Fas-mediated cytotoxicity is not required for rejection of murine nonvascularized heterotopic cardiac allografts

BACKGROUND: Using mice with loss-of-function mutations in the Fas and Fas ligand (FasL) genes (lpr and gld, respectively) in transplantation experiments has resulted in contradictory findings concerning the role of Fas/FasL-mediated cytotoxicity in allograft rejection. The observation that these mutant mice develop an abnormal lymphocyte phenotype with increasing age that is hyporesponsive in vitro led us to examine the possibility that this characteristic might explain seemingly discordant observations in the literature. Therefore, to distinguish between the effects of Fas/FasL pathway disruption and the effects of immune senescence on in vivo cytotoxicity and allograft rejection, we evaluated the survival of cardiac allografts in gld, lpr, and wild-type mice of varying ages. METHODS: Six- to 21-week-old C3H, C3H/HeJ-Fasl(gld), C57B1/6, and B6.MRL-Fas(lpr) recipients were transplanted with heterotopic, nonvascularized cardiac allografts from neonatal Balb/c, C3H, C57Bl/6, and B6.MRL-Fas(lpr) donors. Mixed lymphocyte reactions were performed in naive gld, lpr, and wild-type animals, 6 and 12 weeks of age. Rejected allografts in gld, lpr, and wild-type recipients and functioning syngeneic transplants were evaluated for intragraft apoptosis by a DNA fragmentation detection assay. RESULTS: Graft survival was not significantly different between 6-week-old gld and lpr recipients and their respective wild-type controls. However, allograft rejection was delayed significantly in older (13-week) gld mice compared with age-matched wild-type mice (P=0.02) or young (6-week) gld animals (P=0.04). Similarly, 21-week-old lpr mice exhibited prolonged graft survival compared with 6-week-old lpr animals (P=0.01). Reduced alloreactive proliferative responses in 12-week-old gld and lpr mice were observed when compared with age-matched wild-type strains. Rejecting allografts displayed a similar level of intragraft apoptotic cells regardless of mutant or wild-type phenotype or age of recipient. CONCLUSIONS: The findings of this study confirm that Fas/FasL-mediated cytotoxicity is not required for murine cardiac allograft rejection. Our findings also demonstrate that the observed delayed graft rejection in lpr and gld mice is a consequence of an age-related alteration of the immune system, specific to gld and lpr mice and associated with an in vivo and in vitro hyporeactivity to alloantigens.     

Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.     

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2-/-recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas-T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas-) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas-T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.     

Contribution of Fas ligand to cardiac allograft rejection

Effector mechanisms for allograft injury remain unclear. In the present study, we verified the contribution of Fas and Fas ligand (FasL) to cardiac allograft rejection by utilizing the Fas-deficient lpr or FasL-deficient gld mice as the donor or recipient. Cardiac myocytes prepared from normal mice, but not those from lpr mice, constitutively expressed Fas and were susceptible to FasL-mediated lysis. Survival of cardiac allografts was substantially prolonged when gld or lpr mice were used as the recipient. In contrast, cardiac allografts from lpr mice were normally rejected without a delay. Histological examination of the grafts in the gld or lpr recipients demonstrated a lesser cellular infiltration and much milder myocyte damage. Proliferative response and cytotoxic T lymphocyte induction against the donor-type alloantigens were not impaired in the gld or lpr recipients. These results indicate a substantial contribution of FasL to cardiac allograft rejection, independent of Fas in the grafts. This ralses a possibility that FasL may be more generally involved in tissue damage associated with various diseases than expected from the expression of Fas in the target organs.     

Myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis is chronic/relapsing in perforin knockout mice, but monophasic in Fas- and Fas ligand-deficient lpr and gld mice

The expression and action of Fas/Fas ligand (FasL) in multiple sclerosis has been postulated as a major pathway leading to inflammatory demyelination. To formally test this hypothesis, C57BL/6-lpr and -gld mice, which due to gene mutation express Fas and FasL in an inactive form, were immunized with myelin oligodendrocyte glycoprotein peptide(35-55). Whereas in wild-type C57BL/6 mice, experimental autoimmune encephalomyelitis (EAE), was chronic/relapsing, EAE in lpr and gld mice was characterized by a lower incidence of disease and a monophasic course. This contrasts with C57BL/6 perforin knockout mice, which showed the most severe form of EAE of all mouse strains tested, the course being chronic relapsing. The difference noted cannot be attributed to an involvement of FasL in oligodendrocyte damage since oligodendrocytes are insensitive to FasL-mediated cytotoxicity in vitro, and since in the acute phase of EAE gld mice also show CD4+ T cell infiltrates with associated demyelination in brain and spinal cord. Unlike oligodendrocytes, astrocytes were killed by FasL in vitro. It remains to be established whether this latter finding explains the different disease course of lpr and gld mice compared to wild-type and perforin knockout mice.     

Oxidized LDL activates fas-mediated endothelial cell apoptosis

Oxidized low density lipoproteins (OxLDL) promote chronic inflammatory responses in the vasculature that give rise to atherosclerotic plaques. Fas ligand (FasL) is naturally expressed on the vascular endothelium where it can induce apoptosis in Fas-expressing immune cells as they enter the vessel wall. Although vascular endothelial cells are normally resistant to Fas-mediated cell death, OxLDL were shown to induce apoptosis in cultured endothelial cells and endothelium of arterial explants by a process that could be inhibited with Fas L neutralizing antibodies. OxLDL-induced cell death was also reduced in the aortic endothelium cultured from gld (FasL-/-) and lpr (Fas-/-) mice as compared with wild-type mice. OxLDL acted by sensitizing endothelial cells to death signals from the Fas receptor. Thus, the ability of OxLDL to promote Fas-mediated endothelial cell suicide may be a feature that contributes to their atherogenicity.     

Anterior chamber inoculation of splenocytes without Fas/Fas-ligand interaction primes for a delayed-type hypersensitivity response rather than inducing anterior chamber-associated immune deviation

The inoculation of antigens into the anterior chamber (AC) of the eye induces an antigen-specific immune response that inhibits delayed-type hypersensitivity (DTH). This regulatory response is known as anterior chamber-associated immune deviation (ACAID). The ACAID response appears to be complex, as it can be elicited by a wide variety of soluble and cell-associated antigens, including foreign, self, tumor, and alloantigens. To evaluate the contribution of Fas/Fas ligand (FasL) interaction to the induction of ACAID to alloantigens, gld and lpr mutant mice were used in conjunction with normal C3H, MRL, and BALB/c mice. ACAID was induced by inoculation of non-irradiated splenocytes from donor mice into the AC of various recipients. After 1 week, recipients were primed intradermally with donor splenocytes. One week later DTH was measured by ear swelling. C3Hgld mutants lacking functional FasL did not develop ACAID after the AC inoculation of BALB/c splenocytes. Conversely, the AC inoculation sensitized these mutants. MRL/pr mutants, which lack Fas, developed ACAID following inoculation of BALB/c cells. AC inoculation of lpr splenocytes did not induce ACAID, but sensitized C3H recipients. Treatment of the AC inoculum with an anti-Fas antibody blocked ACAID induction in a transient manner, as the recipients developed ACAID later. These results show that interaction of the Fas and FasL is required to induce ACAID to allogeneic cells. In the absence of Fas expression on donor splenocytes, or FasL expression by the recipient, AC inoculation primes for a DTH response rather than inducing ACAID.     

Delayed kinetics of tumor necrosis factor-mediated bystander lysis by peptide-specific CD8+ cytotoxic T lymphocytes

Mouse CD8+ CTL reactive with an H-2Db presented 9-mer peptide of the human papilloma virus 16 (HPV-16) protein E749-57 (RAHYNIVTF) were generated from the splenocytes of wild-type C57BL/6 (B6), B6.perforin-deficient, B6.gld or B6.TNF-deficient mice. In short-term (4 h) assays, CTL from B6, B6.TNF-deficient and B6.gld mice displayed peptide-specific perforin- and/or Fas ligand (FasL)-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7) or E749-57 peptide-pulsed RMA-S cells, while CD8+ CTL from B6.perforin-deficient mice lysed via FasL exclusively. Rapid and efficient lysis of syngeneic bystander B6 spleen T cell blasts by B6, B6.TNF-deficient or B6.perforin-deficient antigen-activated CTL was mediated apparently exclusively by a FasL/Fas mechanism. By contrast CTL from B6.gld mice did not mediate rapid bystander lysis of B6 blasts. Rather B6.gld CTL delivered delayed bystander lysis after 36-48 h that was mediated by TNF. TNF-mediated bystander lysis of syngeneic blasts appeared to be independent of class I molecules and was mediated at least in part by soluble TNF. By contrast, there was no evidence that soluble FasL-mediated bystander lysis. For the first time, these data indicate that CD8+ CTL may use FasL or TNF in a kinetically and physically distinct fashion to mediate bystander killing.     

