Prevalence and clinical significance of anti-cyclic citrullinated peptide and antikeratin antibodies in palindromic rheumatism. An abortive form of rheumatoid arthritis?

G. Salvador, A. Gomez, O. Viñas1, G. Ercilla1, J. D. Cañete, J. Muñoz-Gomez and R. Sanmarti

Hospital Clínic, Rheumatology and
1 Hospital Clínic, Immunology, Barcelona, Barcelona, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Objective. To analyse the prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies (anti-CCP) and antikeratin antibodies (AKA) in patients with palindromic rheumatism (PR).

Method. Sixty-three patients with PR were included: 33 were defined as pure or persistent PR at the time of serum test measurement, and 30 as associated PR, defined as patients with past history of PR who had developed persistent arthritis at the time of serum test: [21 with rheumatoid arthritis (RA)]. Sixty patients with early RA, and 80 with seronegative spondyloarthropathy were included as control groups. Anti-CCP were determined by a standardized ELISA test and AKA by indirect immunofluorescence in rat oesophagus. Clinical characteristics of these pure PR patients were compared according to the presence or absence of anti-CCP antibodies. A follow-up study was also performed.

Results. Anti-CCP were detected in 18 out of 32 (56.3%) patients with pure PR and 10 out of 30 (33.3%) with associated PR (38.1% in RA-associated PR patients). AKA were detected in 12 patients out of 33, with pure PR (36.4%), and in 9 out of 30 with associated PR (30%) (33.3% in RA-associated PR patients). The prevalence of anti-CCP and AKA in the RA control group was 55% (not significantly different from the pure PR group) and 61.7% (with respect to pure PR patients, P=0.02), respectively. In the spondyloarthropathy group, the prevalence of anti-CCP and AKA was 2.5 and 3.8%, respectively (P<0.001 compared with pure PR patients). No significant clinical differences were observed between pure PR patients with and without CCP antibodies.

Conclusions. Anti-CCP and, to a lesser extent, AKA, were found in a high proportion of patients with PR, suggesting that this syndrome is an abortive form of RA. The predictive value of these antibodies in PR, as markers of progression to an established RA, remains uncertain.

KEY WORDS: Palindromic rheumatism, Anti-CCP antibodies, Antikeratin antibodies, AKA, Rheumatoid arthritis.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Palindromic rheumatism (PR) is a clinical syndrome characterized by short-lasting attacks of acute arthritis, usually involving single joints, with no remnant clinical or radiographic changes. Some of these patients, and especially those with a positive rheumatoid factor (RF), develop a connective tissue disease, mainly rheumatoid arthritis (RA). Several studies have attempted to identify factors that might predict progression to RA, such as the presence of RF, the involvement of hands and genetic markers, but the results are unclear [1]. At present, it is not clear whether PR is a separate entity or is part of the spectrum of RA [2].

High specificities of autoantibodies such as antikeratin (AKA), antiperinuclear factor (APF) and antifilaggrin (AFA) have been described in the sera of patients with RA [3]. All these autoantibodies seem to recognize a similar epitope composed of citrullinated peptides [4]. Citrulline is a post-translational modification of the amino acid arginine by the enzyme peptidil arginine deiminase (PAD) [5]. Recently, an enzyme-linked immunoassay (ELISA) using a cyclic citrullinated peptide (CCP) as the antigenic capture first layer has been developed and the anti-CCP antibodies have proven to be highly specific (98%) for RA [6].

We are unaware of previous studies of the AKA/anti-CCP antibody complex in PR. In a preliminary report by our group, significant proportions (25%) of AKA were observed in a small group of patients with pure PR [7]. In this paper, we report the prevalence and clinical significance of serum AKA and anti-CCP in a large series of patients with PR.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Patients
Sixty-three consecutive patients (46 F/17 M) attending the Rheumatology Unit of the Hospital Clinic of Barcelona were included. In all patients, typical short-lasting acute attacks of short duration were observed by a rheumatologist. Other causes of acute monoarthritis were excluded. All patients diagnosed with PR fulfilled the criteria defined by Guerne et al. (1992) [2]. At the time of clinical evaluation and serum measurements, patients were classified into two groups: (1) pure or persistent PR (n=33), defined as patients with no evidence of associated rheumatic diseases and (2) associated PR (n=30), defined as patients fulfilling criteria for other inflammatory rheumatic diseases [RA (n=21), systemic lupus erythematosus (n=4), and others (n=5) as follows: psoriatic arthritis (n=1), Sjögren's syndrome (n=1), chronic autoimmune hepatitis (n=1), undifferentiated oligoarthritis (n=1) and polymyositis (n=1)], at the time of serum measurement.

