CIX-deficient mice have a subtle defect of the cartilage, which is here indicated by their higher susceptibility to developing spontaneous enthesopathic arthritis. Interestingly, these mice also developed a more severe autoimmune arthritis, as shown with the CIA model. Parts of this effect could be explained by the higher accessibility of CIX-deficient cartilage for binding of arthritogenic CII-specific antibodies.
CIX-deficient mice [6
] have previously been shown to develop microscopic OA at one year of age, with loss of cartilage at the femoral and tibial joint surfaces and increased cartilage and bone formation at the periphery, leading to prominent shape changes. In transgenic mice with truncated CIX [5
], OA was observed in mice up to one year old as a decreased intensity of Safranin O staining and roughening or erosion of the articular surface. In our study we could not detect either gait problems or histological OA. One reason for this might have been that we focused on mice with the CIX deficiency backcrossed to DBA/1 and B10.Q genetic backgrounds, which are not known to be sensitive for spontaneous OA, unlike the C57BL/6 and BALB/c [40
] genetic backgrounds used in the previous studies. The development of spontaneous enthesopathy in DBA/1 mice is an indication of the presence of a good compensatory mechanism to preserve joint homeostasis [29
], resulting in an improved repair mechanism for local injury [41
]. However, enthesopathy in DBA/1 mice is a pathological event, with increased new bone formation in tissues where tendons and ligament attach, with abundant proliferative fibroblasts and chondrocytes. In our SIA experiments the homozygous mice developed a more severe disease, which suggests that the mediators released during stress affect chondrocytes profoundly, and chondrocytes might possibly try to compensate for the CIX deficiency by the overexpression of cartilage components.
To test our hypothesis about whether cartilage disorder could alter the course of arthritis as a result of enhanced accessibility to the immune system, we used our CIX-deficient mice in two different genetic backgrounds in CIA. In both these backgrounds a more severe development of arthritis was observed with CIX deficiency. This phenomenon could not be explained by increased turnover of the fragile matrix and, in turn, priming of autoreactive T cells or efficient tolerance induction, because the disease course was as self-limiting as in the controls. Similarly, there was no change in antibody titers, which argues against de novo priming of T cells. Still, there might have been a difference in the accessibility of cartilage to antibodies, because antibodies are shown to bind complement and to be involved in direct cell interactions via Fc receptors, which would certainly have an effect on disease progression. In fact, this was the case when tested with direct CII antibody injection: homozygous mutant mice developed arthritis much earlier than the controls and had more antibodies attached to the cartilage. This also ruled out the possibility of T-cells as the main promoter during the course of arthritic disease in this model, because antibody-mediated arthritis is T-cell independent.
Because CAIA experiments gave such a rapid response and also an apparent knee inflammation in both the mouse strains, we were prompted to perform in vivo
binding studies with different monoclonal antibodies. Of the different antibodies used, CIIC1 binding to the C1I
epitope showed an increased binding to cartilage. The C1 epitope was identified as a major epitope in generating the antibody response to CII, and the various antibodies thus developed recognized different parts of the epitope: the C1I
epitope 359 to 363, the C1II
epitope 359 to 366 and the C1III
epitope 359 to 369 [35
]. However, all antibodies are dependent on the first part of the epitope, where the CIIC1 antibody binds. The antibody response to the C1 epitope dominates the immune response in CIA in both mice [42
] and rats [43
The CIIC1 antibodies also impaired cartilage formation by cultured chondrocytes [44
], strongly inhibited the self-assembly of CII in vitro
] and caused disorganization of CII fibrils in the extracellular matrix without affecting chondrocyte morphology, along with increased matrix synthesis [46
], and thus the antibodies directed to this epitope can contribute directly to cartilage destruction. Increased binding of CIIC1 to the CIX-deficient cartilage indicates that the C1I
epitope is more exposed for the CIIC1 antibody in the absence of CIX and more antibodies bind per CII molecule in the deeper layers of cartilage.
Interestingly, antibodies against the C1 epitope have also been shown to be associated with RA [47
]. Clearly, the C1 epitope is not only important for its immunodominance but also contributes to matrix component interactions, signaling and stability. In contrast, murine antibodies reacting to the F4 epitope were not arthritogenic in CAIA and were associated with OA rather then RA [47
]. Furthermore EDM1, another subgroup of multiple epiphyseal dysplasia, and pseudochondroplasia are linked to defects in cartilage oligomeric matrix protein (COMP) [48
] and share pathogenesis with EDM2 [49
], suggesting that COMP and CIX are interacting in the large polymeric network of cartilage. Deficiency of CIX might therefore also lead to instability and changed exposure of COMP. Increased serum levels of COMP have not been detected in different human OA, but it would be interesting to study COMP-mediated pathology in CIX-deficient mice.
Using CIX-deficient mice of two well-defined mouse inbred lines B10.Q and DBA/1, which are susceptible to CIA, we have tested the possibility that disordered cartilage alters susceptibility to autoimmune arthritis. We found that the lack of CIX increased the binding of antibodies to the major epitopes on CII in cartilage and led to a higher susceptibility to both CIA and CAIA. RA probably represents a wide variety of diseases with different initiation events. It could in fact include subgroups that have a mild cartilage defect genetically. Such groups could be dormant but the presence of fragile cartilage could expose B-cell epitopes to binding of autoreactive antibodies. The bound antibodies could then initiates an inflammation with the release of cartilage antigens, thereby further driving an autoimmune attack towards cartilage proteins. This study supports the idea that a mild disruption to cartilage increases the severity of arthritis.