Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359–369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising β-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.

IgG anti-collagen type II (CII) antibodies (Ab) can induce arthritis in healthy mice. Here we have investigated if single monoclonal IgG anti-CII Ab can induce arthritis in CIA-susceptible DBA/1 mice and if there is an IgG subclass dependency. The involvement of Fc receptors for IgG (Fc + R) in anti-CII Ab-mediated arthritis was also investigated by comparing the clinical outcome in DBA/1 mice to those in Fc + R-deficient mice. We demonstrate for the first time that single mAb to naive DBA/1 mice can induce persistent arthritis. Histology of the inflamed joints revealed massive cellular infiltrate and cartilage and bone destruction. All IgG subclasses tested (IgG1, IgG2a and IgG2b) were arthritogenic, with the IgG1 and IgG2b isotypes as the dominating arthritogenic Ab. Pathogenicity was dependent on engagement of activating Fc + R, as FcR + -deficient mice were completely resistant to Ab-mediated arthritis. The arthritis induced with the IgG1 and IgG2b Ab was also inhibited by Fc + RIII disruption, whereas arthritis mediated by the IgG2a Ab was not substantially affected. The arthritic response of the IgG1 and IgG2b isotypes, but not of the IgG2a Ab, was further enhanced in mice lacking the inhibitory Fc + RIIB. These results demonstrate that single IgG anti-CII mAb can induce erosive arthritis and that IgG anti-CII Ab mediate arthritis by engagement of Fc + R.

Objective. To investigate the significance and pathogenic potential of a highly conserved major type II collagen triple-helical epitope-specific antibody (U1; amino acids 494-504) in vivo and in vitro in patients with early rheumatoid arthritis (RA) and in experimental animal models of collagen-induced arthritis (CIA).Methods. U1-specific antibodies in sera from patients with early RA (with or without joint erosions) were analyzed. Disease progression in the CIA models in mice and rats with anti-U1 antibodies was compared. The pathogenicity of binding of monoclonal antibodies (mAb) UL1 and CIIF4 to the U1 epitope and the F4 epitope (aa 926-936), respectively, was compared in vivo and on chondrocyte cultures and preformed cartilage in vitro, using Fourier transform infrared microspectroscopy analysis. In addition, UL1-induced proteoglycan depletion in vivo in the presence and absence of the complement factor C5 was analyzed.Results. Increased levels of U1 antibodies were observed in patients with early RA, especially in association with joint erosions. A significant correlation of U1-specific antibodies with disease progression was found in rats and mice with CIA. UL1 mAb induced, whereas CIIF4 mAb inhibited, the progression of arthritis. Similarly, UL1, but not CIIF4, impaired matrix synthesis on chondrocyte cultures and adversely affected preformed cartilage. Furthermore, UL1 induced significant proteoglycan depletion in vivo 3 days after injection, even in the absence of C5.Conclusion. Antibody epitope specificity contributes significantly to the development of arthritis, and the early pathogenic events operate independent of inflammation both in vitro and in vivo.

Objective: To investigate the mode of action of methotrexate (MTX) in different types of models for rheumatoid arthritis (RA) and multiple sclerosis (MS). Methods: Models for RA and MS were selected known to have different pathogenesis-that is, fibroblast induced arthritis in SCID mice, collagen induced arthritis (CIA), anticollagen II antibody induced arthritis (CAIA), and experimental autoimmune encephalomyelitis (EAE) in (Balb/c6B10.Q)F1 and B10.Q mice, and Pristane induced arthritis in DA rats (PIA). The MTX treatment was started 1 day after the onset of disease and continued for 14 days to compare effects on the different models.Results: All models known to be critically dependent on T cell activation (CIA, PIA, and EAE) were effectively down regulated by titrated doses of MTX. In contrast, no effects were seen on fibroblast induced arthritis or CAIA. No effects were seen on the levels of anticollagen II antibodies in the CIA experiment. Conclusion: The data show that MTX has strong ameliorative effect on both classical models of RA, like CIA and PIA, but also on a model for MS, EAE. It also suggests that MTX operates only in diseases which are preceded by, and dependent on, T cell activation. A comparison of CAIA and CIA suggested that MTX operates independently of arthritogenic antibodies. These results demonstrate that different animal models reflect the complexity of the corresponding human diseases and suggest that several models should be used for effective screening of new therapeutic agents.

We have addressed the importance of B cell tolerance to collagen type II, a matrix protein, which is a target in rheumatoid arthritis (RA) and its mouse models. We generated a germline-encoded anti-collagen type II (CII) IgH replacement anti-C1 B cell mouse strain (ACB) to investigate how B cell tolerance to CII, a matrix protein, is subverted and to further understand pathogenesis of RA. Phenotypic analysis revealed that CII-specific B cells were surprisingly neither deleted nor anergized. Instead, they were readily detected in all lymphoid organs. Spontaneously produced autoantibodies could bind directly to cartilage surface without detectable pathology. However, exaggerated arthritis was seen after injection of anti-CII Abs specific for other epitopes. In addition, Abs from CII-specific hybridomas generated from ACB mice induced arthritis. Interestingly, IgH/L chain sequence data in B cell hybridomas revealed a lack of somatic mutations in autoreactive B cells. The ACB model provides the first possibility, to our knowledge, to study B cell tolerance to a matrix protein, and the observations made in the study could not be predicted from previous models. B cell-reactive epitopes on CII are largely shared between human RA and rodent CII-induced arthritis; this study, therefore, has important implications for further understanding of pathological processes in autoimmune diseases like RA.