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Sorting out the multiple roles of Fas ligand
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European Journal of Cell Biology 79, 539 ± 543 (2000, August) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
539
Sorting out the multiple roles of Fas ligand Giovanna Bossia, Jane C. Stinchcombea, Lesley J. Pageb, Gillian M. Griffithsa1) a b
Sir William Dunn School of Pathology, Oxford/UK Imperial Cancer Research Fund Laboratories, London/UK
Fas ligand (CD95 ligand) ± cytotoxic T lymphocyte ± sorting ± secretory lysosome Fas ligand can both be used by the immune system to initiate cell death, and be used by non-lymphoid cells to evade death. Recent work has shown that Fas ligand is differentially sorted in different cell types. Here we present the viewpoint that the differential sorting plays an important part in determining the role of Fas ligand in different cells. Fas ligand (FasL) is a protein with multiple roles. When expressed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, FasL is used to destroy virally infected or tumorigenic targets and plays a critical role in cellular destruction by the immune system (Nagata and Golstein, 1995). However, when expressed by the epithelial cells of the eye, FasL creates an immunologically privileged site by destroying any activated lymphocytes which enter (Griffith et al., 1995) (Figure 1). FasL has been shown to play a critical role in immune regulation by T cell deletion (Lynch et al., 1995) and triggering an inflammatory response by activating polymorphonuclear lymphocytes (PMNs) (Seino et al., 1998). Since FasL can both be used to initiate cell death by the immune system and to protect against cell death by the immune system how do different cell types determine which role FasL is to play? One very simple mechanism for regulating the activity of FasL, which has been overlooked, is the control of FasL appearance on the cell surface. In CTLs and NK cells FasL expression is tightly controlled so that only specific targets are killed. Constant cell surface expression of FasL in CTL and NK cells would be very dangerous, resulting in indiscriminate killing by these cells. However in the epithelial cells of the eye FasL is expressed constitutively on the cell surface in order to maintain the immunological privilege of this site, by destroying any activated cells entering. How is the surface expression of FasL differentially controlled in order to permit such diverse roles for the same protein? Initial studies showed that FasL is transcriptionally upregulated upon T cell activation and results Dr. Gillian M. Griffiths, Sir William Dunn School of Pathology, Oxford, OX1 3RE/UK, e-mail: [email protected], Fax: 44 18 65 27 55 15.
1)
in new protein synthesis (Anel et al., 1995; Suda et al., 1995). The problem with control by new protein synthesis alone is that it provides no mechanism for regulating the FasL appearance on the cell surface or for focusing FasL at the target cell interface during target cell death. So we decided to ask how FasL was sorted to the cell surface in CTLs, NK cells and epithelial cells. We discovered that FasL is differentially sorted in lymphoid versus non-lymphoid cells (Bossi and Griffiths, 1999). We propose that differential sorting in different cell types plays a critical role in controlling the very different functions of FasL.
Intracellular storage of FasL in CTLs and NK cells Newly synthesised FasL can be detected within CTLs and NK cells using an antibody against the extracellular domain (Kayagaki et al., 1995). Since these cells possess a very effective metalloprotease on their cell surfaces any FasL reaching the cell surface immediately loses it s extracellular domain and the antibody no longer recognises the protein. This enabled us to demonstrate that in CTL and NK cells newly synthesised FasL is delivered to the lytic granules, and only released upon degranulation. Increasing evidence indicates that extensive crosslinking of the membrane-bound form of FasL is required for effective induction of apoptosis (Schneider et al., 1998). The granule localisation of FasL in CTLs and NK cells is therefore important in several respects. First the focusing and concentration of FasL at the point of membrane contact (Figure 2) during target cell killing will allow extensive crosslinking upon engagement with target cell Fas. Second, storage of FasL in the granule protects against premature cleavage of the protein by the metalloprotease, ensuring that the effective membrane-bound form will be exposed in response to TcR-mediated recognition of the target. Thirdly, the storage of FasL in the lytic granules means that both perforin and FasL mechanisms of target cell destruction are delivered together. This third point explains why it has often been difficult to entirely separate the contribution of perforin and Fas pathways in target cell death (van den Broek et al., 1996). The fact that the two effector
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3 Fig. 1. Two very different roles for Fas ligand. (Top) FasL is used by T cells to destroy virally infected cells. TcR recognition of the target leads to FasL expression by the T cells. FasL crosslinks Fas and triggers apoptosis in the target. (Bottom) Conversely, FasL is expressed by epithelial cells of the eye to create an immunologically privileged site. FasL on the epithelial cells engages Fas on activated T cells and destroys them before they are able to damage the eye.