Sixty patients with early RA (<2 yr of disease duration) according to 1987 American Rheumatism Association criteria (now ACR), and 80 patients with spondyloarthropathy (SpA) according to ESSG criteria [8] were included as control groups.

Methods
Demographic characteristics and the number, frequency and site of acute attacks were recorded in all patients with PR. The duration of PR until serum measurement was also recorded. In all patients and controls, we determined the presence of rheumatoid factor by nephelometry (normal value: NV <30 UI/l), AKA and anti-CCP. After the initial measurement of AKA and anti-CCP, pure PR patients were followed-up in order to evaluate the development of a secondary connective tissue disease and its relationship with the presence of AKA/anti-CCP at baseline.

AKA measurement. AKA were detected by indirect immunofluorescence, using a modified Young's method [9] with middle-third rat oesophagus specimens as a substrate. Middle-third rat oesophagus cryostat sections were incubated with 1/10 and 1/40 sera dilutions in phosphate-buffered saline (PBS) for 30 min, washed three times in PBS and revealed with fluorescein isothiocyanate (FITC)-conjugated goat antibodies to human IgG, Fc-gamma specific [goat F(ab)'2 anti-human IgG (Fc{gamma} specific) CALTAG Laboratories, Innogenetics diagnostica, SAU, L'Hospitalet (Barcelona) Spain]. Further evaluation using a fluorescence microscope was made by two independent readers, unaware of the clinical context. Sera were considered AKA positive only if titres >=1/10 were found and when the characteristic linear laminated pattern was observed on the stratum corneum of rat oesophagus epithelium. High positive, low positive and negative controls were included for each determination. The sensitivity and specificity of this AKA test for rheumatoid arthritis were 60.2 and 97.5%, respectively [10].

Anti-CCP measurement. Anti-CCP antibodies were tested by ELISA (Immunoscan RA; EuroDiagnostica, distributed by Diasorin, Madrid, Spain). A cut-off value of 50 units was established, as recommended by the manufacturer's protocol. This guarantees a high specificity (97.8%), according to the results of a previous study [6]. Using this cut-off value, the ELISA test had a sensitivity and specificity for RA of 52.9 and 96.6%, respectively, in a previous analysis in our population.

Statistical analysis
Data were analysed using the SPSS 10.0 statistical software package, under licence to the Hospital Clinic of Barcelona. The non-parametric Mann–Whitney U-test was used to compare differences between groups and the {chi}2-test for proportions. The level of statistical significance was established at P <= 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
The demographic and clinical characteristics of patients with PR and controls are shown in Table 1Go. Anti-CCP were found in 18 out of 32 (56.3%) patients with pure PR and in 10 out of 30 patients (33.3%) with associated PR (38.1% in RA-associated PR patients). AKA were found in 12 out of 33 patients (36.4%) with pure PR and in 9 out of 30 (30%) with associated PR (33.3% in RA-associated PR patients). In the RA control group, the proportion of patients with anti-CCP was 55% (not significant compared with pure PR) and with AKA 61.7% (P=0.02 with respect to the pure PR group). In the SpA group, the prevalence was only 2.5 and 3.8%, respectively, (P=0.001 compared with the pure PR group). In the whole palindromic rheumatism group, a positive association between AKA and anti-CCP with RF seropositivity was observed. RF+ was more frequently seen in patients with AKA and anti- CCP antibodies than in those without (71.4 vs 40.5%, P=0.02; and 67.8 vs 35.2 %, P=0.01, respectively). Also the proportion of PR patients with AKA was more prevalent in patients with anti-CCP (+) than those with anti-CCP (-) (67.8 vs 5.9%, P < 0.001). In patients with pure PR and negative rheumatoid factor, the prevalence of anti-CCP and AKA was 42.1 and 31.5%, respectively. All patients with pure PR and positive AKA were anti-CCP positive, but in six anti-CCP-positive patients, AKA were not found in sera. No significant differences in titres of anti-CCP were found between anti-CCP-positive patients with pure PR (mean±S.D.: 671±1095 UI/ml), associated PR (454±907 IU/ml) and early RA (776±1174 IU/ml).


