Fas Ligand Shigekazu Nagata* Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan * corresponding author tel: 81-6-6879-3310, fax: 81-6-6879-3319, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.05005.
SUMMARY
Alternative names
FasL is a type II membrane protein belonging to the TNF family, which induces apoptosis in Fas-bearing cells. FasL can be cleaved off from the membrane by a metalloprotease(s), and the soluble FasL displays a much lower cytotoxic activity than its membranebound form. FasL is one of the effector molecules of cytotoxic T cells and NK cells. FasL is also expressed in stroma cells of the eye, and contributes to the eye's privileged immune status. The murine gld (generalized lymphoproliferative disease) mutation is a loss-offunction mutation of FasL. The mice develop lymphadenopathy and splenomegaly, produce a large amount of immunoglobulin, and develop autoimmune diseases. Administration of FasL into these mice causes hepatitis, and quickly kills them. CTL-induced hepatitis or graft-versus-host disease can be inhibited by soluble Fas or neutralizing anti-FasL antibody, suggesting that various forms of CTL-induced tissue destruction are probably caused by overexpression of FasL.
CD95L.
BACKGROUND
Discovery Fas ligand (FasL) was discovered on the cell surface of cytotoxic T lymphocytes (CTLs) (a mouse-rat T cell hybridoma, d10S clone) as a protein that kills the Fas-bearing cells (Rouvier et al., 1993). The FasL protein was purified from the membrane fraction of d10S cells (Suda and Nagata, 1994), and its cDNA was cloned from the d10S cDNA library by expression cloning (Suda et al., 1993). Human FasL cDNA was subsequently identified in human peripheral blood cDNA library by crosshybridization with the rodent cDNA (Takahashi et al., 1994b).
Structure FasL is a type II membrane protein belonging to the TNF family (Suda et al., 1993; Takahashi et al., 1994b).
Main activities and pathophysiological roles FasL kills the Fas-bearing cells by activating the apoptotic program (Enari et al., 1995; Enari et al., 1996; Nagata, 1997; Suda et al., 1993).
GENE AND GENE REGULATION
Accession numbers Human FasL: U11821 Mouse FasL: M83649
Chromosome location FasL is on human chromosome 1q23 (Takahashi et al., 1994b) and mouse chromosome 1 (Takahashi et al., 1994a).
Relevant linkages The mouse FasL gene is linked to gld mutation (Takahashi et al., 1994a).
450 Shigekazu Nagata
Regulatory sites and corresponding transcription factors T lymphocytes can be activated through the T cell receptor to express the FasL gene. This induced expression of FasL in T cells is mediated by NFB and Egr-3 in the 5' flanking region of the FasL gene (Kasibhatla et al., 1999; Matsui et al., 1998; Mittelstadt and Ashwell, 1998). Exposure of T cells to various stress such as UV radiation and gammairradiation also induces the FasL gene expression (Kasibhatla et al., 1998). This induced expression of FasL is dependent on activation of MEKK1 and JNK (Faris et al., 1998), which leads to binding of c-Jun and ATF2 to the AP-1 site on the 5' promoter region of the human FasL gene. Withdrawal of survival factors activates JNK and p38 via a transient activation of MEKK1 and MAPKKK in neuronal cells such as PC12, which induces FasL in nerve cells (Le et al., 1999).
Cells and tissues that express the gene T cells and NK cells express FasL upon activation (Suda et al., 1995; Vignaux et al., 1995; Tanaka et al., 1996). FasL gene in T cells can also be activated by stress such as UV radiation and gamma-irradiation (Kasibhatla et al., 1998). Large granular lymphocytic leukemia (LGL) of NK or T cell origin, and NK lymphoma cells constitutively express FasL (Tanaka et al., 1996). Normal liver does not express FasL, but when damaged with alcohol, it expresses a high level of FasL (Galle et al., 1995). Immune privileged sites such as the eye and testis constitutively express FasL (Bellgrau et al., 1995; Griffith et al., 1995; Suda et al., 1993). Some carcinoma cells such as those of colon carcinoma, hepatocarcinoma, and melanoma were reported to express FasL, constitutively (Hahne et al., 1996; O'Connell et al., 1996; Strand et al., 1996). However one group could not detect FasL in any of the melanoma cell lines examined (Arai et al., 1997).
PROTEIN
Accession numbers Human: PID g595431 (Alderson et al., 1995), g1345957 (Takahashi et al., 1994b); SwissProt P48043 Mouse: PID g729462 (Lynch et al., 1994; Takahashi et al., 1994a); SwissProt P41047
Figure 1 Amino acid sequence for FasL. Human FasL 1 MQQPFNYPYP 31 PCPTSVPRRP 61 PPLPPLPLPP 91 LVALVGLGLG 121 MHTASSLEKQ 151 GKSNSRSMPL 181 LVINETGLYF 211 VYMRNSKYPQ 241 RSSYLGAVFN 271 ESQTFFGLYK
QIYWVDSSAS GQRRPPPPPP LKKRGNHSTG MFQLFHLQKE IGHPSPPPEK EWEDTYGIVL VYSKVYFRGQ DLVMMEGKMM LTSADHLYVN L
SPWAPPGTVL PPPLPPPPPP LCLLVMFFMV LAELRESTSQ KELRKVAHLT LSGVKYKKGG SCNNLPLSHK SYCTTGQMWA VSELSLVNFE
Sequence See Figure 1.
