Distinct roles for the NF-κB1 (p50) and c-Rel transcription factors in inflammatory arthritis (original) (raw)

The Rel/NF-κB family of transcription factors regulate a wide variety of inflammatory mediators, most of which are found in synovial joints of patients with RA (1, 2). Several immunohistochemical studies have reported Rel/NF-κB activation in synovial tissue from RA patients (28, 29) and in animal models of inflammatory arthritis (30, 31), thereby implicating Rel/NF-κB in disease pathogenesis. In this study we used mice homozygous for null mutations in genes encoding the Rel/NF-κB subunits, c-Rel or p50, to obtain direct evidence for Rel/NF-κB involvement in acute, monoarticular, and chronic polyarticular inflammatory arthritis models. Our results show that Rel/NF-κB is essential for the development of inflammatory arthritis and, to our knowledge, provide the first evidence that selective blockade of Rel/NF-κB subunits may prevent it. We also show that the absence of different Rel/NF-κB subunits can have differential effects suggesting distinct roles, rather than redundancy, for the various Rel/NF-κB subunits in different stages of disease.

An interesting result from this study was the almost complete absence of CIA in c-Rel–deficient mice, but a typical response of these mice in the acute arthritis model. CIA is a widely used model of RA thought to be dependent on the establishment of both cellular and humoral immunity to CII (27), although recent evidence suggests that innate immune responses to CII are also important (4). Both a reduced incidence and clinical severity of CIA were exhibited by c-rel–/– mice. In the few c-rel–/– mice that did develop arthritis (4 of 35) it was limited to isolated digits. In contrast, at the histological level, the pannus formation and bone and cartilage erosion in affected joints was comparable to that seen in arthritic WT mice. Therefore, c-Rel is needed for the polyarticular expression of CIA, but its absence is not absolutely protective against CIA. In contrast, p50-deficient mice were completely protected from CIA when assessed both clinically and histologically.

It was reported previously that c-rel–/– and nfkb1–/– mice had impaired Ab responses (14, 15). Production of anti-CII IgM was essentially normal in c-rel–/– mice, but deficient in nfkb1–/– mice. IgG Abs to CII were markedly suppressed in both knockout mice compared with WT mice. Thus c-Rel is required for the switch from an IgM to an IgG response to CII in vivo, which confirms previous in vitro studies on c-rel–/– B cells (32). The absence of high levels of IgG anti-CII could account for the lack of CIA response in both knockout mice, because IgG anti-CII and fixation of complement C5 are considered essential for CIA (33, 34). However, because some c-rel–/– mice did develop limited CIA, it is also possible that high local IgM anti-CII may have substituted for IgG in affected joints. The complete absence of CIA in nfkb1–/– mice, which lacked an IgM response to CII, would be consistent with this hypothesis. Alternatively, the limited CIA observed in c-rel–/– mice may have resulted from innate immune responses to CII (4) that are dependent on p50 but not c-Rel.

Deficiencies in other immune or inflammatory mechanisms may also limit CIA in c-rel–/– and nfkb1–/– mice. Both knockout mice had reduced T-cell proliferative responses to CII (Figure 3), consistent with previous findings that showed a diminished response to mitogens and/or altered production of cytokines by T cells (14, 15, 35–37). However, antigen-presenting cell defects could also be involved. Whereas c-rel–/– and nfkb1–/– mice exhibited both impaired humoral and cellular immune responses to CII that together could account for the dramatic reduction of CIA, other contributing influences could be the cytokine profile and activation state of macrophage populations (15, 38). Macrophages are important both as antigen-presenting cells (39) and as a source of inflammatory mediators in CIA (40, 41).

In contrast to the response of c-rel–/– mice in CIA, these mice were indistinguishable from WT in the acute inflammatory arthritis model, which is dependent on IL-1 and CD4+ T cells, but not B cells. The knee sections of each showed infiltration of neutrophils and mononuclear cells, pannus formation, soft tissue inflammation, and cartilage and bone erosion. This is consistent with the histological features observed in the few CIA-affected joints of the c-rel–/– mice. Taken together, these results suggest that c-Rel is required for establishment of the immune response in CIA but not for the destructive (effector) phase of inflammatory arthritis. In contrast to c-rel–/– mice, nfkb1–/– mice had a markedly diminished response in the acute arthritis model. There were few inflammatory cells in the joint space and connective tissues and a striking reduction in synovial hyperplasia. Since Rel/NF-κB regulates the transcription of adhesion molecules, prostaglandin-producing enzymes, and chemokines (1), this may explain the reduced cellular trafficking into the mBSA-injected joints of p50-deficient mice.