Fig. 2. FasL is sorted differently in different cell types. (Top) In CTL, NK and mast cells FasL is sorted directly to the secretory lysosomes where it is stored as shown by co-staining FasL (green) and cathepsin D (red) in the human NK cell line YT. (Bottom) In epithelial cells FasL is expressed continuously on the cell surface and maintains the immunological privilege of these cells. This is reflected in the sorting of FasL in the epithelial cell line HeLa transfected with GFP-FasL (green), costained with cathepsin D (red), where FasL is sorted directly to the cell surface. !
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proteins are stored together means that both mechanisms are likely to contribute together in many situations.
Differential sorting of FasL The storage (Bossi and Griffiths, 1999; Lowin et al., 1996; Martinez-Lorenzo et al., 1996) and triggered release (Bossi and Griffiths, 1999) of FasL explains how CTLs and NK cells regulate the surface appearance of FasL, but does not explain how the control differs in cells which use FasL to create an immunologically-privileged environment. The answer lies in the finding that FasL is differentially sorted in haemopoietic and non-haemopoietic cells (Bossi and Griffiths, 1999). FasL is sorted to the secretory granules of haemopoietic cells, but travels directly to the cell surface in most non-haemopoietic cells. When expressed on epithelial cell lines or fibroblasts FasL is found abundantly on the cell surface, but when expressed in a mast cell line or in NK cells FasL is found only intracellularly and not on the cell surface (Figure 2) (Bossi and Griffiths, 1999). This indicates that FasL contains a specific sorting signal which targets it to the secretory granules of haemopoietic cells, but that this signal is not recognised in nonhaemopoietic cells in which FasL follows the default pathway directly to the cell surface. Consequently FasL surface expression is tightly regulated in haemopoietic cells where it is used to kill recognised targets, but constitutive in nonhaemopoietic cells in which constant expression is required to maintain immune privilege.
Molecular basis of differential sorting How does this differential sorting occur? The majority of nonlymphoid cells use separate lysosomes and secretory granules. However in haemopoietic cells these two organelles are combined and these ªsecretory lysosomesº function as both the secretory granules and lysosomes of these cells (reviewed in (Griffiths, 1996)). The finding that FasL is only sorted directly to the secretory lysosomes of haemopoietic cells indicates that it contains a granule/lysosomal targeting signal that is only recognised in cells of this lineage. The sorting signal known to be in the cytoplasmic tail of FasL (Bossi and Griffiths, 1999) has been recently mapped to a sequence enriched in proline in the region proximal to the transmembrane domain (Bossi and Griffiths, unpublished data). Identification of the lineage-specific machinery which recognises this signal would be the next step. Since SH3 domain binds to polyproline sequence (Feng et al., 1994), an SH3 domain-containing protein could be part of the sorting machinery. Other evidence supports the idea that haemopoietic cells use specialised mechanisms for sorting proteins to secretory lysosomes. Perforin and granzyme A, soluble proteins found within lytic granules of CTL (reviewed in (Griffiths, 1997)), can only be sorted correctly to the secretory lysosomes of haemopoietic cells. When expressed in conventional secretory cells they are neither sorted to the lysosome nor the secretory granule (L. J. Page and G. M. Griffiths, manuscript in preparation). Similarly the small number of non-lymphoid cells which possess secretory lysosomes (e.g. melanocytes (Diment et al., 1995), renal tubular cells (Swank and Brandt, 1978) and some
Sorting out the multiple roles of Fas ligand 541
pancreatic cells (Orci et al., 1971)) may also use some distinct sorting mechanisms. Recent work suggests that tyrosinase may also be sorted to the secretory lysosomes of melanocytes via a specialised pathway. Although tyrosinase is targeted to lysosomes when expressed in non secretory cells (Simmen et al., 1999), it does so via a pathway which is at least partially distinct from that used by other lysosomal proteins (M. Marks, personal communication). Taken together these findings suggest that cells possessing secretory lysosomes have sorting pathways which are not found in other cell types. Although the precise molecular details of the differential sorting are not yet known several of the biological mysteries of FasL can now be reconsidered in the light of these findings.
