Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that affects multiple joints, leading to articular damage and bone destruction (1). There is increasing evidence that B cells and secreted antibodies may play an important role in RA, and depleting B cells by use of monoclonal antibodies (mAb) to CD20 (rituximab) ameliorates RA. Since autoantibody levels are not completely depleted, even in RA patients undergoing B cell depletion therapy with rituximab (2), understanding the regulatory mechanisms at the effector level becomes all the more important in designing better treatments for RA patients.
An autoimmune response to native type II collagen (CII) develops in some patients, with IgG antibodies to CII demonstrable in the blood, cartilage, and synovium (3). In collagen-induced arthritis (CIA), which is induced by immunization of animals with CII (4, 5), there is destruction of the articular cartilage matrix as occurs in RA in humans, with accompanying T cell and B cell immune responses to CII that are seen as requisite for disease initiation and development (6). Certain anti-CII mAb produced from mice susceptible to CIA can, on passive transfer to naive mice, cause an acute arthritis known as collagen antibody–induced arthritis (CAIA) (7–10). Since CAIA occurs independently of the direct activity of B cells and T cells, it allows for the study of effector processes without consideration of events in the inductive phase (8).
The articular inflammation that characterizes the effector stage is customarily attributed to the formation and deposition of immune complexes and the activation of complement and Fc receptors (FcR) (11). Interestingly, cleavage of arthritogenic mAb at the hinge region in vivo (12) or selective removal of carbohydrate moieties in the CH2 domain in vitro (13) were shown to completely abrogate the development of antibody-mediated arthritis. Not all CII-specific antibodies are equally arthritogenic, however. CIIF4 mAb is inert after passive transfer and, indeed, is even protective against articular damage in vivo when transferred together with a combination of the normally potently arthritogenic mAb M2139 and CIIC1 (14), but the mechanism of this protection has not yet been identified.
Using in vitro systems based on cartilage explant cultures, we have previously shown that mAb M2139 and CIIC1, which are arthritogenic in vivo, adversely affect cartilage matrix integrity, whereas the nonarthritogenic mAb CIIF4 has no effect (15). In the present study, we extended these observations to show that in vitro, CIIF4 is counterdestructive/protective against the degradative effects of these arthritogenic mAb when used in combination. Notably, in the culture system we used, cartilage damage by antibody occurs independently of immune cells or their small-molecule mediators. Our enquiries were directed toward the mechanism(s) whereby the physical interaction of antibody with a collagen epitope is transduced to matrix degradation, and how mAb CIIF4 could interfere with such processes. We addressed these questions by assessing whether there was a requirement for living chondrocytes in the cultures for these effects of the CIIF4 mAb and whether attachment of CIIF4 to its epitope site might sterically interfere with the binding of matrix metalloproteinase 3 (MMP-3; stromelysin 1) at its catalytic site on CII.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
This study was designed to examine the effects on bovine cartilage explants of 3 mAb to native CII that bind to well-identified and structurally different conformational epitopes (16). Two of these mAb, M2139 and CIIC1, are arthritogenic upon passive transfer in vivo (9, 14), and whether used individually (15) or in combination, they caused progressive denaturation of CII and substantial loss from cartilage of both CII and proteoglycans in vitro. By contrast, the third mAb, CIIF4, which is nonarthritogenic and protective in vivo when given with other mAb (14, 17), had no adverse effect in vitro when used alone on cultured cartilage and countered the adverse effects of arthritogenic mAb, even promoting the regeneration of cartilage matrix components. Thus, this counterdestructive effect of CIIF4 in vitro parallels its antiarthritogenic effects in vivo. It is notable that whereas the destructive effects of the arthritogenic mAb were independent of the presence of viable chondrocytes, the protective effect of CIIF4 required viable chondrocytes, since it was abrogated by freeze-thawing of the cultured cartilage explant.
The effector role of autoantibodies in the development of arthritis has been extensively studied in the context of murine CAIA. The bound antibodies have been shown to trigger and enhance inflammation by activating the complement cascade and FcγR-bearing cells, with release of proinflammatory cytokines by mononuclear cells within the synovium, leading to recruitment of neutrophils and macrophages that amplify the response by the further release of cytokines and tissue-degrading enzymes (31). Moreover, preceding or in parallel with these nonspecific effects of immune complex cellular activation, there is increasing evidence that autoantibodies to CII can have specific destructive effects within the cartilage (15, 16, 17). In addition to the degradative effects on preformed cartilage seen in the present study, the arthritogenic mAb have been shown to impair the synthesis of new matrix and disrupt collagen fibril formation in chondrocyte cultures in vitro (17, 32–34), changes that would directly affect the processes of cartilage repair following damage to the matrix.
