Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset

Abstract

< 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses. A spectrum of autoantibodies is now known to be specifically associated with RA. There continues to be uncertainty as to what stage of the disease each of these autoantibodies develop, and whether they are associated with unique clinical features. To help address these questions, a spectrum of autoantibodies known to be associated with RA in a cohort of patients with early synovitis was evaluated. An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinicially then followed prospectively for 1 year. Patients were classified as having RA on the basis of fulfilling the 1987 criteria. Serum samples collected at the time of the initial evaluation were tested for anti-Sa and anti-RA-33 using immunoblotting, and to (pro)filaggrin (AFA), anti-CCP, and calpastatin (anti-RA-1) using enzyme-linked immunosorbent assay techniques. AKA were detected using immunoflurescence on human epidermal tissue. RF was tested by nephelometry. HLA-DRB1 alleles were determined using sequence specific primers. Initial and 1 year radiographs were evaluated for the presence of erosions. < 0.001). Despite this high level of correlation, of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one, suggesting considerable variability in individual reactivity patterns. , the presence of each of these autoantibodies was significantly associated with having two shared epitope alleles, even when only the RA patients were considered. =0.03). Neither RF nor SE were associated with the disease severity measures, and analyses evaluating all the other autoantibodies failed to reveal a similar trend. Despite a well-documented lack of specificity, RF continues to be a central part of the definition of RA, primarily because of its favourable sensitivity profile. In our cohort, RF had a sensitivity of 66%, a specificity of 87%, and an overall accuracy of 78% for the diagnosis of RA. AFA, anti-Sa, anti-CCP were all highly specific for this diagnosis, and when any of them were present in conjunction with RF, the specificity for RA approached 100%. Potentially of more importance to the clinician is the diagnostic value of these antibodies when RF is not detectable. Our data indicate that only 31% of RF- RA patients had any of AKA, AFA, anti-Sa or anti-CCP, and that anti-Sa was the most specific for this diagnosis. This modest level of sensitivity suggests that testing for this spectrum of autoantibodies carries little advantage over RF alone in diagnosing early RA. AFA, AKA, and antiperinuclear factor (APF) have all been proposed to identify a common antigen present in the skin protein (pro)filaggrin. It has continued to be puzzling why a skin antigen would be targeted relatively specifically in a disorder that is primarily articular. A potential explanation for this may relate to the demonstration that citrulline appears to be an essential constituent of the antigenic determinants recognized by AKA, APF, and AFA. The citrulline rich (pro)filaggrin molecule makes an ideal substrate for detecting this reactivity. Moreover, the SA antigen, which, unlike (pro)filaggrin, is detectable in rheumatoid synovium, has recently been shown to also be citrullinated. It is thus possible that AKA, AFA, APE, and anti-Sa all recognize one or more citrullinated antigens. Despite this possibility, the modest degree of concordance between them in individual patient sera suggests that it is unlikely that a single antigen is involved in generating these responses. This study provides evidence suggesting that anti-Sa antibodies appear to be a marker for a subset of early RA patients whose disease may be more severe and erosive. Moreover, it was determined that anti-Sa, AFA, and anti-CCP were all highly associated with SE, particularly two copies. We examined a spectrum of potential RA severity indicators including the number of swollen joints, CRP level, and presence of early radiographic erosions. Our data indicate that anti-Sa was more highly associated with these measures of RA severity than any other parameter, including the most accepted prognostic indicators, RF and SE. In conclusion, it is demonstrated that antibodies directed against putatively citrullinated antigens including SA, filaggrin, keratin, and CCP are the most specific for RA, and are detectable early in the disease course. It will be of interest to find out whether the cumulative prevalence of specific autoantibody subsets tends to increase over time, as this would suggest that the mechanisms underlying the development of these reactivities continue to evolve over the course of the arthropathy.

Introduction:

A spectrum of autoantibodies is now known to be specifically associated with RA. There continues to be uncertainty as to what stage of the disease each of these autoantibodies develop, and whether they are associated with unique clinical features.