FasL and immune privilege The initial findings that FasL expression could confer immune privilege in the eye and testes, resulted in a host of attempts to use FasL expression to prevent transplant rejection. In some studies FasL-expressing cells conferred protection (Lau et al., 1996) while others did not (Kang et al., 1997; Lau and Stoeckert, 1997). The cell type in which FasL was expressed seemed to be crucial for successful protection from rejection. Allogeneic pancreatic islets co-transplanted with syngeneic myoblasts expressing FasL were protected from rejection, while islet cells expressing FasL themselves were not. Given that FasL is differentially sorted in cells with secretory lysosomes this raises the possibility that the reason some cell types failed to confer protection was due to the fact that the FasL was stored within the cell rather than expressed directly on the cell surface. Interestingly, early studies indicate that lysosomal and secretory products are co-localised in islet beta cells (Orci et al., 1971), raising the possibility that FasL may not reach the surface of these cells without secretory granule release. The whole picture is certainly more complicated, since islets expressing FasL produce a more rapid rejection and insulitis, following neutrophil recruitment which can be induced by soluble FasL (Allison et al., 1997; Lau and Stoeckert, 1997). However, the fact that FasL cell surface expression may require granule release in some cell types clearly becomes an important consideration in preventing rejection of grafts. Another important factor in expression of FasL at the cell surface is the expression of the metalloprotease. In CTLs and NK cells cleavage of FasL by this cell surface protease is so effective that it is impossible to detect cell surface expression of FasL in the absence of protease inhibitors. Since FasL can be detected on epithelial cells involved in immune privilege, then the protease must be lacking. Although the cleavage site of FasL in two different cell types has been determined (Schneider et al., 1998; Tanaka et al., 1998) the identity and distribution of the metalloprotease is not currently known.
CD4 T cells possess secretory lysosomes In CD4 T cells FasL plays a critical role in immune regulation and deletion of autoreactive cells by ªactivation-induced cell deathº (AICD). TcR stimulation of T cells leads to proliferation and activation of primary T cells, but to AICD in previously activated T cells (reviewed in (Kabelitz et al., 1993;
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Lynch et al., 1995)). Consistent with this we found that in CD4 human T cell clones FasL is stored in secretory lysosomes which require TcR activation before they will expose FasL on their surface (Bossi and Griffiths, 1999). Since primary T cells neither express FasL, nor contain secretory lysosomes, then this explains why they do not undergo AICD. Studies on T cell hybridomas and the T cell line Jurkat indicated that FasL/Fas interactions were essential for AICD (Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995). Recent studies have revealed that AICD in non-transformed cells does not proceed in a cell autonomous manner and rather depends upon FasL expression induced on non-lymphoid tissue during the immune response (Bonfoco et al., 1998). The reasons for this discrepancy between the cell lines and the nontransformed T cells in vivo is not immediately obvious. However one possible explanation could be the differential targeting of FasL in transformed and hybridoma cells versus untransformed T cells. For example, cloned murine CTL have been shown to require activation in order to up-regulate Fas ligand on the cell surface, while the T cell lymphoma expresses FasL constitutively on the cell surface (Glass et al., 1996). It is therefore possible that T cell hybridomas, derived by fusion of normal T cells with a thymic lymphoma cell line, may well reflect the lymphoma phenotype and sort FasL to the cell surface. This might then account for the susceptibility to AICD from other T cells.
Tumor evasion of the immune system via Fas and Fas ligand A similar disruption of sorting seems to occur in tumorigenic cells in vivo. Large granular lymphocyte (LGL) and NK lymphomas derived from T and NK cells showed both increased surface expression of FasL and increased serum levels of soluble FasL (Tanaka et al., 1996). This contrasts with T cells from healthy individuals which require activation before expressing FasL on the surface or shedding soluble FasL into the serum. These findings indicate not only that the tumors are constitutively expressing FasL, but that it is now constitutively expressed on the cell surface suggesting that the granule sorting pathway has either been disrupted or overloaded in the tumors. Tumors, derived from other cell types have been shown not only to express FasL (Hahne et al., 1996; O Connell et al., 1996; Strand et al., 1996), but also to have downregulated Fas expression (Owen-Schaub et al., 1998; Strand et al., 1996). So while the absence of Fas allows the tumors to escape FasL mediated cell death by the immune system, the expression of FasL on the surface of the tumors enables them to destroy activated lymphocytes and avoid cell death mediated by alternative effector mechanisms. Escape of the immune system by these cells requires constitutive cell surface expression of FasL on the cell surface. This will be influenced by the sorting machinery and the metalloprotease activity of the cells from which the tumor is derived.
