; J. Corbin for automated blood analysis; B. Helbert and C. Young for genotyping; S. Mihajlovic, E. Tsui, A. Hasanein, V. Babo and K. Weston for histological sections; A. Light and K. O’Donnell for help with antibody measurements; and S. Drake for help with cytokine quantification. We also thank J. Melny (Royal Melbourne Hospital) for measuring anti-DNA antibody levels; A. Banerjee, S. Gerondakis and R. Gugasyan (Burnet Medical Research Institute, Prahran) for antibodies and advice; J. Silke and L. Wong (La Trobe University, Bundoora) for TNFα; P. Morgan for assistance with protein purification; and A. Silva and J. Sharkey for help with animal procedures (Peter MacCallum Cancer Centre). This work was supported in part by the NHMRC (programme numbers 461221 and 454569, a CJ Martin fellowship to N.M.H. and an RD Wright fellowship to L.A.O’R.). We also acknowledge support from the IRIISS (grant numbers 361646 and 257502 to P.B.), the Victorian State Government (an OIS grant), the Leukemia and Lymphoma Society (SCOR grant number 7015), the NIH (grant numbers CA043540-18 and CA80188-6), the JDRF/NHMRC, the Association for International Cancer Research, the Charles and Sylvia Viertel Charitable Foundation (to P.B.) and the Leukemia Research Foundation.

Author Contributions L.A.O’R. planned and performed most experiments and wrote the manuscript. L.T., L.L., E.A.K., S.G., W.D.F., N.M.H., D.M.T., J.-G.Z., G.T.B., M.J.S., P.B. and L.R. contributed to the planning and execution of the experiments and the writing of the manuscript. A.S. conceived the study, planned experiments and wrote the manuscript.

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Authors and Affiliations

  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia ,

    Lorraine A. O’ Reilly, Lin Tai, Lily Lee, Elizabeth A. Kruse, Stephanie Grabow, W. Douglas Fairlie, David M. Tarlinton, Jian-Guo Zhang, Gabrielle T. Belz, Philippe Bouillet, Lorraine Robb & Andreas Strasser

  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia,

    Elizabeth A. Kruse & Stephanie Grabow

  3. Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia ,

    Nicole M. Haynes & Mark J. Smyth

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Correspondence to Andreas Strasser.

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O’ Reilly, L., Tai, L., Lee, L. et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature 461, 659–663 (2009). https://doi.org/10.1038/nature08402

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