Aims:

To help address these questions, a spectrum of autoantibodies known to be associated with RA in a cohort of patients with early synovitis was evaluated.

Methods:

An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinicially then followed prospectively for 1 year. Patients were classified as having RA on the basis of fulfilling the 1987 criteria. Serum samples collected at the time of the initial evaluation were tested for anti-Sa and anti-RA-33 using immunoblotting, and to (pro)filaggrin (AFA), anti-CCP, and calpastatin (anti-RA-1) using enzyme-linked immunosorbent assay techniques. AKA were detected using immunoflurescence on human epidermal tissue. RF was tested by nephelometry. HLA-DRB1 alleles were determined using sequence specific primers. Initial and 1 year radiographs were evaluated for the presence of erosions.

Results:

< 0.001). Despite this high level of correlation, of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one, suggesting considerable variability in individual reactivity patterns.

, the presence of each of these autoantibodies was significantly associated with having two shared epitope alleles, even when only the RA patients were considered.

=0.03). Neither RF nor SE were associated with the disease severity measures, and analyses evaluating all the other autoantibodies failed to reveal a similar trend.

Discussion:

Despite a well-documented lack of specificity, RF continues to be a central part of the definition of RA, primarily because of its favourable sensitivity profile. In our cohort, RF had a sensitivity of 66%, a specificity of 87%, and an overall accuracy of 78% for the diagnosis of RA. AFA, anti-Sa, anti-CCP were all highly specific for this diagnosis, and when any of them were present in conjunction with RF, the specificity for RA approached 100%. Potentially of more importance to the clinician is the diagnostic value of these antibodies when RF is not detectable. Our data indicate that only 31% of RF- RA patients had any of AKA, AFA, anti-Sa or anti-CCP, and that anti-Sa was the most specific for this diagnosis. This modest level of sensitivity suggests that testing for this spectrum of autoantibodies carries little advantage over RF alone in diagnosing early RA.

AFA, AKA, and antiperinuclear factor (APF) have all been proposed to identify a common antigen present in the skin protein (pro)filaggrin. It has continued to be puzzling why a skin antigen would be targeted relatively specifically in a disorder that is primarily articular. A potential explanation for this may relate to the demonstration that citrulline appears to be an essential constituent of the antigenic determinants recognized by AKA, APF, and AFA. The citrulline rich (pro)filaggrin molecule makes an ideal substrate for detecting this reactivity. Moreover, the SA antigen, which, unlike (pro)filaggrin, is detectable in rheumatoid synovium, has recently been shown to also be citrullinated. It is thus possible that AKA, AFA, APE, and anti-Sa all recognize one or more citrullinated antigens. Despite this possibility, the modest degree of concordance between them in individual patient sera suggests that it is unlikely that a single antigen is involved in generating these responses.

This study provides evidence suggesting that anti-Sa antibodies appear to be a marker for a subset of early RA patients whose disease may be more severe and erosive. Moreover, it was determined that anti-Sa, AFA, and anti-CCP were all highly associated with SE, particularly two copies. We examined a spectrum of potential RA severity indicators including the number of swollen joints, CRP level, and presence of early radiographic erosions. Our data indicate that anti-Sa was more highly associated with these measures of RA severity than any other parameter, including the most accepted prognostic indicators, RF and SE.

In conclusion, it is demonstrated that antibodies directed against putatively citrullinated antigens including SA, filaggrin, keratin, and CCP are the most specific for RA, and are detectable early in the disease course. It will be of interest to find out whether the cumulative prevalence of specific autoantibody subsets tends to increase over time, as this would suggest that the mechanisms underlying the development of these reactivities continue to evolve over the course of the arthropathy.

Introduction

] have also been shown to be prevalent in RA patient populations, but not specific for this disease.

].

].