Conclusions The finding that FasL is differentially sorted in different cell types has important implications for future research. First it is no longer sufficient to demonstrate that FasL is expressed by a
cell to imply that it is active on the cell surface. Rather it is important to know where in the cell FasL is found and how it reaches the cell surface. Secondly determining the mechanisms which mediate differential sorting in different cell types will be important in understanding how to regulate many other proteins which may also use this route in haemopoietic cells. Both the route via which FasL is sorted and the presence or absence of the metalloprotease which cleaves membranebound FasL into soluble FasL appear to determine the function of FasL. Given that effective killing by FasL requires extensive crosslinking of the membrane-bound form; while the soluble form is a potent mediator of neutrophil recruitment, then the ultimate function of FasL will be determined by these factors. It appears that to a large extent the different roles played by FasL can be sorted out by the different ways in which it is sorted. Acknowledgements. All authors were supported by grants from the Wellcome Trust (040825 and 050613).
References Allison, J., Georgiou, H. M., Strasser, A., Vaux, D. L. (1997): Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts [see comments]. Proc. Natl. Acad. Sci. USA 94, 3943 ± 3947. Anel, A., Simon, A. K., Auphan, N., Buferne, M., Boyer, C., Golstein, P., Schmitt-Verhulst, A. M. (1995): Two signaling pathways can lead to Fas ligand expression in CD8 cytotoxic T lymphocyte clones. Eur. J. Immunol. 25, 3381 ± 3387. Bonfoco, E., Stuart, P. M., Brunner, T., Lin, T., Griffith, T. S., Gao, Y., Nakajima, H., Henkart, P. A., Ferguson, T. A., Green, D. R. (1998): Inducible nonlymphoid expression of Fas ligand is responsible for superantigen-induced peripheral deletion of T cells. Immunity 9, 711 ± 720. Bossi, G., Griffiths, G. M. (1999): Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells [In Process Citation]. Nature Med. 5, 90 ± 96. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., et al. (1995): Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas [see comments]. Nature 373, 441 ± 444. Dhein, J., Walczak, H., Baumler, C., Debatin, K. M., Krammer, P. H. (1995): Autocrine T-cell suicide mediated by APO-1/(Fas/CD95) [see comments]. Nature 373, 438 ± 441. Diment, S., Eidelman, M., Rodriguez, G. M., Orlow, S. J. (1995): Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells. J. Biol. Chem. 270, 4213 ± 4215. Feng, S., Chen, J. K., Yu, H., Simon, J. A., Schreiber, S. L. (1994): Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science 266, 1241 ± 1247. Glass, A., Walsh, C. M., Lynch, D. H., Clark, W. R. (1996): Regulation of the Fas lytic pathway in cloned CTL. J. Immunol. 156, 3638 ± 3644. Griffith, T. S., Brunner, T., Fletcher, S. M., Green, D. R., Ferguson, T. A. (1995): Fas ligand-induced apoptosis as a mechanism of immune privilege [see comments]. Science 270, 1189 ± 1192. Griffiths, G. M. (1997): Protein sorting and secretion during CTL killing. Semin. Immunol. 9, 109 ± 115. Griffiths, G. M. (1996): Secretory lysosomes ± a special mechanism of regulated secretion in haemopoietic cells. Trends Cell Biol. 6, 329 ± 332. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L. E., Schneider, P., Bornand, T., Fontana, A., Lienard, D., Cerottini,