Death of solid tumor cells induced by Fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressing muscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Evidence for the involvement of Fas ligand and perforin in the induction of vascular leak syndrome

Endothelial cell injury resulting in vascular leak syndrome (VLS) is one of the most widely noted phenomenons in a variety of clinical diseases. In the current study we used IL-2-induced VLS as a model to investigate the role of cytolytic lymphocytes in the cytotoxicity of endothelial cells. Administration of IL-2 (75,000 U/mouse, three times a day for 3 days) into BL/6 wild-type mice triggered significant VLS in the lungs, liver, and spleen. Interestingly, perforin-knockout (KO) mice exhibited a marked decrease in IL-2-induced VLS in all three organs tested. Also, Fas ligand-defective (gld) mice and Fas-deficient (lpr) mice exhibited decreased VLS in the liver and spleen, but not in the lungs. The decreased VLS seen in perforin-KO, gld, and lpr mice was not due to any defect in lymphocyte migration or homing to various organs because histopathologic studies in these mice demonstrated significant and often greater perivascular infiltration of lymphocytes compared with the IL-2-treated wild-type mice. Ultrastructural studies of the lungs demonstrated significant damage to the endothelial cells in IL-2-treated wild-type mice and decreased damage in perforin-KO mice. IL-2 administration caused up-regulation of CD44 in all strains of mice tested and triggered increased LAK activity against an endothelial cell line in wild-type and gld mice, but not in perforin-KO mice. The current study demonstrates for the first time that perforin and Fas ligand may actively participate in endothelial cell injury and induction of VLS in a variety of organs.     

Is epsilon-aminocaproic acid as effective as aprotinin in reducing bleeding with cardiac surgery?: a meta-analysis

Infection of BALB/c mice with Trypanosoma cruzi resulted in up-regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation-induced CD4+ T cell death (AICD), and in Fas: FasL-mediated cytotoxicity. When CD4+ T cells from infected mice were co-cultured with T. cruzi-infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi-infected FasL-deficient BALB gld/gld mice had no detectable AICD in vitro and their activation with anti-TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld/gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild-type mice. Secretion of Th2 cytokines IL-10 and IL-4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti-IL-4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild-type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas: FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2-type immune response to the parasite.     

T-lymphocyte downregulation after acute viral infection is not dependent on CD95 (Fas) receptor-ligand interactions

Infection of mice with lymphocytic choriomeningitis virus (LCMV) causes a major expansion of CD8+ T cells followed by a period of immune downregulation that coincides with the induction of lymphocyte apoptosis in the mouse spleen. CD95 (Fas) and its ligand are important for regulating peripheral T-lymphocyte numbers and can mediate apoptosis of mature T lymphocytes. We infected CD95- and CD95L-deficient mice (lpr and gld, respectively) with LCMV to determine if the immune downregulation that occurred following resolution of the LCMV infection was due to a CD95-dependent apoptotic mechanism. Lymphocytes from LCMV-infected lpr and gld mice were capable of normal T-cell expansion and cytolytic function but were, in contrast to activated cells from normal virus-infected mice, relatively more resistant to T-cell receptor-induced apoptosis in vitro. However, in vivo there were significant numbers of apoptotic cells in the spleens of lpr and gld mice recovering from the infection, and the T-cell number and cytolytic activity decreased to normal postinfection levels. Thus, CD95 is not required for the immune downregulation of the CD8+-T-lymphocyte response following acute LCMV infection.     

Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death

The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.     

Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production

MRL/MP-lpr/lpr (MRL/lpr) mice have a single mutation (lpr) of the fas apoptosis gene. The mutant mice developed significantly smaller lesions than the wild-type mice at the earlier stage of infection with the intracellular protozoan parasite Leishmania major. However, while all the wild-type mice achieved complete lesion resolution, the disease in the mutant mice progressed inexorably. The mutant mice had more IL-12 and nitrite/nitrate in the serum than wild-type mice following infection. Lymphoid cells from infected MRL/lpr mice produced more IFN-gamma but less IL-4 and IL-5 than cells from MRL-+/+ mice. Peritoneal macrophages from the mutant mice also produced more IL-12 and NO after stimulation with LPS. Thus, Fas expression is essential for resistance against leishmaniasis, and Fas-mediated apoptosis may form an integral part of the Th1-mediated microbicidal function.     

Mechanism of radioprotection conferred by the immunomodulator AS101

AS101 [ammonium trichloro (dioxyethylene-0-0') tellurate] is a new synthetic compound previously described by us as having immunomodulating properties and minimal toxicity. Clinical trials are currently in progress with AS101 in AIDS and cancer patients. AS101 has recently been found to have radioprotective effects on hemopoiesis in irradiated mice when administered prior to irradiation. Since the early progenitors, spleen colony-forming units (CFU-S), are the critical cells needed for the reconstitution of the hemopoietic system, the mechanisms of action of AS101 were explored in this study by examining the compound's effect on the recovery of CFU-S, its protective effect on endogenous CFU-S and its effect on self-renewal of CFU-S. We also studied the effect of AS101 on the induction of progenitor cells into the radioresistant S-phase of the cell cycle. On days 1 and 5 after irradiation, the number of CFU-S in the bone marrow and spleen of AS101-treated mice was significantly higher than that of PBS-injected mice. Nine days after sublethal doses of irradiation, the number of endogenous spleen colonies was highest in mice given AS101 every 24 hours or every other day for 1 week prior to irradiation. AS101 administered immediately after irradiation, however, also resulted in an increase in the endogenous CFU-S. The higher number of CFU-S found in each 9-day endogenous spleen colony suggests increased self-renewal of CFU-S in AS101-treated mice. Moreover, we found that AS101 induced a higher number of progenitor cells in the S-phase of the cell cycle. These findings suggest that the radioprotection conferred by AS101 results from induction of progenitor cells in DNA synthesis (S-phase) and from the enhanced stimulation of CFU-S, not only toward proliferation but also toward CFU-S self-renewal.     

Role of spontaneous and interleukin-2-induced natural killer cell activity in the cytotoxicity and rejection of Fas+ and Fas- tumor cells

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)-induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas- tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell-mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2-induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas- targets to poly I:C and IL-2-induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas- tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas- tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas- tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas- (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2-injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas- targets. Also, L1210 Fas+ tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas- tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2-induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.     

Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8(+) and CD4(+) T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8(+) than CD4(+) T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.     

Enhancement of ectopic bone formation in mice with a deficit in Fas-mediated apoptosis

Bone formation is under the control of cytokines as well as growth factors such as bone morphogenetic proteins (BMP). This suggests the possibility that osteogenesis might be modulated by factors which also modulate the immune system. To test whether immune disorders in the host may influence bone formation, we studied BMP-induced bone formation in a C3H/HeJ strain of mice bearing a mutant gene, the lymphoproliferation gene (lpr) or the generalized lymphoproliferative disease gene (gld), both of which are known to be a Fas deletion mutant and a Fas ligand mutant, respectively, and to induce immune disorders via a deficit in Fas-mediated apoptosis. Crude BMP derived from bovine bone were injected into the muscular tissue in the femur of adult C3H/HeJ mice or C3H/HeJ mice bearing an lpr or gld gene. Quantitative analysis of the resulting ectopic bone formation by X-ray photography 2 weeks after injection revealed that the presence of either the lpr or gld gene caused a bone mass significantly larger in dimension than that seen in the wild type mice. Histological examination also revealed the different influence between these mutant genes on the level of bone formation exhibited by hyaline cartilage and bone trabeculae. Based on these results, we discussed the possible mechanisms of the enhanced ectopic bone formation under the deficit in Fas-mediated apoptosis.