Rheumatoid arthritis (RA) is characterized as both an autoimmune reaction initiated by lymphocytes and a proliferation of inflamed synovial membrane accompanied by inflammatory cell infiltration and bone destruction (1, 2). Bone resorption at the site of lesions in RA is caused by osteoclasts, and this site is the so-called bare area (3). Osteoclasts are multinucleated cells that are formed by the fusion of mononuclear cell precursors (4). Osteoclast precursors, which express the receptor activator of NF-κB (RANK) (5–7), recognize the RANK ligand (RANKL) (8) through cell-to-cell interactions with osteoblasts and/or stromal cells, and they differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) (9, 10). RANKL is also expressed on activated T cells, and activated T cells directly induce osteoclast differentiation in vitro (11, 12). Furthermore, a T cell–derived cytokine, interleukin-17 (IL-17), the levels of which are elevated in the synovial fluid of RA patients, is reported to induce osteoclast differentiation (13). These results indicate that in autoimmune arthritis such as RA, bone resorption is regulated by the immune system (14, 15).
The Fas antigen (CD95), a cell-surface receptor that belongs to the tumor necrosis factor receptor/nerve growth factor receptor family, transduces a cell death signal (16). Fas antigen and Fas ligand have been considered to play an important role in the development of autoimmune diseases because a mutation in Fas and Fas ligand leads to immunologic disorders associated with lymphadenopathy and proliferative arthritis in mice and humans (17–20). Fas is expressed on activated lymphocytes and various other cells, including rheumatoid synovial cells (21). Furthermore, stimulation of Fas by Fas ligand or agonistic anti-Fas monoclonal antibody (mAb) induces apoptosis in various cells (21–25). Thus, it is possible that agonistic anti-Fas mAb may have therapeutic effects in RA. In fact, the anti-mouse Fas antibody RK-8 was shown to effectively ameliorate collagen-induced arthritis in the mouse (26). The anti-human Fas antibody CH-11 was also shown to be effective in SCID mice that had been engrafted with human RA tissues (SCID-HuRAg) (27).
One of the serious problems linked with anti-Fas mAb is hepatotoxicity. We have previously reported that our novel humanized anti-human Fas mAb R-125224, which originated from m-HFE7A (28), induced apoptosis in activated lymphocytes but not in hepatocytes. Administration of R-125224 to SCID-HuRAg mice was shown to reduce the number of inflammatory cells (29). Thus, R-125224 could be a candidate therapeutic agent for use in RA.
In the present study, we investigated whether the induction of apoptosis in activated T cells by R-125224 could suppress osteoclastogenesis in vitro and in vivo. Since there are no useful in vivo animal models for monitoring osteoclastogenesis, we established a novel animal model by modifying the SCID-HuRAg model. Dentin slices were engrafted with RA synovial tissues subcutaneously on the back of SCID mice (SCID-HuRAg-pit). Pits formed on the dentin slices 3 weeks after transplantation. Administration of R-125224 to SCID-HuRAg-pit reduced the number of infiltrated lymphocytes in the transplanted tissues and prevented the formation of pits on the dentin slices. These findings suggest that induction of apoptosis in the infiltrated lymphocytes could be a useful therapeutic strategy for RA, in terms of suppressing both inflammation and bone destruction.
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- MATERIALS AND METHODS
In the present study, we used a coculture system to demonstrate that R-125224, a humanized anti-human Fas antibody, inhibited osteoclastogenesis mediated by activated CD4+ T cells. We found that osteoclasts did not express Fas, which indicates that osteoclasts were not the targets of the R-125224–induced apoptosis. CD4+ T cells, on the other hand, expressed Fas and induced osteoclast differentiation from progenitor cells in the coculture system. Thus, the suppression of osteoclastogenesis induced by R-125224 occurred through the induction of apoptosis in infiltrated T cells in the RA synovium.
It was recently reported that activated T cells promote osteoclastogenesis through RANKL expression and regulate bone resorption in autoimmune arthritis (11, 12). Furthermore, other immunomodulatory factors expressed by T cells, such as IL-17, modulate osteoclastogenesis (13). Activated T cells, however, also produce IFNγ, and IFNγ negatively affects osteoclastogenesis (31). Nevertheless, in RA, the production of IFNγ in the synovium is suppressed (32, 33), whereas RANKL expression is enhanced (34, 35). Based on these data, we included anti-IFNγ antibody in our in vitro coculture systems to reflect the clinical situation in RA.
There were no animal models that could be used to evaluate osteoclastogenesis in vivo. We therefore established such an animal model by modifying our SCID-HuRAg mouse (27, 29, 30). The SCID-HuRAg mouse is a model prepared by transplanting human RA synovial tissue into the subcutaneous tissues on the back of SCID mice. Several weeks after the transplantation, neovascularization was observed in the transplanted tissue. The histologic characteristics of the implanted tissue were similar to those of RA in humans, and immunohistologic analysis revealed that cells in the implanted tissue were of human origin (27, 30). Furthermore, potent antirheumatic drugs, such as methotrexate, which are used in clinical settings, were effective in this model (36). These results indicate that the SCID-HuRAg mouse model well reflects the clinical situation.
We had already determined that the cotransplanted bones tended to become necrotic (data not shown), making it difficult to characterize osteoclastogenesis. Pit formation assays on dentin slices are widely used to monitor osteoclast function in vitro, and it has been reported that TRAP-positive multinucleated cells isolated from RA synovium from resorption pits on dentin slices (37, 38). We therefore used our modified SCID-HuRAg model to conduct a pit formation assay on dentin slices in order to monitor osteoclast function in vivo. The dentin slices were covered with human RA synovium and transplanted into the SCID mice. Pits were not observed at 2 weeks after transplantation, but were observed at 3 weeks after transplantation, indicating that the pits on the dentin slices were probably formed by differentiated osteoclasts.
It is known that human osteoclastogenesis requires human M-CSF and RANKL. Suzuki et al (39) developed an in vitro model of bone destruction using osteoclast-like cells derived from a culture of rheumatoid synovial tissues without any inducers (39). We therefore think that M-CSF and RANKL are provided by the transplanted synovial tissues. Thus, with the use of the new SCID-HuRAg-pit mouse model, we were able to evaluate the suppressive effects of R-125224 on osteoclastogenesis through T cell apoptosis.
Intravenous injection of R-125224 resulted in suppression of osteoclastogenesis in the SCID-HuRAg-pit mouse, as judged from the reduced number of pits on the dentin slices. Histologic analysis of the transplanted tissues revealed that the number of infiltrated lymphocytes around the vessels was decreased after intravenous injection of R-125224. Based on the results of our in vitro and in vivo experiments, R-125224–mediated apoptosis of the infiltrated lymphocytes could be a useful therapeutic strategy for RA in terms of suppressing both inflammation and bone destruction. R-125224 is considered to be a promising therapeutic candidate for RA.