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J., Tschopp, J. (1996): Melanoma cell expression of Fas(Apo-1/ CD95) ligand: implications for tumor immune escape [see comments]. Science 274, 1363 ± 1366. Ju, S. T., Panka, D. J., Cui, H., Ettinger, R., el-Khatib, M., Sherr, D. H., Stanger, B. Z., Marshak-Rothstein, A. (1995): Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation [see comments]. Nature 373, 444 ± 448. Kabelitz, D., Pohl, T., Pechhold, K. (1993): Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes. Immunol. Today 14, 338 ± 339. Kang, S. M., Schneider, D. B., Lin, Z., Hanahan, D., Dichek, D. A., Stock, P. G., Baekkeskov, S. (1997): Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction [see comments]. Nature Med. 3, 738 ± 743. Kayagaki, N., Kawasaki, A., Ebata, T., Ohmoto, H., Ikeda, S., Inoue, S., Yoshino, K., Okumura, K., Yagita, H. (1995): Metalloproteinasemediated release of human Fas ligand. J. Exp. Med. 182, 1777 ± 1783. Lau, H. T., Stoeckert, C. J. (1997): FasL ± too much of a good thing? Transplanted grafts of pancreatic islet cells engineered to express Fas ligand are destroyed not protected by the immune system [news; comment]. Nature Med. 3, 727 ± 728. Lau, H. T., Yu, M., Fontana, A., Stoeckert, C. J., Jr. (1996): Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice [see comments]. Science 273, 109 ± 112. Lowin, B., Mattman, C., Hahne, M., Tschopp, J. (1996): Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes. Int. Immunol. 8, 57 ± 63. Lynch, D. H., Ramsdell, F., Alderson, M. R. (1995): Fas and FasL in the homeostatic regulation of immune responses [see comments]. Immunol. Today 16, 569 ± 574. Martinez-Lorenzo, M. J., Alava, M. A., Anel, A., Pineiro, A., Naval, J. (1996): Release of preformed Fas ligand in soluble form is the major factor for activation-induced death of Jurkat T cells. Immunology 89, 511 ± 517. Nagata, S., Golstein, P. (1995): The Fas death factor. Science 267, 1449 ± 1456. O Connell, J., O Sullivan, G. C., Collins, J. K., Shanahan, F. (1996): The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med. 184, 1075 ± 1082. Orci, L., Stauffacher, W., Rufener, C., Lambert, A. E., Rouiller, C., Renold, A. E. (1971): Acid phosphatase activity in secretory
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granules of pancreatic beta cells of normal rats. Diabetes 20, 385 ± 388. Owen-Schaub, L. B., van Golen, K. L., Hill, L. L., Price, J. E. (1998): Fas and Fas ligand interactions suppress melanoma lung metastasis. J. Exp. Med. 188, 1717 ± 1723. Schneider, P., Holler, N., Bodmer, J. L., Hahne, M., Frei, K., Fontana, A., Tschopp, J. (1998): 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. Seino, K., Iwabuchi, K., Kayagaki, N., Miyata, R., Nagaoka, I., Matsuzawa, A., Fukao, K., Yagita, H., Okumura, K. (1998): Chemotactic activity of soluble Fas ligand against phagocytes. J. Immunol. 161, 4484 ± 4488. Simmen, T., Schmidt, A., Hunziker, W., Beermann, F. (1999): The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal. J. Cell Sci. 112, 45 ± 53. Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H., Galle, P. R. (1996): Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligandexpressing tumor cells ± a mechanism of immune evasion? [see comments]. Nature Med. 2, 1361 ± 1366. Suda, T., Okazaki, T., Naito, Y., Yokota, T., Arai, N., Ozaki, S., Nakao, K., Nagata, S. (1995): Expression of the Fas ligand in cells of T cell lineage. J. Immunol. 154, 3806 ± 3813. Swank, R. T., Brandt, E. J. (1978): Turnover of kidney beta-glucuronidase in normal and Chediak-Higashi (beige) mice. Am. J. Pathol. 92, 755 ± 769. Tanaka, M., Itai, T., Adachi, M., Nagata, S. (1998): Downregulation of Fas ligand by shedding [see comments]. Nature Med. 4, 31 ± 36. Tanaka, M., Suda, T., Haze, K., Nakamura, N., Sato, K., Kimura, F., Motoyoshi, K., Mizuki, M., Tagawa, S., Ohga, S., Hatake, K., Drummond, A. H., Nagata, S. (1996): Fas ligand in human serum. Nature Med. 2, 317 ± 322. van den Broek, M. E., Kagi, D., Ossendorp, F., Toes, R., Vamvakas, S., Lutz, W. K., Melief, C. J., Zinkernagel, R. M., Hengartner, H. (1996): Decreased tumor surveillance in perforin-deficient mice. J. Exp. Med. 184, 1781 ± 1790.
