One of the most important serological discoveries in rheumatology in recent years has been the characterization of autoantigens in rheumatoid arthritis (RA) containing the amino acid citrulline. There are many citrullinated proteins in the inflamed RA synovium. Rheumatoid factor (RF), which is the immunologic hallmark of RA, is not specific for RA, as it is found in 5% of healthy individuals and in 10–20% of those over the age of 65 years. RFs are of low titer in early disease stages when a clear diagnosis is often not yet possible; But anti-citrullinated protein antibodies (ACPAs) can be found early in the disease course of RA, even years before the onset of clinical symptoms. The identification of citrullinated epitopes led to the development of the first and later second generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. Anti-CCP2 antibody has shown a specificity of 98% in sera from patients with established RA and 96% in sera from subjects with early RA. Anti-CCP can predict erosive disease, therefore could be a good serological marker for RA diagnosis.
Rheumatoid arthritis (RA) is an autoimmune-inflammatory disease that may lead to joint destruction and disability. Early diagnosis and immediate, effective therapy are crucial in order to prevent joint deterioration, functional disability and unfavorable disease outcome. Numerous studies have shown that substantial irreversible joint damage occurs within the first 2 years. Currently, optimal management of RA is needed within 3–6 months after the onset of disease, thus reliable outcome measures are needed in order to establish prognosis early and to conduct good clinical practice.
The search for autoantigens that might be relevant in both the pathogenesis and diagnosis of RA has led to the characterization of several interesting and novel autoantibodies that appear to be considerably more disease-specific than rheumatoid factors (RF). The identification of citrullinated proteins as autoantigens and the development of new assays detecting anti-citrullinated protein antibodies (ACPAs) is a major breakthrough in the laboratory diagnostics of RA. In this paper, we review recent data on ACPAs with special attention to diagnostic and prognostic value of these antibodies in RA.
History of RA-Associated Antibodies
Rheumatoid factor, the immunologic hallmark of RA, was first mentioned in 1922. RF is an autoantibody that reacts with the Fc (fragment crystallizable) portion of IgG (immunoglobulin G). It may be IgM, IgG or IgA. RFs are found in healthy individuals as part of natural autoimmunity. Low-affinity RFs appear to play an important role in the host response to many infectious organisms. It is a component of the classification criteria for RA published by the American College of Rheumatology (ACR).
Anti-citrullinated protein antibodies (ACPAs)
One of the most important serological discoveries in rheumatology in recent years has been the characterization of autoantigens in RA containing the epitopes with nonstandard amino acid citrulline. Citrulline is the post-translationally modified, deiminated derivative of arginine residue, which is present on certain human proteins.[2, 9] Citrullination is not specific for RA; however, other rheumatologic diseases with synovitis, including inflammatory osteoarthritis, reactive arthritis, undifferentiated arthritis, gout and even trauma, show the presence of citrullinated proteins.
Anti-perinuclear factor (APF)
Anti-perinuclear factor, which has been known since 1964, mostly of the IgG type, is found in 48–86% of serum samples and synovial fluid from patients with RA. Westgeest et al. found that APF titers correlate with the severity of disease, and this is most clearly seen in patients without RF.
Antikeratin antibodies (AKA)
Keratin is significantly expressed in the RA synovial membrane and is a novel substrate for citrullination in this tissue. Citrullinated keratin might be involved in the pathogenesis of RA, including abnormal cell proliferation, apoptosis and autoimmunity, although the exact mechanism remains unclear. AKA were described in 1979, but the antigen targeted by AKA was identified in 1993 as the intermediate filament-aggregating protein filaggrin. Filaggrin is produced during the late stages of terminal differentiation of epithelial cells in mammals and is usually absent from the synovium, physiologically found in keratin. Schellekens et al. showed that the only citrullinated forms of fillagrin were recognized by AKA.[16, 17]
Anti-citrullinated filaggrin antibody (AFA)
The term AFA has been introduced instead of APF and AKA to describe these RA-associated autoantibodies. AFA was found to be highly specific (> 90%) for RA.
