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
]. 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
]. 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 antigen-antibody 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
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 TNFα, IL-1β, IL6, IL8 and granulocyte-macrophage colony-stimulating 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 TNFα in peripheral blood mononuclear cell (PBMC) cultures from healthy donors. When IC from RA serum or healthy serum were used, no elevated levels in TNFα 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 citrulline-containing 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
], 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 α1-antitrypsin [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 with proteinG, but it has the disadvantage of isolating the free immunoglobulins as well. For the identification of the antigens in IC, a sensitive method like mass spectrometry and immunodetection is necessary because of their low abundance.
In this report a broad range proteome approach, by means of mass spectrometry, is used in order to find new antigens in IC. Because of the low abundance of the antigens and the excess of immunoglobulins, it is possible that not all antigens will be detected by this approach, especially antigens that have a molecular weight that correspond to those of immunoglobulins. Therefore, a second, very sensitive method such as immunodetection on 2D-PAGE, was chosen to confirm the results of the broad range proteome approach and to investigate whether known antigens in RA (e.g. fibrinogenβ (Fibβ), fibrinogenγ (Fibγ), fibronectin and vimentin) are present in these complexes. Besides the high sensitivity of immunodetection, Western blot makes it also possible to visualize different isoforms of a certain protein.
Since not only the identification of the antigens, but also their citrullination status was of interest, the choice of antibodies for immunodetection was based on previous reports on citrullinated proteins either in serum, SF or synovial tissue of RA patients. The comparison between citrullinated proteins in serum and SF was already reported by Takizawa et al.
]. In their study, soluble antigens were studied in RA serum, RA SF and osteo-arthritis (OA) SF. They could only identify citrullinated fibrinogen in RA SF. However, two years later, also citrullinated fibronectin and citrullinated vimentin were found as soluble antigens in RA SF and synovial tissue [20
]. Citrullinated fibronectin was also detected in RA SF and synovial exosomes [23
]. Additionally, the presence of citrullinated Fibβ and Fibγ in RA synovium has been reported by Matsuo et al.
]. Based on these findings, immunodetection was performed with anti-Fibβ, anti-Fibγ, anti-fibronectin and anti-vimentin antibodies on 2D-PAGE with IC, followed by anti-modified citrulline (AMC) detection.
The citrullination of the antigens perfectly fits the model for the development and chronic nature of RA proposed by van Venrooij and Pruijn. They divided the process of autoimmunity in RA into five steps: an innocent inflammation in combination with massive apoptosis or impaired clearance can lead to the elevation of cytosolic Ca2+
concentrations (1) followed by the activation of peptidylarginine deiminase (PAD) and the citrullination of proteins (2). When citrullinated antigens are presented to T cells, the production of ACPAs is triggered (3). Immune complexes can be formed if the antigens react with the auto-antibodies (4). These IC stimulate inflammatory processes (5) and cause a vicious circle of inflammation resulting in joint destruction for years [26
This study describes the isolation and characterization of antigens residing in IC of RA patients. We found that circulating IC in the serum of RA patients and healthy controls contain Fibβ and fibronectin, both in a non-citrullinated form. In IC, isolated from RA SF, on the other hand, Fibrinogenβ, Fibrinogenγ, fibronectin and vimentin were identified. More importantly, vimentin and a minor portion of Fibβ were found to be citrullinated in the isolated complexes from RA SF. However, these citrullinated antigens were only 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.