Collagen-induced arthritis (CIA), which results from immunization of susceptible animals with type II collagen (CII), is an inflammatory arthritis with clinical and immunologic features that parallel those in human rheumatoid arthritis (RA) (1). The preferred animal for use in the study of events in this model is the mouse, because of the number of strains that are available for examination of genetic susceptibility to the autoimmune response and ensuing arthritis. In addition, there are well-characterized murine monoclonal antibodies (mAb) to defined epitopes on CII (2, 3), and these can, on passive transfer, result in collagen antibody–induced arthritis (CAIA), which has all the features of CIA (4–6).
CAIA occurs independently of any direct activity of B and T cells; thus, with this model, effector processes can be studied independently of events that occur during disease induction (6). It is not major histocompatibility complex restricted. Most mouse strains are susceptible, but the severity varies, and lipopolysaccharide (LPS) injection may be required for enhancement of arthritis. Articular inflammation and cellular infiltration characteristic of the effector stage are attributable to deposited immune complexes and activation of complement and Fc receptors (FcR) (6, 7). Cartilage and bone destruction follows the activation of macrophages, lymphocytes, and synoviocytes and production of matrix metalloproteinases (MMPs) and cytokines, including tumor necrosis factor and interleukin-1β (7).
Studies of the in vitro effects of mAb to CII have shown that treatment with 2 mAb (M2139 and CIIC1) that are potently arthritogenic in vivo affected cartilage matrix integrity in cartilage explant cultures (8, 9), impaired synthesis of new matrix, and disrupted collagen fibril formation in chondrocyte cultures (10, 11). Notably, in the culture systems used in those in vitro studies, cartilage damage occurred independently of immune cells or their small molecular mediators. These findings suggest that antibodies to CII may participate directly in the cartilage damage that accompanies articular inflammation. The hypothesis for the current study was, therefore, that mice that do not develop the macroscopic inflammation and cellular infiltration characteristic of CAIA would nonetheless show the same microscopic evidence of cartilage damage as that seen in vitro, after receiving arthritogenic mAb in vivo.
To test this we transferred arthritogenic mAb, without LPS, to mice of 2 strains with reduced capacity to mount an inflammatory response. We examined changes in the cartilage on day 3; this time point was chosen to avoid changes secondary to any cellular inflammatory response and to reduce the confounding effects of regeneration and repair seen in healthy cartilage treated with mAb in vitro (9). We used standard procedures to assess histologic changes in articular cartilage and used Fourier transform infrared microspectroscopy (FTIRM) to evaluate changes in the chemistry of collagen. With this approach, we demonstrated that in the absence of macroscopic inflammation or the cellular infiltration characteristic of CAIA, mice nevertheless exhibited loss of cartilage structure including collagen and proteoglycans, and changes to chondrocytes.
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As constituents of immune complexes, autoantibodies to CII are essential for the development of inflammation in CAIA, either by activation of complement or by direct engagement and activation of FcR-bearing inflammatory cells (6, 7), but the detrimental effects of these antibodies on the integrity and function of articular cartilage has rarely been considered. Previously, we developed strategies to isolate deleterious effects attributed to antibody in vitro from those caused by the inflammatory response that occurs in vivo. In those experiments, mAb to CII that were arthritogenic in vivo, in the absence of inflammation in vitro, induced subtle morphologic changes in chondrocytes and abnormalities in collagen fibrils in the newly synthesized matrix (10, 11, 26); in mature cartilage, loss of proteoglycans, denaturation of CII, and overall disruption of the topography and chemistry of articular cartilage in situ were observed (8, 9, 26). The strength of the effects was dependent on the concentration of antibody and duration of exposure, and the balance between breakdown and repair was dependent on the presence of viable chondrocytes (9).
