Numerous studies have suggested that genes regulating apoptosis are involved in the pathogenesis of autoimmune diseases, including RA [26
]. Indeed, the success of anti-TNF therapy points to a major role for this important apoptosis pathway in arthritis development [reviewed [30
In this study, we show that the presence of intact Fas, another important apoptosis pathway, enhances the pathogenesis of CIA induced in DBA mice. Immunization of DBA-lpr/lpr mice and their wild-type littermates with collagen II and CFA leads to the development of CIA in both genotypes. Intact Fas is associated with the higher severity and increased incidence of arthritis but is not essential to disease induction. This is in agreement with previous studies in experimental autoimmune encephalomyelitis in C57Bl/6 mice carrying the lpr mutation. These mice had significantly milder disease than their Fas-expressing littermates [31
Fas could contribute to disease in at least two ways: first, it could promote autoimmunity by blocking peripheral tolerance of autoreactive lymphocytes and inhibiting AICD. The role of the Fas molecule in autoimmunity has been well demonstrated in the MRL-lpr mice, and other animal models such as experimental autoimmune encephalitis. A minority of older MRL-lpr/lpr mice developed mild arthritis [21
]. Fas mutation causes impaired T-cell tolerance and lymphoadenopathy, with accumulation of abnormal cells. Thus, defects in peripheral tolerance may play an important role in the pathogenesis of RA. Secondly, Fas could contribute to disease by destroying target tissues through induction of apoptosis of chondrocytes [33
]. Alternatively, Fas could contribute to synovial hyperplasia by inducing proliferation of Fas-expressing synovial fibroblasts and macrophages. Indeed, there is some evidence suggesting that fibroblasts could be activated through surface Fas [34
] and that Fas expression is higher in RA synovial tissues than in osteoarthritic synovial tissues [35
]. One way to clarify this matter is to examine the T-cell and B-cell responses to collagen in lpr/lpr mice. We found that the Fas-deficient T-cell response to collagen II is significantly stronger than that of normal T cells. Since no change of the collagen-II-specific T-cell precursor frequency was observed, this could reflect an increase in the intrinsic proliferative potential of lpr/lpr cells, or a defect in down-regulating the response due to impairment of AICD, or an alteration of regulatory T-cell function. It has been shown that doubly negative T cells, which are increased in lpr mice, have a regulatory function [36
]. Since the suppression of aggressive T-cell responses mediated by regulatory T cells depends on interaction of Fas and Fas ligand, the Fas-deficient doubly negative T cells could fail to suppress peripheral autoimmune T cells, and this failure could lead to an accumulation of aggressive T cells. The significant increase in T-cell proliferation in response to collagen in lpr/lpr mice was accompanied by significantly higher levels of IFN-γ secretion from these cells. This Th1 cytokine has been shown to be abundantly expressed in arthritic lesions both in mice and in humans [37
]. IFN-γ together with other Th1 cytokines predominate in the acute phase of arthritis [40
]. These results exclude the possibility that the mild clinical disease of CIA in lpr/lpr mice is caused by a lack of generation and priming of collagen-II-specific T cells.
A lack of B-cell response also does not appear to be the reason for the mild clinical arthritis in DBA-lpr/lpr mice, since we saw no significant differences in serum anticollagen II IgG antibody levels at time of onset of arthritis at day 20 or during the chronic phase of disease at day 47 between mutant mice and their wild-type littermates. This is rather surprising, as nonimmunized DBA-lpr/lpr had significantly higher levels of anticollagen antibodies than wild-type mice, which almost lacked detectable antibody levels. This indicates the existence of autoreactive collagen-II-specific B cells in DBA-lpr/lpr mice. In summary, all basic elements of a robust pathological immune response are available in DBA-lpr/lpr mice, i.e., Th1 cytokines, proliferating activated autoreactive T cells, and pathological anticollagen II antibodies. The histopathological examination of the inflamed joints from DBA-lpr/lpr and control mice with CIA reveal less inflammation/joint destruction in DBA-lpr/lpr mice in spite of the same clinical score as that of control mice.
The proinflammatory cytokines including TNF-α and IL-6 have been intensively investigated for their role in the pathogenesis of CIA. It is well known that they play a crucial role in the destruction of joints in CIA [37
]. TNF-α induces synovial fibroblasts to express cytokines (such as IL-6) and other factors such as, e.g., matrix metalloproteinases, which contribute to cartilage and bone destruction.
Surprisingly, these proinflammatory cytokines were found at relatively higher levels in joints of DBA-lpr/lpr mice despite milder arthritis in comparison with the normal DBA mice. The mouse fibroblast cell line NIH3T3 is less sensitive to apoptosis induced by anti-Fas mAb and is accompanied by increased expression of TNF-α and IL-6, suggesting an activating effect by Fas ligation. Fas crosslinking may contribute to cartilage and bone destruction by activating synovial fibroblasts subsequently by production of matrix metalloproteinases, growth factors (such as granulocyte/macrophage-colony-stimulating factor), and chemokines. These results indicate that activation by proinflammatory cytokines is insufficient for full disease manifestation when Fas is deficient. Similar results were obtained with synovial macrophages [45
Taking this into consideration, one could draw the conclusion that the lack of the expected severe disease in DBA-lpr/lpr mice is due to a local attenuating effect of the Fas mutation in pathological processes involving resident joint cells. Fas ligation could also play a role in chondrocyte cell death or in activation of macrophages [45
]. There is evidence indicating that antigen-specific T cells are costimulated through the Fas molecule expressed on the T-cell surface. The involvement of Fas in tissue damage has been shown in other tissue-specific autoimmune diseases, namely autoimmune thyroiditis, multiple sclerosis, and insulin-dependent diabetes mellitus. Thyroid cells obtained from patients suffering from autoimmune thyroiditis were shown to express Fas and FasL in response to cytokines and to be targets of Fas-mediated apoptosis [26
]. Similarly, oligodendrocytes purified from multiple sclerosis patients were targets of Fas-mediated apoptosis [27
]. NOD mice, an animal model of insulin-dependent diabetes mellitus with a mutation of the Fas gene (NOD/lpr mice), do not develop diabetes, pointing to a role of the Fas cell-death pathway in tissue damage in this disease as well [29