European Journal of Cell Biology 79, 539 ± 543 (2000, August) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
539
Sorting out the multiple roles of Fas ligand Giovanna Bossia, Jane C. Stinchcombea, Lesley J. Pageb, Gillian M. Griffithsa1) a b
Sir William Dunn School of Pathology, Oxford/UK Imperial Cancer Research Fund Laboratories, London/UK
Fas ligand (CD95 ligand) ± cytotoxic T lymphocyte ± sorting ± secretory lysosome Fas ligand can both be used by the immune system to initiate cell death, and be used by non-lymphoid cells to evade death. Recent work has shown that Fas ligand is differentially sorted in different cell types. Here we present the viewpoint that the differential sorting plays an important part in determining the role of Fas ligand in different cells. Fas ligand (FasL) is a protein with multiple roles. When expressed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, FasL is used to destroy virally infected or tumorigenic targets and plays a critical role in cellular destruction by the immune system (Nagata and Golstein, 1995). However, when expressed by the epithelial cells of the eye, FasL creates an immunologically privileged site by destroying any activated lymphocytes which enter (Griffith et al., 1995) (Figure 1). FasL has been shown to play a critical role in immune regulation by T cell deletion (Lynch et al., 1995) and triggering an inflammatory response by activating polymorphonuclear lymphocytes (PMNs) (Seino et al., 1998). Since FasL can both be used to initiate cell death by the immune system and to protect against cell death by the immune system how do different cell types determine which role FasL is to play? One very simple mechanism for regulating the activity of FasL, which has been overlooked, is the control of FasL appearance on the cell surface. In CTLs and NK cells FasL expression is tightly controlled so that only specific targets are killed. Constant cell surface expression of FasL in CTL and NK cells would be very dangerous, resulting in indiscriminate killing by these cells. However in the epithelial cells of the eye FasL is expressed constitutively on the cell surface in order to maintain the immunological privilege of this site, by destroying any activated cells entering. How is the surface expression of FasL differentially controlled in order to permit such diverse roles for the same protein? Initial studies showed that FasL is transcriptionally upregulated upon T cell activation and results Dr. Gillian M. Griffiths, Sir William Dunn School of Pathology, Oxford, OX1 3RE/UK, e-mail: [email protected], Fax: 44 18 65 27 55 15.
1)
in new protein synthesis (Anel et al., 1995; Suda et al., 1995). The problem with control by new protein synthesis alone is that it provides no mechanism for regulating the FasL appearance on the cell surface or for focusing FasL at the target cell interface during target cell death. So we decided to ask how FasL was sorted to the cell surface in CTLs, NK cells and epithelial cells. We discovered that FasL is differentially sorted in lymphoid versus non-lymphoid cells (Bossi and Griffiths, 1999). We propose that differential sorting in different cell types plays a critical role in controlling the very different functions of FasL.
Intracellular storage of FasL in CTLs and NK cells Newly synthesised FasL can be detected within CTLs and NK cells using an antibody against the extracellular domain (Kayagaki et al., 1995). Since these cells possess a very effective metalloprotease on their cell surfaces any FasL reaching the cell surface immediately loses it s extracellular domain and the antibody no longer recognises the protein. This enabled us to demonstrate that in CTL and NK cells newly synthesised FasL is delivered to the lytic granules, and only released upon degranulation. Increasing evidence indicates that extensive crosslinking of the membrane-bound form of FasL is required for effective induction of apoptosis (Schneider et al., 1998). The granule localisation of FasL in CTLs and NK cells is therefore important in several respects. First the focusing and concentration of FasL at the point of membrane contact (Figure 2) during target cell killing will allow extensive crosslinking upon engagement with target cell Fas. Second, storage of FasL in the granule protects against premature cleavage of the protein by the metalloprotease, ensuring that the effective membrane-bound form will be exposed in response to TcR-mediated recognition of the target. Thirdly, the storage of FasL in the lytic granules means that both perforin and FasL mechanisms of target cell destruction are delivered together. This third point explains why it has often been difficult to entirely separate the contribution of perforin and Fas pathways in target cell death (van den Broek et al., 1996). The fact that the two effector
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3 Fig. 1. Two very different roles for Fas ligand. (Top) FasL is used by T cells to destroy virally infected cells. TcR recognition of the target leads to FasL expression by the T cells. FasL crosslinks Fas and triggers apoptosis in the target. (Bottom) Conversely, FasL is expressed by epithelial cells of the eye to create an immunologically privileged site. FasL on the epithelial cells engages Fas on activated T cells and destroys them before they are able to damage the eye.
Fig. 2. FasL is sorted differently in different cell types. (Top) In CTL, NK and mast cells FasL is sorted directly to the secretory lysosomes where it is stored as shown by co-staining FasL (green) and cathepsin D (red) in the human NK cell line YT. (Bottom) In epithelial cells FasL is expressed continuously on the cell surface and maintains the immunological privilege of these cells. This is reflected in the sorting of FasL in the epithelial cell line HeLa transfected with GFP-FasL (green), costained with cathepsin D (red), where FasL is sorted directly to the cell surface. !
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proteins are stored together means that both mechanisms are likely to contribute together in many situations.
