Complement activation produces the 74-residue protein C5a, which is the most potent of the anaphylatoxins (1). C5a mediates numerous immune and inflammatory functions, including chemotaxis and activation of inflammatory cells, increased vascular permeability, spasmogenesis, immune regulation, and the release of a variety of inflammatory cytokines and mediators (1–3).
Rheumatoid arthritis (RA) is an immune complex disease involving the local activation of inflammatory cells, predominantly in the smaller peripheral joints. The complement system, and in particular the factor C5a, has long been identified as a likely contributor to the pathogenesis of RA. Elevated levels of C5a have been found in the plasma and inflamed joints of patients with RA (4, 5). The number of C5a receptors on synovial mast cells is also increased in RA (6). Most important, the degree of complement activation in the joint and circulation correlates with the severity of the disease, suggesting that C5a plays a central role in disease pathogenesis (7–10).
Several animal models of RA have been used to elucidate the role of complement and C5a in this disease. Depletion of complement with cobra venom factor has been shown to delay the disease onset in a number of laboratory models of RA, including adjuvant arthritis, collagen-induced arthritis (CIA), and antigen-induced arthritis (AIA) in rats (11–13). Administration of recombinant soluble complement receptor 1 (sCR1) or anti-C5 antibodies, either intraperitoneally (IP), intravenously (IV), or intraarticularly (IA), has been shown to reduce disease progression in anti-CD59 antibody–induced models of acute arthritis (14), CIA (13, 15), and AIA (12). However, the role of C5a as a pathogenic factor in these models has been difficult to prove due to the lack of availability of a specific C5a antagonist.
This issue was addressed recently with the use of a peptidic C5a receptor antagonist, MeFKPdChaWr, in a model of membrane attack complex (MAC)–dependent anti-CD59 antibody-induced arthritis (14). Administration of the antagonist, either IV or IA, failed to inhibit the development of disease parameters (14). Explanations for the apparent lack of C5a involvement in this animal model include the possibility of impaired clearance of MAC rather than activation of the complement cascade, failure of pathology to be affected by complement depletion, and resolution of the lesion within 3 days (16).
Current drug therapies for RA remain relatively ineffective at retarding disease progression and are associated with significant side effects (for review, see ref. 17). Drugs such as the nonsteroidal antiinflammatory drugs (NSAIDs), which include ibuprofen, provide palliative relief of symptoms, but have little beneficial effect on the pathology and progression of disease (17, 18). Patients with developing RA require an effective, orally active therapy that will moderate the symptoms as well as arrest the progression of the destructive joint pathology.
Researchers in our laboratory have recently developed a series of small molecule antagonists of the human C5a receptor (19, 20). One of these is a cyclic peptide, AcF-[OPdChaWR], which is orally active and has been shown to effectively reduce the severity of diseases in various rat models (21–23). In the present study, we tried to use this specific C5a receptor antagonist to determine the relative contribution of C5a in the pathogenesis of immune-mediated monarticular arthritis and assess the effectiveness of the antagonist in treating the disease symptoms and pathology. This compound has been shown to be a potent antagonist of both human and rat C5a receptors on polymorphonuclear leukocytes (PMNs) (24). It is also a potent antagonist of C5a receptors on human macrophages (25). We therefore hypothesized that any demonstration of anti-arthritic activity in rats would bode well for future testing in human arthritic conditions. The data herein demonstrate that oral administration of AcF-[OPdChaWR], either before or after induction of the disease, is effective at preventing the development of pathology and moderating disease progression. These findings confirm the importance of C5a in this disease model and suggest a future role for antagonists of the C5a receptor in the treatment of immune-mediated arthritis and other inflammatory diseases.
- Top of page
- MATERIALS AND METHODS
Current drug treatment of RA involves the inhibition of cytokines and other mediators thought to be involved in its pathogenesis. The complement factor C5a is also recognized as a very important proinflammatory mediator in RA (6). Levels of C5a in the plasma and synovial fluid of patients with RA are higher than those found in patients with osteoarthritis (5). The pathology of RA includes the recruitment and accumulation of neutrophils and monocytes in the synovial tissues, with the down-regulation of C5a receptors in monocytes possibly contributing to chronicity of the disease (26, 27). Synovial effusions contain high levels of C5a, which is a powerful chemoattractant for neutrophils and monocytes and invokes microvascular plasma leakage within the tissues (4, 26). Inhibitors of activation of the complement system, or of the formation or action of C5a, have therefore been proposed to be of potential use in the treatment of RA. Anti-C5 monoclonal antibodies have been shown to block the development of CIA in rats as well as reduce the progression of established pathology (15). A recombinant protein inhibitor of complement activation, sCR1, has been suggested as a potential therapy for RA because efficacy with this compound has been shown in various animal models of arthritis (12, 13, 28).
The model of an antigen-induced monarticular Arthus reaction produces a discrete lesion of highly reproducible severity in a single joint, leaving the contralateral joint available for comparison. It has been suggested that the changes in the joint in clinical RA are due to this reaction (29). Neutrophils predominate in the synovial fluid in RA, particularly in the early stages, and this is mirrored in this rat model (30). Mononuclear cells were the predominant inflammatory cells in the rat synovium; this is also the case in RA (31). Additionally, the presence of the proinflammatory cytokines TNFα and IL-6 in the arthritic knee joint and TNFα in the serum of rats in the 14-day study correlates with the presence of these cytokines in the joints of arthritis patients (32).