Description of protein A single transmembrane region (22 amino acids) divides the molecule into an N-terminal cytoplasmic region of 80 amino acids, and a C-terminal extracellular region of 179 amino acids. The N-terminal cytoplasmic region is extremely rich in proline residues (about 40% amino acids in this region are prolines) (Suda et al., 1993; Takahashi et al., 1994b). A metalloproteinase cleaves the membrane-bound FasL to produce its soluble form (Kayagaki et al., 1995; Tanaka et al., 1995, 1996). The soluble FasL exists as a homotrimer (Tanaka et al., 1995).
Discussion of crystal structure The crystal structure is not known. However, the amino acid sequence of FasL predicts an elongated, antiparallel -pleated sheet sandwich with a `jelly-roll' topology, based on its similarity to that of TNF (Suda et al., 1993; Peitsch and Tschopp, 1995; Schneider et al., 1997).
Important homologies The amino acid sequence of extracellular region of FasL is significantly related to that of other TNF family members (Suda et al., 1993).
Posttranslational modifications Human FasL has three potential N-glycosylation sites (Asn184, Asn250, and Asn260). N-glycosylation
Fas Ligand 451 seems to be essential for expression of FasL on the cell surface (Schneider et al., 1997; Tanaka et al., 1997).
Regulatory molecules: Inhibitors and enhancers
CELLULAR SOURCES AND TISSUE EXPRESSION
A decoy receptor (DcR3), a soluble receptor belonging to the TNF receptor family, binds to FasL with a Kd of 1 nM, and antagonizes the function of FasL (Pitti et al., 1999). The DcR3 gene is amplified in 50% of cases of human lung carcinoma and colon carcinoma.
Cellular sources that produce T cells and NK cells produce FasL upon activation (Vignaux et al., 1995; Arase et al., 1995; Suda et al., 1995; Tanaka et al., 1996). Stroma cells in the eye constitutively produce FasL (Griffith et al., 1995). Large granular cell leukemia (LGL) cells, NK cell lymphoma cells, and some other cancer cells also constitutively express FasL (Hahne et al., 1996; O'Connell et al., 1996; Strand et al., 1996; Tanaka et al., 1996). Recombinant glycosylated soluble FasL can be produced in a large scale in the yeast Pichia pastoris (Tanaka et al., 1997).
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators
Bioassays used FasL kills Fas-bearing cells, which can be assayed by MTT or [3H]thymidine-incorporation. FasL-induced apoptosis can be directly assayed by staining the cells with fluorescence-labeled Annexin V, by quantitating DNA fragmentation on agarose gel, or by measuring the caspase activity using fluorescent substrates.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles
RECEPTOR UTILIZATION
FasL expressed on the cell-surface of activated T cells kills T cells (activation-induced suicide of T cells) (Russell et al., 1993; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995), as well as the activated B cells (Fukuyama et al., 1998), thus leading to downregulation of immune reaction (Nagata and Golstein, 1995). FasL on cytotoxic T lymphocytes (CTLs) or NK cells works as an effector of these cytotoxic cells (Arase et al., 1994; Ju et al., 1994; Kojima et al., 1994; KaÈgi et al., 1994; Lowin et al., 1994; Oshimi et al., 1996; Bradley et al., 1998; Halaas et al., 1998; Zamai et al., 1998), and kills the cells infected by virus, or cancerous cells. FasL expressed in the stroma cells of the eye counterattacks CTLs and neutrophils infiltrated into the eye, thus contributing to the immuneprivileged state of the eye (Griffith et al., 1995).
FasL specifically binds to its receptor Fas (Kd of about 1 nM).
Species differences
The activation stimuli of T cells such as PMA and ionomycin (Suda et al., 1993), and high doses of IL-2 stimulates FasL gene expression (Suda et al., 1995). The oncogene c-myc activates FasL gene expression in T cells, while TGF inhibits the FasL expression by inhibiting c-myc expression (Genestier et al., 1999). Anticancer drugs and gamma-irradiation were reported to stimulate FasL gene expression in some cancer cells (Friesen et al., 1996; Kasibhatla et al., 1998; Muller et al., 1997).
IN VITRO ACTIVITIES
There is no species-specificity between rat, mouse, and human (Takahashi et al., 1994b).
In vitro findings
Knockout mouse phenotypes
FasL kills cells by activating apoptotic program in Fas-bearing cells.
No FasL-knockout mice have been developed. The spontaneous mouse mutation gld is a loss-of-function
452 Shigekazu Nagata mutation of FasL (Lynch et al., 1994; Takahashi et al., 1994a). FasL in gld mice carries a point mutation in the extracellular region of FasL that causes aggregation of FasL and blocks its binding to the Fas receptor (Schneider et al., 1997). The gld mice develop lymphadenopathy and splenomegaly, produce autoantibodies, and develop autoimmune diseases such as nephritis (Cohen and Eisenberg, 1991).