A promising candidate antigen for autoimmune response in RA is fibrin, which is present in the synovium of RA patients. In inflamed RA synovia the oxygen metabolism is in disequilibrium, which leads to generation of reactive oxygen species and then hypoxia which can cause synovial tissue micro-infarctions. At these sites, plaques of extravascular fibrin can be found. It has recently been shown that antifilaggrin and anticitrulline antibodies recognize citrullinated fibrin. A follow-up study showed that citrullinated fibrin is not specific for RA synovium but is also found in spondylarthropathy and inflamed osteoarthritis synovium.
Anti-citrullinated fibrinogen antibodies (ACF)
Fibrinogen can also be regarded as citrullinated antigen. RA is characterized by excessive generation and breakdown of fibrinogen. Citrullinated fibrin(ogen) is certainly one of the most abundant and important antigens. It was detected as a soluble autoantigen in RA synovial fluids.
Scott et al. and Sanchez-Pernaute et al.[22, 25] reported that most fibrin aggregates in the RA synovium contain fibronectin (Fn). Fn comprises a large family of isomeric glycoproteins characterized by repeated amino acid units that form domains (including fibrin, gelatin, collagen, cell, vascular endothelial growth factor [VEGF] and heparin-binding domains). Chang et al. show that Fn in synovial tissue and plasma of RA is citrullinated and after citrullination, Fn increases its binding activity to VEGF but decreases its affinity to integrin β1 and the ability to stimulate apoptosis. Shelef et al. recently in their study concluded that citrullination of Fn alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of RA.
Anti-citrullinated vimentin/Anti-Sa antibodies
Citrullinated vimentin has been described as a relevant autoantigen expressed in synovial tissue. Vimentin is an intermediate filament protein and thus is located in the cytosol. It is specifically citrullinated in the macrophages after Ca2+ (calcium) influx.
An RA-specific autoantibody was discovered in 1994 named after a patient as anti-Savoie (anti-Sa). It was shown that the placental Sa antigen corresponds to citrullinated vimentin, and that the RA-specific anti-Sa antibodies recognize citrullinated (but not unmodified) vimentin in vitro. A study by Hueber et al. showed that anti-Sa was ~ 98% specific for RA.
Both citrullination and mutation can influence the antigenicity of vimentin. Recombinant mutated citrullinated vimentin (MCV) has been suggested to be a more sensitive autoantigenic target of ACPA. To detect antibodies to citrullinated vimentin, a new enzyme-linked immunosorbent assay (ELISA) system has recently been developed. This assay utilizes genetically MCV to improve the performance of the test.
Immunopathogenesis of RA
It has been recently shown that several RA-associated genetic factors may be functionally linked to RA via modulation of the production of citrullinated proteins or the antibodies directed at them. Only a small percentage of the general population carries ACPAs. Smoking promotes the citrullination of synovial proteins and thus ACPA production. Recent data suggest that cigarette smoking establishes a higher risk for ACPA-positive RA. Higher body weight at birth, the use of oral contraceptives and excessive caffeine consumption (10 cups per day) conferred higher risk to develop ACPA-positive RA.
The normal, uninflamed synovium do not contain high amounts of citrullinated proteins. The association of high titers of the ACPAs with an erosive disease outcome suggests a possible role in the pathophysiology of the disease.
It is more likely that RA patients exhibit an abnormal humoral response to citrullinated proteins that may be present in any form of (synovial) inflammation. In those individuals who are able to present citrullinated fragments of proteins to T cells via certain human leukocyte antigen (HLA) molecules, an immune response to citrullinated antigens might be generated, resulting in the production of high-affinity IgG ACPAs. Masson-Bessière et al. have recently shown that these anti-citrullinated protein antibodies are locally produced by plasma cells in the synovium, and thus are likely to be triggered by a citrullinated substrate that is present in the RA synovium.