The findings reported here show that an orally active, small molecule antagonist of the C5a receptor reduces the disease severity in a rat model of immune-mediated arthritis. Given on a daily basis, either before the initiation of arthritis or after symptoms were detectable, the drug reduced both joint swelling and gait symptoms. In the latter treatment case, drug therapy was started either 2 or 4 days after the lesion was stimulated in order to mimic the human clinical situation where a patient would present with an acute progressive lesion. In the 14-day study, PMNs were the predominant cell recovered in joint lavage fluid, whereas in the 28-day study, macrophages were the principal cell type found. In these shorter-term studies, cartilage erosion did not occur. In order to stimulate erosion, it was necessary to prolong the duration of the study and rechallenge the rats with antigen. Under these conditions, the C5a antagonist was again effective in reducing the degree of structural change in the joint.
These results show that this class of drug has multiple activities at different stages of the disease process. In previous short-term studies involving endotoxic shock and the Arthus reaction in the peritoneum and the dermis, the C5a antagonist was very effective at inhibiting the inflammatory challenges (21, 22, 33). The results of the present study demonstrate the efficacy of this class of drugs in a model of chronic immune complex–mediated inflammation, following daily oral administration of the drug.
The destruction of cartilage in osteoarthritis results from the IL-1–stimulated degradation of proteoglycans and inhibition of chondrocyte proteoglycan synthesis (18). NSAIDs protect the joint from swelling and cellular infiltration, but have little effect on disease progression, while glucocorticoids normalize proteoglycan synthesis (17, 18). The NSAID ibuprofen diminishes the responses in the rat as measured by joint swelling and disturbance of gait, and these findings equate well with the response to most NSAIDs in the clinic (17, 34). Ibuprofen is less successful in reducing the structural pathology in the rat joint, and this is also similar to human clinical findings (17, 34). In contrast, the C5a receptor antagonist used in this study significantly reduces the degree of structural pathology in the joint as well as other signs of the disease in the rats. This ability to moderate structural changes in the joint is a clear advantage over most of the NSAIDs.
The C5a antagonist used in the present study is orally active, with peak circulating plasma levels around 0.1–0.3 μM following a single oral dose of 3 mg/kg (22). The drug has a high affinity for human C5a receptors, and is an insurmountable antagonist active at low nanomolar concentrations (25). The drug was developed by structural analysis of the effector portion of C5a, and is a cyclic peptide that is resistant to metabolic degradation in the gut and plasma (20). In rats, a single oral dose of 10 mg/kg inhibits the neutropenia response to IV C5a for up to 24 hours (22), and oral treatment for 7 days at a dose of 1 mg/kg completely blocks the binding of C5a to circulating PMNs. In the present study, administration of 1 mg/kg/day was also an effective oral dose for reducing the expression of the arthritis symptoms and joint pathology. This C5a antagonist does not inhibit the formation of MAC at concentrations up to 100 μM (Strachan AJ: unpublished observations), indicating that, at least in the rat model of monarticular arthritis, inhibition of C5a alone is sufficient for effective reduction of symptoms and pathology. Similar results have been found in the same rat model of arthritis for the protein sCR1, which inhibits the formation of C3a, C5a, and MAC (12). This is consistent with our hypothesis that C5a is a major pathogenic mediator of disease pathology in this model. The precise role of MAC in antigen-induced monarticular arthritis, however, remains to be fully established.
In this study, combination drug therapy with ibuprofen and the C5a antagonist was found to be no more effective than therapy with the C5a antagonist alone. C5a causes the release of eicosanoids when administered in vivo to a variety of animal species, and the effects of C5a on blood pressure are blocked by cyclooxygenase inhibitors (35–37). The lack of additional efficacy on all parameters with the combination therapy may be due to the fact that the eicosanoid cascade is blocked early on by the C5a antagonist. The increased efficacy of the C5a antagonist over ibuprofen for joint pathology may be due to the additional inhibition of expression of proinflammatory cytokines, such as TNFα and IL-6. The importance of TNFα in the pathology of RA has been clearly demonstrated in a number of studies and has led to the development of a soluble TNFα receptor–based treatment (etanercept) in RA (38, 39). It has previously been shown that inhibitors of C5a reduce the expression of TNFα and IL-6 in other disease models in vivo (21–23, 40, 41). The ability of the C5a antagonist to inhibit the expression of TNFα indicates an early role for C5a in the inflammatory cascade, and suggests a pivotal role for the therapeutic use of C5a antagonists in RA. The disease-modifying properties of the C5a antagonist in the present study may be due to its capacity to inhibit formation of both these cytokines and eicosanoids, rather than to the inhibition of eicosanoids alone.
In summary, this study demonstrates for the first time that a small molecule C5a receptor antagonist, given orally, prevents some principal signs of arthritis and significantly reduces the joint damage caused by an immune-mediated monarticular arthritis in rats. The disease-modifying effects of this antagonist suggest a potential use for C5a antagonists as antiarthritic agents in the clinic.