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AUTOIMMUNITY

Autoantibodies in rheumatoid arthritis

One of the most severe and most destructive of all joint diseases, rheumatoid arthritis (RA) is characterised serologically by the presence of a large number of autoantibodies with different antigenic specificities and varying diagnostic sensitivities and specificities. This article reviews the characteristics of the principal RA autoantibodies, highlighting those that are most useful diagnostically. In addition to their diagnostic use, a deeper understanding of RA autoantibodies should give greater insight into the cellular and molecular processes underlying the disease itself. by Dr Gnter Steiner
Rheumatoid arthritis (RA) is the most severe and most destructive of all joint diseases affecting approximately 1% of the world-wide adult population. It is also the most common systemic autoimmune disease and is characterised by the presence of various autoantibodies in serum and synovial fluid. This distinguishes RA from other joint diseases such as psoriatic arthritis, reactive arthritis or osteoarthritis where autoimmune phenomena are rarely observed. Rheumatoid factor (RF) was the first autoreactive element identified in the sera of RA patients and was later shown to be an autoantibody specific for immunoglobulin G (IgG). Since the initial description of RF by Emil Waaler in 1940, numerous additional autoantibodies have also been found in the sera of patients with RA. The targets of these autoantibodies are quite diverse and include collagen and other cartilage proteins, heat shock proteins, enzymes, nuclear proteins and, most importantly, citrullinated proteins such as fibrin or vimentin [Table 1]. Among the antibodies detectable in the sera of RA patients, anti-citrullinated protein antibodies (ACPA) show the highest disease specificity, while all other antibodies including RF may occur also in other diseases and even in healthy individuals. Nevertheless, RF is still the most widely used serological marker of RA and one of the seven classification criteria of the American College of Rheumatology for the disease. Even though most antibodies cannot be used for diagnostic purposes, they may contribute to the pathological processes characteristic of RA such as chronic synovitis and erosiveness [1-4]. epithelial cells. Interestingly, filaggrin forms the target of antikeratin antibodies that had been described previously to be highly specific for RA [6]. Since filaggrin is not expressed in the joint, it presumably represents a cross-reacting antigen rather than the primary target structure of ACPA. One attractive candidate antigen is fibrin and its precursor fibrinogen, since citrullinated forms of these proteins have been demonstrated to be present in the synovial tissue of patients with RA and other arthritides. Citrullinated fibrinogen appears to be a dominant B cell antigen that is targeted by the majority of RA patients with established disease [7]. Another antigen of great interest is the cytoskeleton protein vimentin that occurs in various citrullinated isoforms. ACPA can be measured by ELISA which may use citrullinated proteins such as filaggrin, vimentin or fibrinogen or citrullinated peptides as antigen. The currently most widely used assays employ a cyclic citrullinated peptide (CCP); ACPA determined by such assays (available commercially from several manufacturers) are usually referred to as anti-CCP antibodies. It is now definitely established that ACPA are the most specific serological marker antibodies for RA. Moreover, as with RF, they are present early in the course of the disease and may even precede clinical onset [4,7].ACPA have a specificity for RA of at least 95% and a sensitivity that is comparable to that of RF, reaching approximately 75% in established RA. However, as with RF, the sensitivity in early RA is lower, ranging between 40% and 55% [Table 3]. Significant correlation has been established in several studies between the presence of ACPA and radiographic disease progression (i.e bone damage).
Diagnosis RF20U/mL 66%* 62% 27% 44% 18% 10% 25% 13% RF50U/mL (RF50) 46% 52% 10% 8% 0% 4% 4% 0% 46% 88%

Antigen Immunoglobulin G Citrullinated proteins (fibrin, vimentin, filaggrin) Heterogeneous nuclear ribonucleoprotein A2 (RA33) Collagen II Stress proteins Glucose-6 phosphate isomerase (GPI)

Antibody Rheumatoid factor ACPA, anti-CCP anti-RA33 anti-collagen anti-BiP, anti-hsp90 anti-GPI

Table 1. Autoantigens of potential pathogenetic relevance in rheumatoid arthritis.