PAD enzyme and citrullination
The process of citrullination in mammalian cells has been studied by only a few groups, and involves the enzymatic conversion (deimination) of protein-contained arginine residues (Fig. 1). The result of this conversion is a very small change in molecular mass (somewhat less than 1 Da) and the loss of one positive charge. The consequence of the latter might be a change (loss or gain) in its ability to interact with neighboring proteins. The enzyme responsible for the citrullination is PAD (peptidylarginine deiminase). PAD enzyme was first described in 1977. Normally, PAD enzymes are present intracellularly (either in the cytosol or, in the case of PAD4, in the nucleoplasm).
Five PAD isoforms (PAD1, PAD2, PAD3, PAD4–5, PAD6) are currently distinguished. The PAD4 catalyzes citrullination of polypeptides.
Inflammatory leukocytes including synovial T and B cells, macrophages, neutrophils, as well as fibroblast-like synoviocytes express two isoforms of this enzyme, PAD2 and PAD4. The most noticeable difference between the different PAD isotypes is their tissue-specific expression. PAD2 is the most widely expressed type of PAD. The most abundant expression is observed in skeletal muscle, brain, spleen and secretory glands.
Concurrently, the association of functional haplotypes of the gene encoding citrullinating enzyme of PAD4 with susceptibility to RA was reported and it was also demonstrated that PAD4 affected levels of the antibody to citrullinated peptides in sera from patients with RA. Calcium ions are required for activity of the PAD enzymes, but the cytosolic and nucleoplasmic Ca2+ concentration under normal physiological conditions is much too low (about 100-fold) for PAD activity. During apoptosis (or necrosis) the plasma membrane Ca2+ pump (PMCA) is cleaved by caspases, so that influxing calcium can no longer be efficiently cleared by PMCA, which results in Ca2+ overload. When there is massive apoptosis, for example due to a toxicant or infection, or a (genetic) defect in the clearance system, some apoptotic cells may become necrotic. At this time they release the intracellular citrullinated proteins (for example, histones, vimentin) and the activated PAD enzymes. PAD enzyme isoforms contain highly conserved cysteine in their active site, which plays a crucial role in the catalysis process. It has been shown that agents acting on cysteine sulfhydryl groups via binding them covalently can inactivate the enzyme, while reduced compounds can enhance its activity.
The CCP1 and CCP2 systems
The identification of citrullinated epitopes as targets for anti-filaggrin antibodies led to the development of the first and later second generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. The first ELISA systems detecting antibodies against citrullinated antigens utilized synthetic, filaggrin-derived linear peptides. Since filaggrin is not expressed in the synovium, it is most likely not the natural antigen for the anti-citrullinated protein antibodies. Nowadays more sensitive, second generation anti-CCP2 assays are used. Second generation ELISAs use synthetic peptides as antigens, with a ring structure due to intramolecular disulfide. Use of these CCPs has improved their specificity between 96% and 98%, without changing the sensitivity. Anti-CCP antibodies, the most important members of the family of ACPAs so far, are specific diagnostic and prognostic markers in RA.
Local presence of citrullinated auto antigens
Irrespective of the site of B cell activation, there is experimental evidence that ACPAs are produced in RA joints and may mediate tissue injury. It has been noted that novel IgM-producing B cells are continuously recruited to the inflamed RA joint, demonstrating that the ACPA response is continuously reactivated during the course of arthritis. When citrullinated proteins are present in the inflamed RA synovium, PAD enzymes must also be present. Citrullinated proteins were identified in the synovial lining layer, the sublining layer, and in the extravascular fibrin deposits in the RA joint. It is also reported that PADs can autodeiminate themselves, due to their molecular structure, which might be changed profoundly and new epitopes may arise. To easily understand ACPAs see Table 1.