]. In the current study, we sought to determine the prevalence and diagnostic value of the RA associated autoantibodies in this heterogeneous cohort of patients with early synovitis. Sera obtained within 12 months of symptom onset were tested for these antibodies, and their presence was related to disease features and subsequent clinical course over a 1 year period. Our data indicate that AFA, anti-Sa, and anti-CCP are all highly specific for early RA, but demonstrate only a modest degree of concordance in the sera of individual patients. Anti-Sa identifies a subset of male RA patients with severe disease.

Patients

Two hundred and thirty-eight patients were recruited to an early synovitis study at the National Institutes of Health (protocol 94-AR-194). The study patients had persistent synovitis (>6 weeks) of at least one peripheral joint, which had been present for less than 1 year. Patients with traumatic, septic, and crystal induced arthritis were specifically excluded.

] on at least one visit. If a patient had met ACR criteria, but with follow up was unequivocally diagnosed as having another well-characterized rheumatic disease, this individual was not included in the definition of RA.

Detection of autoantibodies

]. Control peptides had an unmodified arginine rather than citrulline. The cutoff level for positivity (OD, 0.3) was determined on the basis of generating 100% specificity for RA in previous assays using local normal controls. AKA were detected using immunoflurescence. Sections of human breast epidermis were used, and the slides were read independently by two observers (AR and CZ). Any slides in which the observers did not agree were reread, and a consensus agreed. In all cases, the detected autoantibodies were of the IgG class. The results were not corrected for total IgG levels.

HLA typing

Statistical analysis

Patient groups were compared using analysis of variance of the Kruskal-Wallis test for continuous variables, and using the Chi-squared test for proportions. The significance level for associations between individual autoantibodies and HLA-DRB1 alleles was adjusted for multiple comparisons using the Bonferroni method. Statistical analysis was performed using Epi Info statistical software (Center for Disease Control, Atlanta, GA, USA: http://www.cdc.gov/epo/epi/epiinfo.htm).

Prevalence of the autoantibodies in the patient cohort

<0.01), but there were no differences in the prevalence of the other autoantibodies between these two patient groups. In total, 90/106 (85%) of the RA patients and 75/132 (57%) of the nonRA patients had at least one of the RA associated antibodies.

Diagnostic value of antibodies for RA

. RF had the highest sensitivity at 66%, and all the other antibodies were individually less than 50% sensitive. AFA, anti-Sa and anti-CCP were greater than 90% specific for RA, while RF and AKA were 80-90% specific, and anti-RA-1 was not specific for this diagnosis. The data further indicate that adding any one of AFA, AKA, anti-Sa, or anti-CCP to RF increases the specificity for RA from 80 to 90%. In the absence of RF, the presence of one or more of these antibodies carried a sensitivity of only 31% for RF- RA, with anti-Sa being the most specific at 98%. At the completion of the study, of the 37/132 (28%) nonRA patients positive for one of AFA, AKA, anti-Sa, or anti-CCP, 12 had some form of spondylarthropathy, 4 had a connective tissue disease, and 21 continued with a diagnosis of 'undifferentiated arthritis'.

Correlation between antibodies in patient sera

=0.09). In total, 101 patients had at least one of the four antibodies, 64% of whom had RA. Of these 101 patients, 57 were in fact positive for only one antibody (AKA = 20, AFA = 12, anti-Sa = 6, anti-CCP =19). Only seven patients were positive for all four antibodies.

= not significant).

RF, Sa, AFA, and CCP are highly associated with shared epitope in early RA

0401 were positive for at least one of these autoantibodies.

Anti-Sa antibodies identify a male predominant subset of RA patients with severe disease

further indicates that neither RF nor SE was associated with the disease severity measures, and analyses evaluating all the other autoantibodies failed to reveal a similar trend (data not shown). Of interest, only six RA patients demonstrated the presence of nodules, and this feature was more associated with RF and SE than with anti-Sa. Analysis of the nonRA patients did not reveal any clinically meaningful associations with any of the autoantibodies.

Discussion

]. Although the duration of follow up in this study was shorter, we found that almost all of the patients who had met the RA criteria at the completion of the study period had done so on their initial visit.