Transgenic overexpression No FasL transgenic mice have been developed.
Pharmacological effects A high dose of FasL (500 mg, i.v.) causes hemorrhagic necrosis in the liver (Tanaka et al., 1997). When the mice are pretreated with Propionibacterium acnes, they become very sensitive to FasL and administration of 30 mg of FasL will kill the mice within 4 hours by causing hepatitis. The agonistic anti-Fas antibody (Jo2) has been reported to have a stronger cytotoxic activity in vivo than the soluble FasL: an i.p. administration of 50 mg of the antibody kills the mice within 3 hours by causing hepatitis (Ogasawara et al., 1993).
Endogenous inhibitors and enhancers Treatment of cells with IFN and/or TNF sensitizes the cells for killing by FasL or agonistic anti-Fas antibody (Yonehara et al., 1989; Itoh et al., 1991; MoÈller et al., 1993).
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects The serum level of soluble FasL in healthy persons is less than 50 pg/mL (Tanaka et al., 1996). Patients with NK-LGL leukemia, T-LGL leukemia, or NKlymphoma have 0.3±7.0 ng/mL of sFasL in their serum. Treatment of the patients with chemotherapy reduces the serum level of FasL (Sato et al., 1996). Neutropenia and hepatitis, often observed in LGL
patients seems to be due to the FasL produced by leukemia cells. Patients with systemic lupus erythematosus, rheumatoid arthritis, SjoÈgren's syndrome, lymphohistocytosis, and myocarditis, and alcoholic liver disease also show a high level of the soluble FasL in the serum (Nozawa et al., 1997; Hasegawa et al., 1998; Toyozaki et al., 1998). The joints of patients with severe rheumatoid arthritis contain a high level of FasL (Hashimoto et al., 1998).
Role in experiments of nature and disease states Infection of eyes of the mice expressing no functional FasL (gld mice) with herpes simplex virus causes inflammation and severe damage of the eye (Griffith et al., 1995), symptoms that do not occur in the wildtype mice, indicating that FasL blocks the inflammation by killing the inflammatory cells infiltrated into the eye. It has been suggested that exogenous expression of FasL in organ transplants induces tolerance by killing the cytotoxic T cells (Lau et al., 1996). In contrast, the FasL-expressing grafts were rapidly rejected by neutrophils recruited to the grafts (Allison et al., 1997; Kang et al., 1997; Seino et al., 1997; Miwa et al., 1998). Diabetes in NOD mice is not developed in mice lacking Fas (lpr mice) (Chervonsky et al., 1997; Itoh et al., 1997), suggesting that FasL expressed in CTLs is involved in killing cells in the pancreas. Development of experimental allergic encephalomyelitis (EAE) is also significantly reduced in lpr mice (Sabelko et al., 1997; Waldner et al., 1997), suggesting a role of FasL in various forms of CTLinduced tissue destruction.
IN THERAPY
Preclinical ± How does it affect disease models in animals? Administration of an agonistic anti-Fas antibody (anti-Apo1), which has a function similar to that of FasL, kills lymphomas growing in nude mice. This suggested a use for the agonistic antibody or FasL for cancer therapy (Trauth et al., 1989). However, the strong cytotoxicity of FasL or agonistic anti-Fas antibody, e.g. on liver (see above), suggests that a method of delivering FasL specifically to the tumor should be developed before it is used for cancer patients. Soluble Fas (Fas-Fc, the extracellular region fused to the Fc portion of human IgG) or neutralizing
Fas Ligand 453 anti-Fas ligand antibody prevents the development of CTL-induced hepatitis in a mouse model (Kondo et al., 1997). Graft-versus-host disease caused by allogenic bone marrow transplantation can also be blocked by antiFasL antibody, indicating that FasL expressed in alloreactive CTLs is at least in part responsible for development of graft-versus-host disease (Hattori et al., 1998). Adenovirus carrying FasL gene was used to infect tumor cells in vivo (Arai et al., 1997). The tumor cells expressing Fas were killed directly by FasL/Fas interaction. The tumor cells that do not express Fas were also eliminated by inflammatory cells recruited by FasL.
Toxicity Administration of FasL or agonistic anti-Fas antibody to mice kills the mice by causing hepatitis (Ogasawara et al., 1993; Tanaka et al., 1997).
Clinical results No clinical trials with FasL or the antibody against FasL have been carried out.
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LICENSED PRODUCTS Hamster monoclonal antibodies against human FasL (clones 4H9 and 4A5) (Tanaka et al., 1996) are available from Medical & Biological Laboratories (MBL, Nagoya, Japan). Mouse monoclonal antibodies against human FasL (clones NOK1 and NOK2) (Kayagaki et al., 1995) are also available from PharMingen (San Diego). Mouse monoclonal antibody against mouse FasL (clone KAY10) is available from PharMingen (San Diego). Several other anti-FasL polyclonal and monoclonal antibodies are available from various commercial sources. However, some of them may not be specific, and should be used with cautions (Fiedler et al., 1998; Stokes et al., 1998).