Rheumatoid factors (RF)

These interact with the Fc portion of IgG. The major isotype is IgM but IgG-RF and IgA-RF are also present in the serum and synovial fluid of patients with RA and may provide additional diagnostic information. RF can be measured by various methods including agglutination techniques such as the classical Waaler-Rose test, laser nephelometry and ELISA, with the latter being able to distinguish between RF subtypes. Transient increases of IgM-RF are part of the normal immune response and are commonly observed during infections, probably in reaction to immune complexes containing microbial antigens. Thus, low-affinity IgM-RF can be found in 10-15% of (apparently) healthy individuals. In contrast, chronic persistence of high-titered IgM-RF (as well as the presence of IgG and IgA subtypes) is a

characteristic feature of RA which is less often seen in other autoimmune conditions. IgM-RF can be detected in 60-80% of RA patients with established disease, whereas the prevalence in patients with early RA is considerably lower. IgM-RF are not specific for RA and they can also be detected at high titers in most patients with primary Sjgren's syndrome or mixed cryoglobulinaemia and also at lower titers in other rheumatic diseases. However, the specificity of RF for RA increases considerably with higher titres, and RF>50 IU/mL (RF50) is quite specific for RA [Table 2]. However, RF50 is present in only about 50% of patients with early RA when the diagnostic criteria for RA are often not yet fulfilled [Table 3]. RF of all subtypes may be present already in the earliest stages of the disease and can even precede the onset of RA by several years. Interestingly, several studies suggest IgA-RF to be a more specific marker antibody for RA than IgMand IgG-RF subtypes. Importantly, high titer IgMRF and IgA-RF have also considerable prognostic value because they are associated with the severe forms of RA, such as radiological erosions, more rapid disease progression, worse outcome and extra-articular manifestations [2, 3, 5].

Anti-Citrullinated Protein Antibodies (ACPA)

One of the most exciting scientific findings in autoimmune research in the last decade has been the identification in the sera of patients with RA of autoantibodies which are specific for epitopes containing the unusual amino acid citrulline that is generated by post-translational deimination of arginyl residues by the enzyme peptidyl arginine deiminase [Figure. 1]. Autoantibodies recognising citrullinated epitopes are now generally named anti-citrullinated protein antibodies (ACPA). Citrullinated epitopes were first identified in filaggrin, a protein expressed exclusively in squamous

Rheumatoid arthritis Sjgren's syndrome Systemic lupus erythematosus Scleroderma Poly/dermatomyositis Reactive arthritis Osteoarthritis Healthy controls

Sensitivity for established RA(%) 66% Specificity for established RA (%) 78%

Table 2. Sensitivity and specificity of IgM rheumatoid factor for rheumatoid arthritis in patients with established disease. RF was determined by nephelometry in sera of more than 300 patients with various rheumatic diseases as well as in 30 healthy controls. * Percentage of positive patients

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Sensitivity (%) RF (>20 IU/ml) RF50 (50 IU/ml) ACPA (anti-CCP) Anti-RA33 55 45 41 28 Specificity (%) 89 96 98 90 PPV (%) 84 92 96 74

Issue 7 November 2006

Table 3. Diagnostic value of RF, ACPA and anti-RA33 autoantibodies in patients with early arthritis. RF was measured by nephelometry, ACPA by ELISA (anti-CCP) and anti-RA33 by immunoblotting in a cohort of 200 patients with early untreated arthritis of less than 3 months duration [8]. A final diagnosis of RA was made in 102 patients, 98 patients developed other arthritides such as reactive arthritis, osteoarthritis or undifferentiated arthritis. ACPA (anti-CCP) and RF50 (50 IU/mL) both showed high specificity for RA and high positive predictive values (PPV), while RF<50 IU/mL proved of little diagnostic value. There was a highly significant correlation between ACPA and RF50 (p<0.0001), whereas anti-RA33 was not associated with either antibody characterising a subset of patients with relatively mild disease [see also Figure 2].

with SLE or mixed connective tissue disease where they are significantly associated with anti-Sm and anti-U1 RNP antibodies which are not targeted in RA. Anti-RA33 antibodies have a specificity of approximately 90% for RA which is lower than the specificity of ACPA or RF50 but much better than that of low titer RF (i.e. RF<50 IU/mL). As with ACPA and RF, anti-RA33 antibodies may be present already in the initial stages of the disease [Table 3]. Since they do not correlate with RF or ACPA they represent useful additional marker antibodies, particularly in patients negative for ACPA and RF. Furthermore, they are not associated with radiographic progression and seem rather to characterise patients with a more favourable prognosis [8].

autoimmunity plays a major role in the pathogenesis of human RA. The prevalence of anti-collagen II antibodies in RA sera has been reported as being between 30% and 70%; levels of anti-collagen II seem to be particularly high in early disease and to decline as the disease progresses. Most studies did not find a significant correlation between autoimmunity to collagen II and disease duration, activity and severity of RA. Because of this and due to their apparent lack of disease specificity anti-collagen antibodies are not considered as useful diagnostic markers. Antibodies to heat shock proteins Stress or heat shock proteins are upregulated under conditions of cellular stress and protect cells from severe damage and premature death. They are evolutionarily highly conserved from bacteria to man and are among the most immunodominant microbial antigens. This has led to speculations about potentially pathogenetic cross-reactivities that might arise in the course of infections. Antibodies to stress proteins, which are highly expressed in

Other autoantibodies
Anti-collagen antibodies Antibodies to collagen II are present in RA synovial fluids and are presumably produced locally in the joint. Although arthritis can be induced in susceptible mouse strains by immunisation with collagen II, there is little evidence that anti-collagen

Furthermore, ACPA were found to be significantly associated with the presence of RA associated HLADR alleles ("the shared epitope"). ACPA positive carriers of the shared epitope appear to be at particularly high risk for developing severe forms of RA. Thus, ACPA are currently the most valuable marker antibodies whose determination is particularly useful in patients with early arthritis when the clinical criteria for RA are not fulfilled.