In the present study 2 strains of mice with limited capacity to develop CAIA were injected with arthritogenic mAb to determine whether autoantibodies to CII cause similar damage unrelated to inflammation in vivo. B10.Q mice may develop arthritis without additional LPS treatment but not as early as within 3 days (5), and B10.QC5δ mice lack C5 and do not develop CAIA (16). We examined the joints on day 3, before inflammatory sequelae were anticipated and before an injection of LPS is usually given. The mAb used bind cartilage both in vitro in explant cultures, where penetration of mAb increases as matrix is destroyed (8), and in vivo, where injected biotinylated mAb bind even more readily than on cartilage in vitro (27, 28). Despite the absence of any overt macroscopic or histologic evidence of inflammation, cartilage damage occurred in the mAb-injected mice, with proteoglycan loss and associated histologic changes including disruption of the cartilage surface, thinning of the cartilage in small joints, and evidence of chondrocyte hyperplasia and activation and/or empty chondrons. Changes demonstrated by FTIRM included denaturation and loss of collagen and abnormalities consistent with cellular activation and new collagen synthesis. The changes in vivo were similar to those seen previously in vitro in cartilage explants (8, 9, 26). There were no significant differences between the parent and congenic animals, indicating that the effects were not due to complement activation.
Overall, the most prominent feature observed in the joints of the mice injected with arthritogenic mAb was loss of proteoglycan from the cartilage. Proteoglycan loss is an early marker of cartilage disruption in both human OA and RA, and is usually attributed to the activation of MMPs and aggrecanases that are released after disruption of the molecular interactions between matrix constituents (29, 30). As in OA, the proteoglycan loss was accompanied by evidence of compensatory repair, including chondrocyte clusters and increased matrix synthesis, and areas of further damage marked by areas of empty chondrons (31, 32), changes that also occur in vitro (8, 9, 26). The empty chondrons may reflect the loss of CII, as the mAb are not directly cytotoxic (10, 11, 26), but increased chondrocyte death is associated with lack of CII in transgenic mice (33).
Although the mAb to CII do not react directly with proteoglycans, CII is the major protein of the extracellular matrix, forming the fibrillar skeleton within which are enmeshed chondrocytes, proteoglycans, and other matrix proteins (34). The importance of such interactions for the maintenance of cartilage integrity is highlighted by our previous observation that the arthritogenic mAb CIIC1 penetrates more readily and deeply into cartilage that lacks the important structural protein type IX collagen (35). The epitopes on CII are sites of interactions with other matrix components that are critical for the structural integrity of the cartilage matrix (3, 11, 36), and disruption of these interactions by mAb would enhance mechanical damage and proteoglycan loss. This loss of proteoglycans which provide “cushioning” of the cartilage in the joint would result in greater susceptibility to damage from compressive forces and greater penetration of degradative molecules, whether these be enzymes released by inflammatory cells or the arthritogenic mAb used in the present study.
The apparent thinning of the cartilage in the small joints at the tips of the paws correlates with the observation of in vitro collapse of the cartilage matrix that follows loss of proteoglycans and the denaturation of collagen demonstrated by FTIRM (8). In cartilage explant cultures, penetration of the mAb extended as the cartilage matrix was destroyed. In vivo, disruption of the matrix would result in direct mechanical loss of cartilage protein that could then be readily taken up into the surrounding synovial tissue. Such uptake has been shown for citrullinated CII (37) as well as for native triple-helical CII (38), with fragments of CII being detectable by immunohistochemistry in both synovial fluid and synovial tissue in human RA. Such release could provide a further antigenic stimulus, and would contribute to the development of CAIA by facilitating immune complex deposition. In this study the inflammatory changes that normally accompany immune complex deposition and complement activation were abrogated, but in most mouse strains, particularly following LPS injection, the proteoglycan loss induced by the arthritogenic mAb would enhance immune complex deposition within the joint and potentiate the degradative response.