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TABLE 1. Demographic and serological characteristics of patients with PR, RA and SpA

 
Clinical and serological differences in pure PR patients with and without anti-CCP are shown in Table 2Go. There were no significant differences in the number of affected joints and the frequency of attacks between the two groups. Hand involvement was commonly seen in both anti-CCP-positive and -negative patients (88 and 85.7%, respectively). As expected, RF was significantly more frequent (P=0.05) in anti-CCP-positive patients. Disease duration until serum measurement was higher in anti-CCP-negative than in anti-CCP-positive patients. After a mean follow-up time of 35.1 months (range 2–102), only four patients with an initial diagnosis of pure PR developed other connective tissue diseases: three RA and one SLE. The three patients who evolved to RA were RF positive, and two were both AKA and anti-CCP positive. The patient who developed SLE was RF negative, and AKA and anti-CCP negative.


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TABLE 2. Clinical and serological characteristics in pure PR patients with and without anti-CCP

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
This study found a high frequency of anti-CCP in the sera of patients with palindromic rheumatism. Until now, these antibodies were considered to be highly specific for RA. In this study, the proportion of pure PR and early RA patients with anti-CCP was very similar, with no significant differences (56.3 and 55%, respectively). This proportion is quite similar to the sensitivity (41–60%) of these antibodies in the majority of studies of RA populations [3, 11]. No significant differences were found in titres of anti-CCP between the two populations. As expected, the frequency of anti-CCP in patients with spondyloarthropathy was very low (2.5%).

The prevalence of AKA in pure PR was also high (36.4%), although significantly lower than in patients with early RA (61.7%), and with a lower sensitivity than anti-CCP in the pure PR population. As expected, a close, but not absolute, concordance was observed between both autoantibodies, suggesting that they might represent different spectrums of autoimmune response in RA, identifying related, but probably not identical, antigenic substrates [5].

No clinical differences between pure PR patients with and without anti-CCP were observed. As expected, an overexpression of serum AKA and RF was observed in anti-CCP-positive patients, as previously described in patients with established RA [11]. Anti-CCP were also found in 42.1% of PR patients without serum RF, a proportion similar to that found in the seronegative RA control group (38.5%), and even higher than that reported in other series with seronegative RA, which ranged from 14 to 35% [3, 11].

Progression to other connective tissue diseases, mainly RA, has been found in up to 62% of patients with PR at long-term follow-up in different series [2, 12, 13]. In a recent series of patients with PR, a positive RF and the early involvement of wrist and interphalangeal proximal joints (hand involvement) identified PR patients at increased risk of developing RA [1]. Whether anti-CCP and/or AKA might predict the development of RA in patients with PR better than RF is unknown. It is well established that the presence of RF in healthy subjects is a risk factor for the development of RA, in particular in those subjects with high RF titres [14]. Studies in pre-illness sera of patients with RA have also demonstrated that AKA, antiperinuclear factor or both have been observed in up to one-third of these patients [15]. Similar findings are reported in a recent study with anti-CCP antibodies; in this preliminary report, anti-CCP antibodies predict the development of RA much better than RF, although arthritis symptoms may occur up to 9 yr after anti-CCP determination [16]. In our study, only four out of 33 patients with pure PR at the time of AKA and anti-CCP measurement developed a connective tissue disease during the follow-up (three RA and one SLE). Two had anti-CCP and AKA at baseline; however, one patient with negative anti-CCP and AKA developed RA. The three patients who developed RA were RF+, and early hand involvement was also observed in all three. These results, together with the small number of patients and the limited follow-up period (mean: 2.9 yr), mean it is not possible to ascertain whether the presence of anti-CCP or AKA may be predictive of RA in our group of patients.

In conclusion, similar proportions of anti-CCP antibodies are observed in PR and RA, even in negative RF patients. AKA are also observed in a considerable proportion of these patients, but the proportion is significantly lower than in RA patients. Given that these antibodies are considered the most specific markers of RA and usually appear at early stages of the disease, it may be surmised that PR and RA represent a different spectrum of the same syndrome, and that PR should be considered as an abortive form or incomplete expression of RA.


    Conflict of interest
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
The authors have declared no conflicts of interest.


    Acknowledgments
 
We are grateful to L. Rubio, M. Romera, A. Hernández, M. C. Anton and Y. Machuca for their technical assistance.


    Notes
 
Correspondence to: R. Sanmarti, Hospital Clínic, Rheumatology, Barcelona, Barcelona, Spain. E-mail: sanmarti{at}clinic.ub.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 

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Submitted 7 August 2002; Accepted 8 January 2003