The mechanism by which CIIF4 exerts its effects is currently unknown. The nonarthritogenic mAb CIIF4 was derived in a manner similar to that of the arthritogenic mAb from a DBA/1 mouse immunized with CII, and it is an IgG2a mAb, as is the arthritogenic mAb CIIC1. It binds strongly to CII, as demonstrated by ELISA, as well as to cartilage, both in vitro and in vivo. Hence, CIIF4 should activate complement and bind to FcγR-bearing cells to trigger immune complex–mediated inflammation. Nonetheless, it is not arthritogenic, but is actually protective in vivo, reducing the arthritis in mice injected with otherwise arthritogenic mAb (14, 17). CIIF4 is reactive with a conformational epitope at the COOH-terminus of the CII triple helix (aa 932–936), and within the assembled collagen fibrils, it is close to one of the cleavage sites of MMP-3 (stromelysin 1) (14) that is located within the NH2 telopeptide region in the collagen fibril (30). Moreover, its effects differ from those of other mAb, in that they were only seen when viable chondrocytes remained in the cartilage.
Our hypothesis was that CIIF4 exerts its protective effect by steric hindrance, blocking the cleavage of CII by MMP-3, which is produced by chondrocytes and by synovial fibroblasts, and is associated with cartilage degradation in osteoarthritis and in RA (35, 36). MMP-3 primarily targets proteoglycans and is not a conventional collagenase, but it acts as a telopeptidase by cleaving CII at a site inside its NH2-telopeptide crosslinking residue (30) and most likely also cleaving off the C-telopeptide. MMP-3 plays an essential role in the degradation of not only aggrecan, but also collagen fibrils in the cartilage (35), so that MMP-3–knockout mice are resistant to cartilage degradation in antigen-induced arthritis (37). The use of a broad-spectrum MMP inhibitor to block MMP-3 activity in the cultures, however, did not mimic the effect of the addition of the mAb CIIF4. Thus, the MMP inhibitor did not prevent cartilage damage as assessed histologically, and there were no apparent differences in the cultures that contained living or dead chondrocytes, indicating that the protective effect of CIIF4 was not merely a result of steric hindrance of MMP-3 activity, although steric hindrance of the binding of one or other of the arthritogenic mAb could not be discounted.
The use of the MMP inhibitor GM6001 in culture provided further information about the likely mechanism of damage by the arthritogenic mAb. By FTIRM, it appeared that the MMP inhibitor prevented both the denaturation of collagen and the loss of proteoglycans seen in cartilage cultured with the arthritogenic mAb, indicating that MMPs do play a role in the cartilage degradation induced by the antibodies. The fibrillar nature of the cartilage damage in the presence of the inhibitor suggests that the initial effect of the binding of the arthritogenic mAb on the surface of the collagen fibrils was to cause disaggregation of the collagen fibrils, but the lack of collagen denaturation or proteoglycan loss in the presence of the MMP inhibitor indicates that these enzymes play an important role in further cartilage degradation. The results suggest that the disruption of the collagen matrix causes the release of the MMPs known to be sequestered in the intact matrix (38, 39), and these MMPs are responsible for the ongoing loss of proteoglycans and collagen degradation, without any requirement for further MMP inhibitor synthesis.
A striking observation from these studies is the importance of living chondrocytes in the maintenance of cartilage integrity in the presence of arthritogenic mAb and in the protective effect of CIIF4. In each case, the arthritogenic mAb caused more damage to the freeze-thawed cartilage, as judged by the greater shift in the position of the amide 1 peak and the loss of proteoglycans on both day 7 and day 14. This augmented damage was also seen in the freeze-thawed cartilage even in the presence of CIIF4. The appearance of chondrocyte dimers, or clumps of cells in the antibody-treated cartilage, which were absent in the freeze-thawed cartilage, suggested that cell division was occurring in response to the treatment, and the appearance of an additional peak at 1632–1635 cm–1 in the living cartilage was consistent with cellular activation. Taken together, these observations suggest that there is a balance between damage and regeneration within the cartilage.
The use of FTIRM, a technique that allows the examination of localized chemical changes within tissue without any requirement of a priori knowledge of the likely mechanism has allowed definitive detection of the protective effect of CIIF4. Determination of the mechanism of the protective effect of CIIF4 will require the use of different techniques and is beyond the scope of the current study, but we are investigating the gene expression profiles in chondrocytes treated with CIIF4 and other antibodies.
The major question that arises from these studies is whether there is any connection with the pathogenesis of RA. RA is generally considered to be an immune complex–mediated disease, and its features are well accounted for by the formation and deposition in joint structures of immune complexes that are operative by complement activation and by Fc binding and Fcγ activation. In CIA in animals, the antigenic component is clearly CII, but in RA in humans, there is uncertainty (noting that an antibody response to ALL of the epitopes recognized by the mAb in this study also occur in RA). If antibodies to CII of the same specificity as the arthritogenic mAb also occur in human sera (14), the mechanisms of joint damage that occur in CAIA could also occur in RA. Interestingly, both proteoglycan and CII destruction and secretion into the synovium occur in RA (40–42). It is striking that antibodies to CII of the same specificity as CIIF4 also occur in human sera, but they are associated with osteoarthritis rather than rheumatoid arthritis (14), and it is tempting to speculate that such antibodies may also prevent the inflammatory damage seen in human RA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Rowley had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Croxford, McNaughton, Holmdahl, Rowley.
Acquisition of data. Croxford, Crombie, Rowley.
Analysis and interpretation of data. Croxford, Crombie, McNaughton, Nandakumar, Rowley.