Table 1. Different kinds of anti-citrullinated protein antibodies
Keratohyalin granules in the cytoplasm, profilaggrin
Detected in early RA
Filaggrin in keratin
Detected in early RA
Detected in early arthritis, for radiographic progression after 1 year
Synovial tissue, plasma
Alters synovial fibroblast behavior
Vimentin, Recombinant MCV
RF and anti-CCP2 seronegative patients
Positivity is associated with higher serum anti-CCP levels
Citrullination and ACPA response
Although all citrullinated proteins are not specifically recognized by ACPAs, the presence of citrullinated proteins with proven antigenic affinity for ACPAs, such as citrullinated (pro)filaggrin in the skin, is not sufficient to initiate the antibody response. The greater presence of citrullinated proteins will not necessarily lead to chronic inflammation because in 99% of individuals the citrullinated proteins are degraded without a (humoral) reaction of the immune system.
It is well known that in vivo ACPAs are directed against various citrullinated epitopes in which not only the citrulline residue, but also the flanking amino acids, make up the antibody recognition site. Different citrullinated substrates yield different ACPA sensitivities and specificities for RA when used in the same patient cohorts (Table 2).
Table 2. Specificity and sensitivity of anti-citrullinated protein antibodies and RF
The development of an autoimmune response against citrullinated epitopes is facilitated by a specific genetic predisposition namely, the presence of particular HLA-DRB1 alleles (‘shared epitope,’ SE). The presence of HLA-DRB1 SE alleles in RA patients are primary risk factors for the generation of anti-CCP antibodies. Disease progression has been related to anti-CCP production and SE positivity. HLA-DR genes exert a major influence on the (cluster differentiation) CD4+ αβ T-cell repertoire, and SE alleles are thought to efficiently present self-peptides to CD4+ T cells in the thymus.
Cantaert et al. explained two interesting mechanisms that could be involved in the restriction of the immune response toward citrullinated proteins. First, not only the presence, but also the amount, of antigen presented to the immune system determines the immune response, by breaking a putative tolerance threshold. Suzuki et al. proposed that increased PAD4 messenger RNA (mRNA) stability in RA could lead to an increase in the expression of the deiminating enzyme, which in turn could augment protein citrullination and contribute to breaking the tolerance threshold.
Although the HLA–DR shared epitope is involved in the ACPA response, the MHC background cannot explain alone the specificity of ACPAs for RA.
Diagnostic Potential of ACPAs
Rheumatoid arthritis is diagnosed based on a set of clinical, serological and radiological criteria. In many early cases of RA, clinical symptoms are milder and nonspecific, and patients will not fulfill ACR classification criteria for RA. Therefore, the detection of a disease-specific autoantibody like anti-CCP could be of great diagnostic and therapeutic importance. Early diagnosis and immediate, effective therapy are crucial in order to prevent joint deterioration, functional disability and unfavorable disease outcome.[1, 18] RF have a modest RA disease specificity (up to 66%) and occur with less frequently or are of low titer in early disease stages when a clear diagnosis is often not yet possible; but ACPAs can be found early in the disease course of RA, even years before the onset of clinical symptoms. ACPAs are detected in approximately 80% of RA patients at a specificity of 98%. AKA can be detected in early RA, as well as in RF-negative patients. The overall sensitivity of anti-CCP2 assays is similar to that of RF (60–80%), but importantly, anti-CCP2 antibody is positive in 20–30% of RF seronegative patients. Anti-CCP2 antibody ELISA testing has shown a specificity of 98% in sera from patients with established RA and 96% in sera from subjects with early RA. APF has been detected in 50–70% of RA patients, even in the early phase of the disease. It has a high specificity (90%) but rather low sensitivity. Some reports mention raised IgA-RF levels as parameters for disease activity; but it can also be detected in sera of patients with other autoimmune diseases, infectious disorders, as well as in the healthy elderly population. Due to its high specificity for RA, anti-CCP can be used to differentiate RA from other forms of arthritis that may be confused with RA because they can be erosive and RF-positive. At the same time, the presence of ACPAs is associated with more severe joint destruction and greater disease activity.