The mechanical instability associated with loss of proteoglycan from cartilage contributes to development of noninflammatory arthritis, and OA can be induced in animals by intraarticular injection of papain, which causes proteoglycan degradation (39–41). Changes in the knees of mice 3 days after injection of papain, described by van der Kraan et al (41), closely resembled those in the joints of the mice on day 3 in the present study, and included pronounced proteoglycan loss, damage at the cartilage surface, and chondrocyte proliferation and/or cell death. Residual chondrocytes were often surrounded by a pericellular halo of strongly staining matrix. Notably, the cartilage damage after papain injection progressed without further treatment, and by day 42 was typical of early OA, including cartilage fragmentation and osteophyte formation. However, there was no inflammation. Possibly mAb to CII could induce changes akin to those of OA in the longer term, but in the animal models of CIA and CAIA, and in human RA, the inflammatory response dominates the pathology. The prospect of ongoing antibody-mediated damage leading to changes in the absence of inflammation is, however, important in light of the increasingly successful therapy of human RA with new biologic agents targeting inflammation. Extension of the current work to include longitudinal investigations in mice that did not develop CAIA after injection with mAb to CII might be informative, but was beyond the scope of the present study.
Antibodies to CII are found in the serum, cartilage, and synovial fluid of patients with RA (42–44). However, despite a prevailing view that they occur in only a minority of patients (36), several studies have shown a high frequency, particularly in early RA (45–47), with frequency decreasing as the disease progresses (46), and these antibodies have been eluted from cartilage (44). Their later disappearance from serum may reflect binding to the cartilage matrix and immune complex formation as more epitopes are exposed due to cartilage damage. Possible explanations for variable assay results include contamination of the purified CII by bound matrix molecules, which block the epitopes, or by pepsin, which may increase background levels in the assay. Use of purified antigens might increase sensitivity. For example, the frequency of antibodies detected by enzyme-linked immunosorbent assay increased from 24% when intact CII was used to 88% when the CB10 fragment of CII was used (48), and the use of synthetic triple-helical peptides of CII resulted in an even higher frequency (49). Since antibodies of the same epitope specificity as those in mice also occur in RA (49), the detrimental effects on the integrity and functions of articular cartilage observed in the present study may well contribute to the perpetuation and chronicity of the human disease.
Apart from anti-CII antibodies, a wide array of autoantibodies (50), some of which may function similarly to CII-specific antibodies in disrupting the architecture of articular cartilage, have been found in patients with RA. In particular, antibodies to citrullinated protein antigens are clearly important in disease pathogenesis, being particularly associated with erosive arthritis. Citrullinated CII occurs in the joints of RA patients, and antibodies reactive with it may mediate inflammation by formation of immune complexes (51). However, citrullination involves the modification of arginine, and the major B cell epitopes of CII contain arginine and are surface exposed on the collagen fibril (3). At least some antibodies to citrullinated CII react with the collagen fibril in the same regions as the antibodies to CII, and hence could contribute to cartilage breakdown in the same way as do CII antibodies. Indeed, mAb to the citrullinated C1 epitope on CII have been derived and shown to bind cartilage and to induce or enhance arthritis in mice (37), although their effects on cartilage have not been tested in vitro.
In essence, the importance of our findings lies in the implication that the autoimmune response in RA originates and persists in articular cartilage and there is a balance between disruption and repair regulated by chondrocytes. Many of the sequelae that develop in RA are a consequence of the inflammatory response associated with immune complex formation. It has been shown that CII-containing immune complexes obtained from RA patient sera induced the production of proinflammatory cytokines from peripheral blood monocytes via FcγRIIA (44), suggesting that this could be one of the possible detrimental mechanisms by which immune complexes induce inflammation. Therapeutic suppression of inflammation may restore quality of life for patients with RA but does not address the underlying problem of the autoimmune reaction and the balance between disruption and repair. Collectively, the present observations suggest the importance of understanding autoimmune responses and their consequences in order to design effective treatments to suppress the specific autoimmune response causing damage, and to promote repair and healing.