Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, cartilage damage, and bone erosion. The hallmark of RA is the formation of tumor-like inflammatory tissue that has a propensity to invade bone. Upon initiation of disease, the synovial membrane becomes hyperplastic due to the accumulation of fibroblasts and cells of hematopoietic origin. This alteration of the synovial membrane is followed by the destruction of neighboring structures, such as bone and articular cartilage, due to invasive properties of synovial tissue. This latter property of inflamed synovial tissue is based on a tight interplay between synovial fibroblasts, lymphocytes, and osteoclasts.
This complex series of pathologic events leading to full-blown arthritis is governed by various proinflammatory cytokines that allow communication between inflammatory cells, culminating in concerted actions such as tissue invasion. Several of these proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6, are of particular clinical importance, because their blockade ameliorates RA (1–3). Most importantly, the role of TNF in chronic arthritis has been convincingly demonstrated in many ways, as follows: in the setting of RA, TNF is overexpressed by synovial membrane cells (4), systemic overexpression of TNF leads to an RA-like disease in rodents (5), and the 3 TNF blockers currently approved for the treatment of RA are highly effective in reducing signs and symptoms of RA and retarding joint destruction (3, 6, 7). Moreover, TNF itself induces proinflammatory cytokines, enhances the generation of matrix metalloproteinase (MMP), and supports the formation of osteoclasts, which are essential tools for generating inflammatory bone loss (8).
The proinflammatory and destructive potential of TNF is mediated through activation of multiple intracellular signal transduction pathways. Among these molecules, p38 MAPK is considered to be one of the most important signals for TNF-mediated inflammatory responses. This supposition is based on 4 important observations: 1) p38 MAPK is strongly activated in RA synovial membrane but not in the synovial membrane of patients with degenerative joint disease (9); 2) active p38 MAPK is predominantly expressed in endothelial and lining layer cells, which represent 2 regions of significant importance when considering the necessity of transendothelial migration by blood-derived cells in the course of inflammation as well as the role of the lining layer in the formation of the destructive pannus (10); 3) synthetic blockers of p38 MAPK have potent antiinflammatory properties and inhibit experimental arthritis in rodents (11); and 4) p38 MAPK is involved in regulating the expression of many proinflammatory cytokines, including TNF (12). Thus, p38 MAPK is pivotally involved in TNF expression induced by lipopolysaccharide or IL-1 and is, therefore, thought to play a major role in inducing TNF expression during inflammatory disease in humans (13). This regulatory function of p38 MAPK on TNF expression is yet another important reason for considering inhibitors of p38 MAPK as promising future antiinflammatory drugs.
So far, however, all of the evidence suggesting p38 MAPK to be importantly involved in joint destruction is circumstantial and indirect, and it is unclear whether activation of p38 MAPK downstream of TNF is a truly relevant factor for the induction of inflammatory—and especially destructive—joint disease in vivo. To investigate this issue, we tested the capacity of 2 specific inhibitors of p38 MAPK to block arthritis in human TNF–transgenic mice. This animal model of arthritis is based on stable transgenic overexpression of TNF leading to inflammatory arthritis. The effects of the two p38 MAPK inhibitors were assessed with respect to interference with synovitis as well as cartilage damage and bone erosion. Based on the important involvement of p38 MAPK in osteoclast differentiation (14), we specifically addressed the ability of this therapeutic approach to block osteoclast differentiation and protect the structural integrity of the inflamed joints.
- Top of page
- MATERIALS AND METHODS
In this study, we investigated the impact of p38 MAPK inhibition on TNF-induced inflammatory arthritis. The results of this proof-of-concept study suggest that activation of p38 MAPK is of central importance in transducing the deleterious effects of TNF on cells of the synovial membrane. We showed that inhibition of p38 MAPK by 2 different specific inhibitors not only decreased the severity of synovial inflammation and cartilage damage but also led to a dramatic protection against bone destruction, when applied in the setting of chronic arthritis induced by the overexpression of TNF. These effects were associated with reduced expression of proinflammatory cytokines, such as IL-1, in the synovial membrane, a decrease of proteoglycan loss from articular cartilage, as well as an impaired differentiation of osteoclasts in inflamed synovial tissue.
The MAPK family member p38 is well known as a major signaling molecule of inflammation. Importantly, p38 MAPK plays a major role in proinflammatory cytokine production by activating transcription factors binding to the promoter regions of many proinflammatory cytokines, including TNF and IL-1 (21). For instance, endotoxin-induced production of TNF is regulated by p38 MAPK activation (22). These observations have stimulated research on inhibitors of p38 MAPK as potential antiinflammatory drug therapy. Indeed, p38 MAPK inhibitors have been successfully tested in animal models of arthritis, such as collagen-induced arthritis, and have also passed early-phase clinical trials (23, 24). Many of these studies have supported the concept that p38 MAPK is importantly involved in the production of proinflammatory cytokines upon initiation of inflammatory arthritis. In addition, however, p38 MAPK itself is also regarded as an important signaling molecule of proinflammatory cytokines. Among 4 different isoforms of p38 MAPK, the α and β isoforms are mainly involved in signaling of cytokines, whereas the δ isoform, which is also expressed in RA synovial tissue, is activated by cytokine-independent mechanisms such as retrotransposable viral sequences termed L1 elements (25).
