Rheumatoid arthritis (RA) is a heterogeneous disease of multifactorial etiology and unpredictable outcome (1, 2). Early diagnosis and treatment are essential in order to prevent erosive joint destruction, and therefore, efforts are being made to search for reliable diagnostic and prognostic markers. Recently, the diagnosis of RA was substantially improved by the introduction of standardized immunoassays for the detection of autoantibodies against citrullinated antigens (3–5). Despite the excellent performance of these immunoassays, anti–cyclic citrullinated peptide (anti-CCP) antibodies provide only a sensitivity comparable with that of rheumatoid factor (RF), and analysis of the correlation with RA disease activity yielded conflicting results (6, 7). Moreover, this artificial antigen of a so-far-undisclosed composition is not expressed in the affected tissue and is therefore presumably not directly involved in the pathogenesis of RA.
Anti-Sa antibodies have been characterized as a specific marker for RA, including early disease manifestations (8–10). However, the sensitivity of anti-Sa was relatively low compared with that of RF. Immunoprecipitation experiments revealed that vimentin, an abundant structural component of the intermediate filaments, represents the target of anti-Sa antibodies (11). Importantly, the antigenic properties of vimentin were substantially triggered by citrullination, and furthermore, citrullinated peptides of vimentin were found to be presented via the HLA–DR4 shared epitope (12). Several other investigations linked vimentin by its known function, such as modification during apoptosis and secretion from macrophages in response to tumor necrosis factor α signaling, to the pathogenesis of RA (13–15). However, the antibody response against citrullinated vimentin shows a diagnostic sensitivity of only 40% for RA (16, 17).
In the present study, modifications of vimentin were investigated under pathogenic conditions (e.g., oxidative stress) in more detail. A mutated isoform of vimentin was identified by mass spectroscopy analysis of purified vimentin from a human fibroblast cell line and from synovial fluid (SF) samples from RA patients. Subsequently, mutated vimentin was expressed and citrullinated for standardized detection of the respective antibodies in RA patients and in patients with rheumatic diseases other than RA by enzyme-linked immunosorbent assay (ELISA).
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we investigated the expression of vimentin in synovial sites under pathologic conditions. As an important result, we identified in RA patients an isoform of vimentin modified not only by citrullination, but also by mutation. Using this potentially relevant alternative antigen, we developed the first ELISA for the detection of autoantibodies against human MCV. Surprisingly, the antigenic properties of this molecule were strongly influenced by modification of the sequence. Compared with established markers, determination of anti-MCV antibodies provided the highest specificity and sensitivity for the diagnosis of RA in our cohort.
A decade ago, disease-specific autoantibody reactivity against the Sa antigen was described in patients with RA, and vimentin was subsequently identified as the target antigen, using antibodies against citrullinated epitopes. Therefore, antigen recognition by these RA-specific autoantibodies is critically dependent on the presence of the unusual amino acid citrulline, formed by a posttranslational modification of arginine residues by the enzyme PAD. Interestingly, a predominant expression of PAD isoforms has been described in macrophages infiltrating the synovial tissue; moreover, vimentin was found to represent a predominant substrate of these enzymes, especially in activated and dying macrophages in patients with RA (26–28). It was shown that proteolysis of vimentin by caspases promotes apoptosis via a proapoptotic cleavage product (29, 30). In fact, the occurrence of citrullinated vimentin in synovial membranes and its secretion by macrophages in response to growth factors and proinflammatory cytokines make it a relevant joint-associated autoantigen in the pathogenesis of RA (11, 13). However, the prevalence of anti–citrullinated vimentin antibodies was relatively low, and this marker was not suitable to substantially improving the procedures for diagnosing RA.
Vimentin intermediate filaments are dynamic structures, and their flexible organization is important for the regulation of mechanical stress between chondrocytes and the surrounding matrix tissue. Furthermore, a role for vimentin was described in the regulation of T cell activation. As a posttranslational modification, citrullination of vimentin occurs under circumstances that are not unique to inflamed synovial tissue in RA (31). Recently, it was reported that different vimentin isoforms resulted from phosphorylation, degradation, or glutathionylation (32). In the present study, we identified transcriptional modifications of relevant epitopes of vimentin, providing a novel marker for the diagnosis and prognosis of RA.
Recently, it was shown that presentation of antigen variants to the immune system triggers the recognition of normally silent epitopes and can enhance the generation of adaptive immune responses to self (33). The assumption of existing isoforms of vimentin was based on the growing evidence of abundant microsatellite instability and suppression of mechanisms that limit genomic DNA damage in RA synovium (24). Moreover, overexpression of, and functional mutations in, key genes were shown for the rheumatoid synovium (e.g., for the p53 tumor suppressor gene) (34). As a relevant pathologic mechanism, it was assumed that persistent oxidative stress might be involved in DNA damage. In this context, an increased susceptibility to oxidative stress of the guanine base (G) must be taken into account due to the lowest oxidation potential. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A (for review, see refs. 35 and36). This pairing may cause mutations of glycine residues to arginine residues. Although the number of G/C transversions is increased under exposure to oxygen radicals, the molecular basis of G/C transversions is not completely understood.
In the present investigation, we identified mutated glycine residues within the vimentin DNA caused by at least 1 single-nucleotide polymorphism. Moreover, it was shown that mutated vimentin is also citrullinated in SF of patients with RA. Our data indicate that citrullination by PAD is influenced by amino acid residues that flank arginine, resulting in a nonrandom modified protein. However, despite citrullination, mutation of vimentin represents an independent trigger of the antigenic properties of the antigen in RA.
Using the mutated and citrullinated recombinant human antigen vimentin for the diagnosis of RA in a standardized ELISA, we clearly documented a preserved high diagnostic specificity of the antigen. Of note, weak antibody reactivities against MCV were observed in a small subset of patients with SLE and primary SS. Therefore, the diagnostic significance of anti-MCV antibodies must be confirmed in a multicenter study. Importantly, the assay showed better sensitivity than the anti-CCP ELISA. Furthermore, anti-MCV titers correlated with disease activity in our cohort and, accordingly, may allow the stratification of RA. This raises the possibility that this novel marker may be suitable for both monitoring and diagnosing RA. However, even larger series will be necessary to fully appreciate the very important correlation between disease activity and anti-MCV levels, and such studies are currently being conducted by a European network. Our results strongly indicate that mutations and citrullinations of vimentin trigger the autoantibody response. The breakage of self tolerance to this antigen and the potential pathogenic significance of modified vimentin will be investigated in future studies.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Burmester 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 design. Bang, Egerer, Gauliard, Fredenhagen, Berg, Burmester.
Acquisition of data. Bang, Egerer, Gauliard, Lüthke, Fredenhagen, Berg, Burmester.
Analysis and interpretation of data. Bang, Egerer, Feist, Burmester.
Manuscript preparation. Bang, Egerer, Feist, Burmester.
Statistical analysis. Rudolph.