Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Van Steendam et al. Arthritis Research & Therapy RCesietarrcuh lalritincleated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins Katleen Van Steendam 0 Kelly Tilleman 0 Marlies De Ceuleneer 0 Filip De Keyser 1 Dirk Elewaut 1 Dieter Deforce 0 0 Laboratory for Pharmaceutical Biotechnology, Ghent University , Harelbekestraat 72, B-9000 Ghent , Belgium 1 Department of Rheumatology, Ghent University Hospital , De Pintelaan 185, B-9000 Ghent , Belgium Introduction: Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods: IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results: Circulating IC in the serum of RA patients and healthy controls contain fibrinogen and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogen and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogen were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions: Citrullinated fibrinogen and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA. - Introduction Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by chronic inflammation of the peripheral joints. It is a complex multifactorial pathology, in which genetic and environmental factors, like smoking, can play an important role in the onset of disease and the progression of the joint damage [1,2]. The presence of immune complexes (IC) in serum and synovial fluid (SF) of RA patients is likely to contribute to the pathogenesis of the disease and to articular damage, since they are responsible for the activation of complement, the stimulation of phagocytes through their Fc receptor and the release of chemotactic factors, cytokines, metalloproteinases and reactive oxygen intermediates [3-6]. The formation of IC as such is not specifically related to autoimmune pathologies as it is a natural process, completing an immune response in the body. The antigenantibody complexes are usually effectively removed by phagocytosis. However, it is known that an impaired clearance of these complexes can elicit or sustain an inflammatory response [7,8]. The pathological nature of IC has been suggested by several groups based on in vitro studies. The effect of the SF IC from juvenile RA patients on healthy PBMCs was studied by Jarvis et al. They found that especially the high molecular weight IC, separated by size exclusion chromatography from the other immunoglobulins and low molecular weight IC, were responsible for inducing a spectrum of pro-inflammatory cytokines, such as TNF, IL-1, IL6, IL8 and granulocyte-macrophage colonystimulating factor (GM-CSF) [9]. A comparison between IC from SF of RA patients, serum of RA patients and serum of healthy persons was made by Schuerwegh et al. They demonstrated that IC isolated from RA serum and RA SF, in contrast to IC from healthy persons, had an effect on chondrocyte growth, NO production and apoptosis, thereby contributing directly to cartilage destruction in RA [10]. Mathsson et al. showed that polyethylene glycol (PEG) precipitated IC from RA SF induced the production of the pro-inflammatory cytokine TNF in peripheral blood mononuclear cell (PBMC) cultures from healthy donors. When IC from RA serum or healthy serum were used, no elevated levels in TNF could be seen [11]. These reports show the relevance of IC in the joint destruction and the pathogenesis of RA. The best known IC in RA is the rheumatoid factor (RF) bound to its antigen, the Fc domain of IgG. The RF, which is mainly IgM [12], is used in diagnostic tests for RA and has a sensitivity of 78.6% and a specificity of 80.8% [13]. The RF factor is also found in other diseases such as systemic sclerosis (20 to 30%) [14] and occasionally in healthy persons (1.3 to 4%) [5]. Besides the RF, immunoglobulins and complement factors, other components can also be present in IC from serum of RA patients. Indeed, recently, it has been shown that fibrinogen and citrullinecontaining fibrinogen are present in the IC of RA patients [15]. Because of the pathogenic nature of IC in RA, it is important to identify the antigens in these complexes. After identification of these antigens, a better understanding of the immunological process in the affected joints can be achieved. Since anti-citrullinated protein/peptide antibodies (ACPA) are very specific for RA (specificity of 98%, sensitivity 68%) [16] and high amounts of citrullinated proteins, like fibrinogen, have been detected in the joint of RA patients, it is likely that some antigens in IC of RA patients are citrullinated. The isolation of IC and subsequent identification of the antigens is therefore of great importance in the understanding of RA. The isolation of IC from biological matrices has been tackled by many different techniques such as PEG precipitation [10,11,17], C1q ELISA [15] and immunoprecipitation [18]. PEG precipitation is broadly used for the isolation of IC but the IC-fraction also contains a considerable amount of non immune complex (IC)related proteins, such as albumine, haptoglobin and 1antitrypsin [17]. C1q ELISA will isolate IC that are bound to the C1q component of the complement and this method is gaining interest because of the high throughput possibilities. However, to capture IC by C1q ELISA, C1q must be present and accessible in the IC. IC from serum and SF can be isolated with a high purity by means of immunoprecipitation (...truncated)