Rheumatoid arthritis (RA) is characterized as a chronic and progressive inflammatory process that leads to systemic immunologic abnormalities of the joints (1). It has been suggested that proinflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-6, and IL-8, which are linked in a cascade, are important in the etiology of RA (2–5). Histopathologic characterization of bone erosions in patients with RA and in animal models of inflammatory arthritis has provided strong evidence that osteoclasts play an important role in focal, marginal, and subchondral bone loss in inflammatory arthritis (6). Recently, the roles of receptor activator of nuclear factor κB ligand (RANKL), a central regulator of osteoclast recruitment and activation, and TNFα have been shown to be crucial in the pathogenesis of rheumatoid joint destruction (7–11).
Although no conventional medications effectively suppress such joint destruction, TNFα blockers (soluble TNFα receptor fusion protein and TNFα blocking antibody), IL-1 blockers (soluble IL-1 receptor and IL-1 receptor antagonist), and monoclonal antibodies that neutralize IL-6 have all successfully decreased the intensity of synovitis and prevented or retarded the progression of cartilage destruction, both in experimental models and in human trials (12–26). However, these protein products are limited, due to host immune response, rebound of symptoms, a short half-life, and cost (27–29).
FR167653 was first discovered as a potent inhibitor of TNFα and IL-1β production in lipopolysaccharide-stimulated human monocytes and phytohemagglutinin-M–stimulated human lymphocytes (30, 31). FR167653 inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by suppressing the phosphorylation of p38 MAPK, preferentially in the α isoform, but not in the γ isoform (32–34). In addition, p38 MAPK is involved in production of IL-6 and IL-8 induced by TNFα and IL-1β (1, 35).
Collagen-induced arthritis (CIA) is a widely used experimental model of polyarthritis that has many histopathologic features in common with RA. TNFα, IL-1β, IL-6, and IL-8 also play an important role in the pathogenesis of CIA (5, 36–39). In this study we subcutaneously injected FR167653 into rats with CIA. This study is the first to demonstrate that this p38 MAPK inhibitor effectively prevents the onset and progression of CIA in rats.
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Because p38 MAPK regulates inflammatory cytokines such as TNFα and IL-1β, several researchers have used inhibitors of p38 MAPK in inflammatory disease models in vivo, including adjuvant-induced arthritis, and their results have confirmed the effectiveness of these inhibitors (30–34, 45–49). However, the role of p38 MAPK in the CIA model has not been previously investigated. The current study is the first to elucidate the effects of FR167653, a potent p38 MAPK inhibitor, which completely prevented the onset of CIA and also markedly improved the symptoms of inflammatory changes even after the onset of arthritis, with significant reductions in radiographic and histologic degrees of joint injury.
By what mechanism does FR167653 protect the joint against inflammatory injury? There is evidence that the proinflammatory cytokines TNFα and IL-1β help to propagate the extension of a local or systemic inflammatory process. We confirmed that the inflammatory process in the untreated CIA rats led to substantial increases in the serum levels of TNFα and IL-1β and in the ankle-joint concentration of IL-1β. The serum and ankle-joint concentrations of these proinflammatory cytokines were significantly lower in the rats treated prophylactically with FR167653, suggesting that FR167653 inhibits the polyarticular inflammation process and joint destruction by inhibiting the production of TNFα and IL-1β.
The involvement of CD8+ T cells in autoimmune disease is multifaceted. CD8+ T cells and major histocompatibility complex class I participate in disease onset in murine and rat autoimmune disease models (50–55). CD8+ T cells may play an important role in initiating CIA (56), but their role is not fully understood (57, 58). In the present study, we found a significant increase of CD4−,CD8a+ T cells in the local bone marrow of untreated CIA rats when compared with FR167653-treated rats and normal rats in the prophylactic treatment model. These findings suggest that CD4−,CD8+ T cells play an important role in initiating CIA, and that FR167653 may possibly inhibit the accumulation of inflammation initiators, such as CD4−,CD8a+ T cells, in local bone marrow.
Since macrophage inflammatory protein 1α (MIP-1α) and monocyte chemoattractant protein 1 (MCP-1), which are chemokines that may be responsible for CD8a+ T cell and macrophage infiltration (59, 60), are regulated by cytokines such as IL-1α, IL-1β, and TNFα (61), these chemokines may be involved in the mechanism of CD4−,CD8a+ cell accumulation in bone marrow. Therefore, FR167653 may possibly inhibit cytokines via the inhibition of IL-1β and/or TNFα, result-ing in inhibition of inflammatory cell infiltration. How-ever, in our immunohistochemical study, the difference in local expression of MIP-1α and MCP-1 in the periarticular region was marginal between the groups (data not shown).
TNFα and IL-1β are potent inducers of osteoclastic bone resorption. Accordingly, FR167653 inhibition of TNFα and IL-1β production may suppress the periarticular osteolysis in RA. It was recently reported that activation of the p38 MAPK pathway plays an important role in RANKL-induced and TNF-mediated osteoclast differentiation of mouse bone marrow cells (62, 63). In the present study, we found that the number of osteoclastic TRAP-positive multinuclear cells in both the prophylactic and therapeutic FR167653-treated rats was significantly smaller than that in their respective untreated CIA groups. Furthermore, in the rat bone marrow culture assay, an FR167653 concentration of 10−6M almost completely inhibited the differentiation and maturation of osteoclast-like cells induced by both sRANKL and TNFα. This concentration is equivalent to the serum concentration in rats 8 hours after receiving 32 mg/kg daily by subcutaneous injection (Fujisawa Pharmaceutical: unpublished data). Thus, FR167653 multilaterally inhibits joint destruction by suppressing joint inflammation, reducing serum osteoclastic cytokine levels, and directly inhibiting osteoclast formation and maturation.
In addition to FR167653, other inhibitors of p38 MAPK, SB203580 and SB242235, have been used in several inflammatory disease models, and obvious adverse events were not seen (30–34, 45–49). In our in vivo study, we found no adverse events caused by 32 mg/kg/day of FR167653, a dosage that was used in another inflammation model and found to be safe and effective (64). Although most of the in vivo investigations found no adverse effects, one study demonstrated increased plasma creatine levels and lactate dehydrogenase levels in rats (65). The toxicity of FR167653 should be studied extensively.
In conclusion, FR167653, a potent p38 MAPK inhibitor, not only prevents the onset of arthritis by prophylactic treatment, but also suppresses the progression of joint destruction by therapeutic treatment of rats with CIA. FR167653 appears to protect the joints from inflammation injury through the inhibition of TNFα and IL-1β production, recruitment of CD4−,CD8a+ T cells, and osteoclastic bone resorption. These findings suggest that p38 MAPK is a potential therapeutic target for RA.