The effect of p50 deletion on T-cell function is unclear. There are conflicting reports on nfkb1–/– T-cell proliferation and cytokine production (15, 36, 37). The p50-deficient mice were protected in a T cell–dependent model of allergic airway inflammation (37); antigen-specific T-cell responses were maintained in these mice, but IL-5 production was reduced. It is possible that different T-cell cytokine profiles in the c-rel–/– and nfkb1–/– mice may account for responses in the acute arthritis model. Both c-rel–/– and nfkb1–/– mice had antigen-specific T-cell proliferative responses, but these were reduced — to a similar degree — compared with WT mice (Figure 3). In spite of this, T cells from c-rel–/– mice were able to elicit a normal response in the T cell–dependent acute arthritis model, but nfkb1–/– mice showed almost no disease.

A recent study showed Rel/NF-κB inhibition enhanced synovial apoptosis in a rat model of arthritis (42), consistent with the potential involvement of Rel/NF-κB transcription factors in synovial hyperplasia. Once formed, pannus is thought to mediate cartilage and bone destruction in RA through matrix metalloproteinase (MMP) production (43). The transcription of MMPs appears to be regulated in part by Rel/NF-κB (44). Inhibition of Rel/NF-κB in human rheumatoid synovial cell cultures reduced proinflammatory cytokine production, together with MMP-1 and MMP-3 (45). In IL-1β–stimulated rabbit synovial fibroblasts, p50 bound to a regulatory element in the distal promoter of MMP-1 (46). These findings, together with the results presented here, suggest p50 is involved in pannus formation and perhaps in regulating joint tissue destruction through effects on MMP gene transcription.

The importance of p50 in synovial cell gene transcription was confirmed by EMSAs, which showed p50/p65 heterodimers and p50 homodimers in nuclear extracts of cells isolated from the acutely inflamed joints of WT and c-rel–/– mice (Figure 5). In contrast, c-Rel was not involved in the activated synovial cell response. Because primary cells were used, the Rel/NF-κB complexes could have been derived from macrophage-like (type A) or fibroblast-like (type B) synovial cells, or both. Using immunohistochemistry, p50 and p65 have been found in type A cells of rheumatoid synovium (28, 29) and in passaged rheumatoid synovial fibroblasts by EMSA (47, 48). We have now also identified p50 homodimers in synovial cell nuclei. Whereas regulation of gene transcription by p50-containing dimers is complex — indeed it has been suggested that p50 homodimers can function as transcriptional repressors (11) — our data clearly demonstrate that the net effect of p50 deletion is a marked reduction of inflammatory joint disease.

In summary, our results show that Rel/NF-κB is essential for the development of inflammatory arthritis and provide strong evidence that selective blockade of Rel/NF-κB subunits may prevent major components of the disease. We also show that the absence of different Rel/NF-κB subunits can have differential effects in acute inflammatory arthritis suggesting distinct roles, rather than redundancy, for the various Rel/NF-κB subunits. Several strategies have been used to block global Rel/NF-κB activity in various disease models (31, 42, 49–51). However, targeting a specific Rel/NF-κB subunit, especially within a given anatomical compartment, may cause fewer side effects than those that inhibit all Rel/NF-κB functions. Such a specific approach with p65 antisense phosphorothioate oligonucleotides was used to prevent experimental colitis (52). Our results support several previous studies that indicate total Rel/NF-κB blockade would prevent inflammatory arthritis (31, 42, 49). However, we have demonstrated that specific Rel/NF-κB subunits may play distinct roles at different stages of disease. Consequently, local inhibition of p50 may prevent joint inflammation and the synovial tissue responses that lead to joint destruction. We conclude that p50 and genes regulated by p50 are targets for rational drug design in inflammatory arthritis.