Anti-RA33 autoantibodies
These are directed to the heterogeneous nuclear ribonucleoprotein (hnRNP) A2 which is involved in various posttranscriptional processes including pre-mRNA splicing, mRNA transport and translation. Remarkably and of potential relevance for pathogenetic involvement, the hnRNP is highly over-expressed in inflamed synovial tissue, while expression in normal joints is rather low [2].
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Figure 1. Deimination (citrullination) of an argininyl residue by peptidylarginine deiminase (PAD). Enzymatic arginyl to citrullyl conversion is a post-translational modification that changes the charge and biochemical properties of proteins. Citrullination is predominantly observed in proteins of the cytoskeleton such as cytokeratin, vimentin or filaggrin, but also in other proteins and seems to represent a general regulatory mechanism occurring particularly during apoptosis.

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Anti-RA33 antibodies can be measured by a commercially available ELISA or by the somewhat more sensitive immunoblotting method which, however, can be performed only in a few specialised laboratories. Occurring in approximately one third of RA patients, anti-RA33 antibodies are less common than RF or ACPA but, importantly, they are rarely detected in other arthritides such as osteoarthritis, reactive arthritis, ankylosing spondylitis or psoriatic arthritis [1, 2]. They may also be present in patients

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References
1. Steiner G, Smolen J. Arthritis Res Ther 2002; 4 Supple 2:S1-5. 2. Steiner G. In: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH, editors. Rheumatology. London: Elsevier Sciences; 2003: p. 833-42. 3. Dorner T et al. Curr Op Rheum 2004; 16(3): 246-53. 4. Vossenaar ER & van Venrooij WJ. Arthritis Res Ther 2004; 6(3): 107-11. 5. Scott DL. Rheumatology 2000; 39: 124-9. 6.Youinou P & Serre G. Int Arch Allergy Immunol 1995; 107(4): 508-18. 7. Vincent C et al. Autoimmunity 2005; 38(1): 17-24. 8. Nell V et al. Ann Rheum Dis 2005; 64(12): 1731-6. 9. Panayi GS & Corrigall VM. Autoimmunity Reviews 2006; 5(2): 140-2. 10. van Eden W. Human Immunology 2006; 67(6): 446-53. 11. Benoist C & Mathis D. Arthritis Res Ther 2000; 2(2): 90-4. 12. Kamradt T & Schubert D. Arthritis Res Ther 2005; 7(1): 20-8.

early presence is significantly associated with the development of bone erosions. Anti-RA33 on the other hand appears to be associated with mild disease and a relatively benign outcome. RF50 and ACPA are strongly correlated, while anti-RA33 occurs independently from these two antibodies [Figure 2]. Therefore sequential determination of RF (IgM), ACPA and antiRA33 is recommended as efficient and costeffective strategy for routine diagnostics [8].
Figure 2. Associations between RF50, ACPA and antiRA33 antibodies in patients with early RA. RF50 and ACPA overlapped significantly (p<0.0001), co-occurring in 28% of the patients. Thus, although RF50 was present in 45% of the patients and ACPA in 41% of them, only 58% were positive for RF50 and/or ACPA; these patients had a highly increased risk for developing erosive disease. AntiRA33 was the only detectable antibody in an additional 13% of the patients who had significantly milder disease and a more favourable prognosis. Data are adapted from Nell et al [8]. See also Table 3.