Differential sorting of FasL The storage (Bossi and Griffiths, 1999; Lowin et al., 1996; Martinez-Lorenzo et al., 1996) and triggered release (Bossi and Griffiths, 1999) of FasL explains how CTLs and NK cells regulate the surface appearance of FasL, but does not explain how the control differs in cells which use FasL to create an immunologically-privileged environment. The answer lies in the finding that FasL is differentially sorted in haemopoietic and non-haemopoietic cells (Bossi and Griffiths, 1999). FasL is sorted to the secretory granules of haemopoietic cells, but travels directly to the cell surface in most non-haemopoietic cells. When expressed on epithelial cell lines or fibroblasts FasL is found abundantly on the cell surface, but when expressed in a mast cell line or in NK cells FasL is found only intracellularly and not on the cell surface (Figure 2) (Bossi and Griffiths, 1999). This indicates that FasL contains a specific sorting signal which targets it to the secretory granules of haemopoietic cells, but that this signal is not recognised in nonhaemopoietic cells in which FasL follows the default pathway directly to the cell surface. Consequently FasL surface expression is tightly regulated in haemopoietic cells where it is used to kill recognised targets, but constitutive in nonhaemopoietic cells in which constant expression is required to maintain immune privilege.
Molecular basis of differential sorting How does this differential sorting occur? The majority of nonlymphoid cells use separate lysosomes and secretory granules. However in haemopoietic cells these two organelles are combined and these ªsecretory lysosomesº function as both the secretory granules and lysosomes of these cells (reviewed in (Griffiths, 1996)). The finding that FasL is only sorted directly to the secretory lysosomes of haemopoietic cells indicates that it contains a granule/lysosomal targeting signal that is only recognised in cells of this lineage. The sorting signal known to be in the cytoplasmic tail of FasL (Bossi and Griffiths, 1999) has been recently mapped to a sequence enriched in proline in the region proximal to the transmembrane domain (Bossi and Griffiths, unpublished data). Identification of the lineage-specific machinery which recognises this signal would be the next step. Since SH3 domain binds to polyproline sequence (Feng et al., 1994), an SH3 domain-containing protein could be part of the sorting machinery. Other evidence supports the idea that haemopoietic cells use specialised mechanisms for sorting proteins to secretory lysosomes. Perforin and granzyme A, soluble proteins found within lytic granules of CTL (reviewed in (Griffiths, 1997)), can only be sorted correctly to the secretory lysosomes of haemopoietic cells. When expressed in conventional secretory cells they are neither sorted to the lysosome nor the secretory granule (L. J. Page and G. M. Griffiths, manuscript in preparation). Similarly the small number of non-lymphoid cells which possess secretory lysosomes (e.g. melanocytes (Diment et al., 1995), renal tubular cells (Swank and Brandt, 1978) and some
Sorting out the multiple roles of Fas ligand 541
pancreatic cells (Orci et al., 1971)) may also use some distinct sorting mechanisms. Recent work suggests that tyrosinase may also be sorted to the secretory lysosomes of melanocytes via a specialised pathway. Although tyrosinase is targeted to lysosomes when expressed in non secretory cells (Simmen et al., 1999), it does so via a pathway which is at least partially distinct from that used by other lysosomal proteins (M. Marks, personal communication). Taken together these findings suggest that cells possessing secretory lysosomes have sorting pathways which are not found in other cell types. Although the precise molecular details of the differential sorting are not yet known several of the biological mysteries of FasL can now be reconsidered in the light of these findings.
FasL and immune privilege The initial findings that FasL expression could confer immune privilege in the eye and testes, resulted in a host of attempts to use FasL expression to prevent transplant rejection. In some studies FasL-expressing cells conferred protection (Lau et al., 1996) while others did not (Kang et al., 1997; Lau and Stoeckert, 1997). The cell type in which FasL was expressed seemed to be crucial for successful protection from rejection. Allogeneic pancreatic islets co-transplanted with syngeneic myoblasts expressing FasL were protected from rejection, while islet cells expressing FasL themselves were not. Given that FasL is differentially sorted in cells with secretory lysosomes this raises the possibility that the reason some cell types failed to confer protection was due to the fact that the FasL was stored within the cell rather than expressed directly on the cell surface. Interestingly, early studies indicate that lysosomal and secretory products are co-localised in islet beta cells (Orci et al., 1971), raising the possibility that FasL may not reach the surface of these cells without secretory granule release. The whole picture is certainly more complicated, since islets expressing FasL produce a more rapid rejection and insulitis, following neutrophil recruitment which can be induced by soluble FasL (Allison et al., 1997; Lau and Stoeckert, 1997). However, the fact that FasL cell surface expression may require granule release in some cell types clearly becomes an important consideration in preventing rejection of grafts. Another important factor in expression of FasL at the cell surface is the expression of the metalloprotease. In CTLs and NK cells cleavage of FasL by this cell surface protease is so effective that it is impossible to detect cell surface expression of FasL in the absence of protease inhibitors. Since FasL can be detected on epithelial cells involved in immune privilege, then the protease must be lacking. Although the cleavage site of FasL in two different cell types has been determined (Schneider et al., 1998; Tanaka et al., 1998) the identity and distribution of the metalloprotease is not currently known.