Despite a well-documented lack of specificity, RF continues to be a central part of the definition of RA, primarily because of its favorable sensitivity profile. In our cohort, RF had a sensitivity of 66%, a specificity of 87%, and an overall accuracy of 78% for the diagnosis of RA. AFA, anti-Sa and anti-CCP were all highly specific for this diagnosis, and when any of them were present in conjunction with RF, the specificity for RA approached 100%. Potentially of more importance to the clinician is the diagnostic value of these antibodies when RF is not detectable. Our data indicate that only 31% of RF- RA patients had any of AKA, AFA, anti-Sa or anti-CCP, and that anti-Sa was the most specific for this diagnosis. This modest level of sensitivity suggests that testing for this spectrum of autoantibodies carries little advantages over RF alone in diagnosing early RA.

]. The finding that anti-Sa antibodies are associated both with male gender and with severe early RA further emphasizes the importance of gender differences in the clinical expression of this heterogeneous disease.

]. Together, these data are most consistent with the hypothesis that individual RA patients respond to unique antigenic determinants, that may preferentially be citrullinated, but that are likely distinct from those of targeted by other RA patients.

]. It should be pointed out that the testing for anti-RA-33, including that performed in the current study, was all performed in the same laboratory (GS). The reasons for this discrepancy are unclear, and may represent inherent differences in the populations studied. Alternatively, it is possible that the development of anti-RA-33 reactivity increases as the diseases progresses. Longitudinal follow up of early RA cohorts, such as the present one, will help to further clarify this issue.

In the current study, it was demonstrated that antibodies directed against putatively citrullinated antigens including Sa, filaggrin, keratin, and CCP are the most specific for RA, and are detectable early in the disease course. It will be of interest to find out whether the cumulative prevalence of specific autoantibody subsets tends to increase over time, as this would suggest that the mechanisms underlying the development of these reactivities continue to evolve over the course of the arthropathy.

Acknowledgements

We would like to acknowledge the invaluable contributions of Marianna Crane and Dr Thurayya Arayssi, Dr Jose Pando, Dr Percio Gulko, Dr Richard Siegel, Dr Michael Froncek and Dr Robert Ortmann, NIH fellows who performed the clinical evaluations of the patients. We would like to thank Dr Dimitios Boumpas for his thoughtful review of the manuscript, and also acknowledge the help of David Smith II, Curtiland Deville in the analysis of the data. The authors also wish to acknowledge Dr Peter Lipsky for his thoughtful review of this manuscript.

Figures and Tables

Presenting clinical features and prevalence of autoantibodies in rheumatoid factor positive rheumatoid arthritis (RF + RA), RF-negative RA (RF-RA), and nonRA patients

<0.01 compared with nonRA. CRP, C-reactive protein; ANA, antinuclear antibodies; AFA, antifilaggrin antibody; RNP, ribonucleoprotein; CCP, cyclic citrullinated peptide; AKA, antikeratin antibody.

Association of autoantibodies with shared epitope (SE) alleles

<0.05 by Chi-square after Bonferroni adjustment for multiple comparison, RF, Rheumatoid factor; AFA, antifilaggrin antibody; CCP, cyclic citrullinated peptide; AKA, antikeratin antibody.

Diagnostic value of individual autoantibodies for rheumatoid arthritis

Includes antifilaggrin antibody (AFA), anti-Sa, cyclic citrullinated peptide (anti-CCP) and antikeratin antibody (AKA). PPV, positive predictive value; NPV, negative predictive value; RF, rheumatoid factor.

Sa is associated with severe rheumatoid arthritis (RA)

One hundred and two out of 106 (96%) of the patients had 1 year radiographs available for analysis. Multiple erosions included all patients with two or more erosions that involved at least two separate joint areas. RF, Rheumatoid factor; SE, shared epitope; CRP, C-reactive protein; DMARD, disease modifying antirheumatic drug.

Keywords

• autoantibodies
• early synovitis
• human leukocyte antigen
• rheumatoid arthritis
• spondylarthropathy