The combination of RF and ACPA, especially antibodies against CCP, is generally accepted by the majority of rheumatologists and recommended by the European League of Arthritis and Rheumatism (EULAR). The new RA classification criteria proposed by a joint task force of the ACR and EULAR at the ACR/ARHP (Association of Reproductive Health Professionals) 2009 Annual Meeting in Philadelphia also include both RF and anti-CCP as serological critera.
Anti-CCP antibodies constituted a higher proportion of IgG in synovial fluid than in serum. An ELISA based on MCV has been commercially available for the diagnosis of RA. A significant correlation has been established between anti-MCV antibody titers and both the severity of the RA and the disease activity score. Szekanecz et al. showed that the anti-MCV test is able to identify a number of RA patients who are tested seronegative by IgM RF or anti-CCP2 assays. The combined application of anti-CCP2 and anti-MCV assays can improve the laboratory diagnostics of RA.
Prognostic Value of ACPAs
A significant amount of available data suggests that genetic factors and autoimmune-inflammatory markers could be good indicators for RA outcome. ACPA has a prognostic role during the progression of RA and it is also associated with more pronounced structural damage of the joints indicated by radiographic progression. The determination of ACPA in association with genetic and environmental factors at the onset of the disease may serve as an outcome measure in RA. Hayem et al. who found the incidence of ACPAs in RA patients with severe destructive disease, showed that the anti-Sa antibodies may be of prognostic benefit. Anti-Sa antibodies provide a high specificity of > 98%, but a limited sensitivity of 22–40% for patients with RA. Investigators have recently confirmed that anti-Sa positivity is associated with higher serum anti-CCP levels. ACF antibodies are good predictors of diagnosing RA at 1 year in patients with early arthritis, and for radiographic progression after 1 year. There is not yet a standardized commercial test for the immunoassay to citrullinated fibrinogen which can be used in routine diagnostic testing.
A study using the CCP2 assay found progression from undifferentiated polyarthritis to RA in 93% of anti-CCP-positive patients but only in 25% of anti-CCP-negative patients after 3 years of follow-up. The anti-CCP status rarely (5%) changes during the disease course in established RA.
In a comparative study, anti-CCP had higher positive predictive value for erosive RA than RF, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) or matrix metalloproteinase-3 (MMP-3).
The presence of autoantibodies suggests that a diagnosis of RA in the absence of RF-positive sera can potentially be made. RF analysis should be accompanied by other autoantibody assays, particularly when the RF titer is low or absent, or when the diagnosis is uncertain. According to current knowledge, diagnosis of RA should not be solely based on the determination of RF. Although RFs are not highly specific for RA, the persistence of high-titer RF may predict the development of erosive RA. Persistent production of high levels of IgM RFs is typical for RA and contributes to disease progression. Anti-CCP antibodies could be detected in predisease serum samples up to 14 years before onset of the first symptoms of RA and IgM-RF up to 10 years. To date, the anti-CCP with IgM-RF and IgA for profiling provides a remarkable diagnostic sensitivity of 88.2%. The additional determination of anti-MCV and anti-filaggrin increases the sensitivity to more than 90.1%.
Considerable evidence exists for the pathogenetic involvement of RF; therefore, a potential role for autoantibodies in the disease process of RA should not be ignored.
Identifying biomarkers, suggesting the transition of undifferentiated polyarthritis to RA are of utmost importance. The determination of ACPA in association with genetic and environmental factors at the onset of the disease may serve as a notable index in RA.
The sensitivity of CCP was increased by the second test generation CCP2 and is now comparable with the sensitivity of IgM-RF. The CCP can be used as a screening test as well. Because anti-CCP is extremely specific for RA, is present early in disease and predicts the erosive states of disease, therefore it could be a good serological marker for RA. It is likely that as the range of RA-associated autoantigens expands, and as the repertoire of citrullinated target autoantigens is defined, multiplex assay of serum autoantibodies will play an increasingly important role in the diagnosis and prognosis of subsets of inflammatory arthritides such as RA.