CD4 T cells possess secretory lysosomes In CD4 T cells FasL plays a critical role in immune regulation and deletion of autoreactive cells by ªactivation-induced cell deathº (AICD). TcR stimulation of T cells leads to proliferation and activation of primary T cells, but to AICD in previously activated T cells (reviewed in (Kabelitz et al., 1993;
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542 G. Bossi, J. C. Stinchcombe et al.
Lynch et al., 1995)). Consistent with this we found that in CD4 human T cell clones FasL is stored in secretory lysosomes which require TcR activation before they will expose FasL on their surface (Bossi and Griffiths, 1999). Since primary T cells neither express FasL, nor contain secretory lysosomes, then this explains why they do not undergo AICD. Studies on T cell hybridomas and the T cell line Jurkat indicated that FasL/Fas interactions were essential for AICD (Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995). Recent studies have revealed that AICD in non-transformed cells does not proceed in a cell autonomous manner and rather depends upon FasL expression induced on non-lymphoid tissue during the immune response (Bonfoco et al., 1998). The reasons for this discrepancy between the cell lines and the nontransformed T cells in vivo is not immediately obvious. However one possible explanation could be the differential targeting of FasL in transformed and hybridoma cells versus untransformed T cells. For example, cloned murine CTL have been shown to require activation in order to up-regulate Fas ligand on the cell surface, while the T cell lymphoma expresses FasL constitutively on the cell surface (Glass et al., 1996). It is therefore possible that T cell hybridomas, derived by fusion of normal T cells with a thymic lymphoma cell line, may well reflect the lymphoma phenotype and sort FasL to the cell surface. This might then account for the susceptibility to AICD from other T cells.
Tumor evasion of the immune system via Fas and Fas ligand A similar disruption of sorting seems to occur in tumorigenic cells in vivo. Large granular lymphocyte (LGL) and NK lymphomas derived from T and NK cells showed both increased surface expression of FasL and increased serum levels of soluble FasL (Tanaka et al., 1996). This contrasts with T cells from healthy individuals which require activation before expressing FasL on the surface or shedding soluble FasL into the serum. These findings indicate not only that the tumors are constitutively expressing FasL, but that it is now constitutively expressed on the cell surface suggesting that the granule sorting pathway has either been disrupted or overloaded in the tumors. Tumors, derived from other cell types have been shown not only to express FasL (Hahne et al., 1996; O Connell et al., 1996; Strand et al., 1996), but also to have downregulated Fas expression (Owen-Schaub et al., 1998; Strand et al., 1996). So while the absence of Fas allows the tumors to escape FasL mediated cell death by the immune system, the expression of FasL on the surface of the tumors enables them to destroy activated lymphocytes and avoid cell death mediated by alternative effector mechanisms. Escape of the immune system by these cells requires constitutive cell surface expression of FasL on the cell surface. This will be influenced by the sorting machinery and the metalloprotease activity of the cells from which the tumor is derived.
Conclusions The finding that FasL is differentially sorted in different cell types has important implications for future research. First it is no longer sufficient to demonstrate that FasL is expressed by a
cell to imply that it is active on the cell surface. Rather it is important to know where in the cell FasL is found and how it reaches the cell surface. Secondly determining the mechanisms which mediate differential sorting in different cell types will be important in understanding how to regulate many other proteins which may also use this route in haemopoietic cells. Both the route via which FasL is sorted and the presence or absence of the metalloprotease which cleaves membranebound FasL into soluble FasL appear to determine the function of FasL. Given that effective killing by FasL requires extensive crosslinking of the membrane-bound form; while the soluble form is a potent mediator of neutrophil recruitment, then the ultimate function of FasL will be determined by these factors. It appears that to a large extent the different roles played by FasL can be sorted out by the different ways in which it is sorted. Acknowledgements. All authors were supported by grants from the Wellcome Trust (040825 and 050613).
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