TNF activates the p38 MAPK pathway through the type I TNF receptor, and this downstream activation of p38 MAPK allows TNF to transduce and communicate inflammatory signals to cells of the involved organ (e.g., the synovial membrane) (26). Because signaling of type I TNF receptor is rather complex and, apart from p38 MAPK, involves other MAPK families as well as NK-κB, the impact of p38 MAPK signaling on the systemic inflammatory effects of TNF is rather poorly understood. In this model, we made use of the stable overexpression of TNF due to a transgene to investigate the impact of p38 MAPK as a downstream mediator of the proinflammatory effects of TNF. Thus, this model does not address the production of TNF by p38 MAPK but rather investigates the importance of this signaling molecule as a downstream mediator of continuous high amounts of TNF. In this model, inhibition of p38 MAPK mitigated clinical signs and all major histopathologic features of chronic arthritis, such as synovial inflammation, cartilage damage, and bone loss. This finding suggests that activation of p38 MAPK is a key signaling step in TNF-mediated pathology in the joint.
Upon inhibition of p38 MAPK, TNF-induced formation of inflamed synovial tissue was generally reduced, and no qualitative effect on specific cell populations was noted; rather, the quantity of synovial cellularity was significantly reduced. This points to a general inhibition of synovial inflammation by p38 MAPK blockade rather than specific effects on single cell types, and suggests that p38 MAPK activation is a critical factor in the severity of synovial inflammation. It is compatible with our previous finding of overexpression of p38 MAPK in endothelial cells, allowing us to speculate that p38 MAPK blockade may affect transendothelial migration (27). Furthermore, the observed reduction in the expression of downstream cytokines such as IL-1 and RANKL is also in accordance with the reduction in the inflammatory response. The efficacy of p38 MAPK inhibition was demonstrated by reduced activation of the downstream target MAPKAP-2, whereas phosphorylation of p38 MAPK itself, which is accomplished by upstream kinases, was not altered.
Apart from synovial inflammation, p38 MAPK inhibition beneficially affected cartilage damage. Proteoglycan loss from articular cartilage adjacent to inflamed synovial tissue was significantly reduced, and the efficacy of p38 MAPK inhibition was similar to that observed for synovial inflammation. The protection of articular cartilage might be an indirect effect due to lower expression of proinflammatory cytokines, especially IL-1, which is a key inducer of metalloproteinases. This notion is supported by the observation that matrix synthesis did not increase upon p38 MAPK inhibition.
A reduction in TNF-mediated bone loss was the major hallmark of p38 MAPK inhibition. Tissue invasion into juxtaarticular bone was almost completely abolished with use of p38 MAPK inhibitors. In keeping with this effect, a dramatic reduction in the number of mature osteoclasts was noted. However, this was accompanied by a significant reduction in the number of in vivo osteoclast precursor cells, suggesting that impaired osteoclastogenesis is a consequence of blocking signaling through p38 MAPK. This was further confirmed in studies of in vitro osteoclastogenesis. These observations are consistent with 2 important principles, as follows. First, activation of p38 MAPK is a key signal for osteoclastogenesis. Thus, activation of p38 MAPK is important for the differentiation of mature osteoclasts from their precursors (28); this observation is also relevant for TNF-mediated osteoclastogenesis in relation to bone loss as a result of the inflammatory process involved in arthritis. The second principle is based on the role of osteoclasts in inflammatory bone destruction. Osteoclasts invade juxtaarticular bone in close contact with synovial fibroblasts and T cells and are essential players in this bone resorption. Complete absence of osteoclasts results in blockade of bone resorption but not synovial inflammation, and osteoclast-targeted therapies strongly inhibit bone loss in arthritis (29). Thus, unraveling p38 MAPK inhibition as a potent tool to reduce synovial osteoclast formation and retard inflammatory bone damage is consistent with the above-mentioned concepts.
In summary, blockade of p38 MAPK inhibits arthritis caused by overexpression of TNF. This suggests that p38 MAPK is an important signaling molecule downstream of TNF, and that its inhibition might be a potent tool to interfere with the deleterious effects of TNF on the joint. Most strikingly, inflammatory bone loss depends on activation of p38 MAPK, because formation of osteoclasts in the inflamed synovium depends on intact activation of the p38 MAPK signaling pathway. Effective blockade of p38 MAPK might therefore be regarded as an interesting therapeutic option to protect joints from the destructive attack of inflamed synovial tissue.