Pathogenetic considerations
Despite many years of intensive research the role of autoimmune reactions in the pathogenesis of RA is still incompletely understood. The search for antigens that might induce or modulate the disease has led to the identification of novel autoantigens and the characterisation of the autoimmune responses directed to them. As with RF, the majority of these responses are not particularly specific for RA. In contrast, ACPA are almost exclusively present in RA patients showing the highest disease specificity for RA of all autoantibodies identified so far. Nevertheless, the pathogenetic role of ACPA is still incompletely understood. It is assumed that the binding to their targets (e.g. fibrinogen) in the synovial tissue has proinflammatory effects by virtue of immune complex formation leading to B and T cell stimulation. This is most likely a self-maintaining process which may be partly responsible for the chronicity of the disease [4, 7]. Since fibrin deposition occurs after the onset of joint inflammation, ACPA might be initially induced by another antigen such as citrullinated vimentin and only secondarily cross-react with citrullinated fibrin. Concerning joint specific antigens such as collagen II, the inflammatory and destructive processes lead to an abundant release of materials which may give rise to autoimmunisation of the patient via phagocytosing macrophages functioning as antigen presenting cells. Autoimmune reactions to other proteins may arise due to over-expression, post-translational modification and aberrant processing of the antigens induced and maintained by the proinflammatory milieu within the joint as observed for stress proteins or hnRNP-A2 (RA33). Thus, depending on the genetic background (HLADR, cytokines, etc.) an increasing number of autoimmune reactions may be generated in the course of disease all of which may somehow contribute to the pathophysiology of RA. Taken together, autoantibodies can certainly no longer be regarded just as interesting epiphenomena some of which may be quite useful as diagnostic markers. The identification of new autoantigens, particularly citrullinated proteins, and the characterisation of the cellular and molecular processes underlying the pathological autoimmune reactions against them has provided new insights into the pathogenesis of RA. A better understanding of the disease process will finally make possible the development of novel therapeutic concepts that may allow the disease to be treated more effectively in its initial stages where the chances are highest to stop the disease process and bring patients into complete remission.

inflamed synovial tissue, can be found in many pathological conditions and may also occur in healthy individuals. They do not show specificity for any disease, even though they may be elevated in the sera of RA patients as compared to patients with osteoarthritis. Therefore, antibodies to stress proteins are only of modest diagnostic value. On the other hand heat shock proteins may contribute to the pathogenesis of RA by virtue of T cell reactivities directed to them. Remarkably, T cell autoreactivity to heat shock proteins seems to be of beneficial (i.e. anti-inflammatory and/or immunosuppressive) nature since in children suffering from juvenile chronic arthritis the presence of such T cell reactivity was associated with a better prognosis. Based on these findings and observations made in animal arthritis models, a role has been suggested for heat shock proteins in the control of immune regulation in inflammatory diseases [9, 10]. Antibodies to glucose-6 phosphate isomerase (GPI) Glucose-6 phosphate isomerase (GPI) is a highly conserved glycolytic enzyme that was identified as the arthritogenic autoantigen in the KRNxNOD mouse model of RA in which a transgenic T cell receptor induces arthritis closely resembling human RA (11). Moreover, immunisation with GPI has recently been shown to induce arthritis in susceptible mouse strains (12). Although anti-GPI antibodies may be present in sera and synovial fluids of RA patients, their incidence is low and they are not specific for RA. Interestingly, however, they have been found with increased frequency in RA patients suffering from extra-articular manifestations (Feltys syndrome) which may be indicative of a potential pathogenetic role in human disease. Diagnostic value of autoantibodies Among the various autoantibodies described in RA only RF, ACPA and anti-RA33 have so far proven useful for diagnostic purposes. As outlined in this review ACPA and RF50 (i.e. RF 50 IU/mL) both show high specificity for RA and considerable prognostic value since their

The author
Gnter Steiner, Ph.D., Department of Rheumatology, Internal Medicine III, Medical University of Vienna Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria Contact details: Department of Rheumatology, Internal Medicine II Medical University of Vienna Vienna General Hospital Waehringer Guertel 18-20, A-1090 Austria Tel +43 1 40400-4301 e-mail: guenter.steiner@meduniwien.ac.at

Novel marker for RA uses synthetic citrullinated peptides of human immunoglobulin G

A number of studies within recent years have demonstrated that the citrullination of arginine residues is a general characteristic of RA-specific antibody response: citrulline is a common epitope of RA-specific antibodies in a number of proteins such as keratin, filaggrin or vimentin. Anti-citrullinated protein antibodies (ACPA) have been established as the most specific serological marker antibodies for RA. They are particularly useful in early arthritis. Commercially available ELISA tests have adopted this property for serological detection based on coating with citrullinated peptides. It was demonstrated that short citrulline containing peptide sequences are sufficient for antibody recognition. As antibodies of RA patients always react with all sorts of citrullinated peptides and proteins occurring in the body, it was considered prudent to develop ELISA tests based on synthetic citrullinated peptides. The German company AESKU.DIAGNOSTICS succeeded in developing a novel ELISA test, AESKULISA RA/CP Detect, using synthetic citrullinated peptides of human immunoglobulin G for coating. The assay is a most sensitive and specific serological approach to the diagnosis of RA, combining the high specificity of the citrullin antigen with the sensitivity of a rheumatoid factor ELISA test. Showing comparable sensitivities and specificities AESKULISA RA/CP Detect is an effective and cost-effective alternative to the known CCP ELISA tests.