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Rheumatology 2001; 40: 1126-1134
© 2001 British Society for Rheumatology

Original Papers

Autoantibodies to the 27 C-terminal amino acids of calpastatin are detected in a restricted set of connective tissue diseases and may be useful for diagnosis of rheumatoid arthritis in community cases of very early arthritis

O. Vittecoq1,2,, V. Salle1, F. Jouen-Beades1, K. Krzanowska2, J. F. Ménard3, A. Gayet4, P. Fardellone5, P. Tauveron6, X. Le Loët1,2 and F. Tron1

1 Unité INSERM 519 et Institut Fédératif de Recherche Multidisciplinaire sur les Peptides (IFRMP 23), Faculté de Médecine et de Pharmacie, Rouen,
2 Service de Rhumatologie, Centre Hospitalier Universitaire de Rouen,
3 Unité de Biométrie-Biostatistique, Centre Hospitalier Universitaire de Rouen,
4 Collège des Rhumatologues de Haute-Normandie, Rouen,
5 Service de Rhumatologie, Centre Hospitalier Universitaire d'Amiens and
6 Service de Rhumatologie, Centre Hospitalier Universitaire de Tours, France


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Calpastatin is the natural inhibitor of calpains, a protease that is overexpressed in rheumatoid synovial tissue and plays a key role in cartilage destruction. Autoantibodies to calpastatin (ACAST) were recently detected in rheumatoid arthritis (RA). Our aim was to determine their prevalence and their clinical significance.

Methods. ACAST were detected in a solid-phase enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the 27 C-terminal amino acids of calpastatin (CAST-C27) as the antigen. All sera reacting with this peptide also bound to purified erythrocyte calpastatin in an ELISA and/or an immunoblot assay. The frequencies and clinical significance of ACAST-C27 were assessed in sera from a well-documented population of 102 community-recruited patients (76 females; mean age 50 yr) with RA that had been evolving for <5 yr (median 2 yr) (group 1), 109 healthy blood donors, 289 patients with non-RA rheumatic disease and 88 community cases of very early (median 4 months) arthritis, i.e. 58 RA and 30 non-RA patients (group 2).

Results. The sensitivity of ACAST-C27 for RA was 19.5% (20/102) in group 1 and 10.3% (6/58) in group 2. These antibodies were also found in patients with anti-double-stranded DNA-positive systemic lupus erythematosus (SLE) (15.5%) and patients with anti-Ro-positive Sjögren's syndrome (18.5%). However, they were not detected in cases of rheumatism resembling early RA, i.e. peripheral spondylarthropathies. ACAST-C27 were not detected in the 30 non-RA patients of group 2. They were predominantly of immunoglobulin isotype G3 and exclusively expressed {lambda} chains. Among ACAST-C27-positive sera, eight out of 20 (group 1) and four out of six (group 2) were negative for rheumatoid factor and anti-keratin antibodies/antiperinuclear factor. No relationship was found between ACAST-C27 and clinical, biological or radiological findings.

Conclusion. ACAST-C27 are detected only in a restricted set of connective tissue diseases and therefore appear to be specific for RA when antibodies that are usually associated with SLE or primary Sjögren's syndrome are negative. Because of their presence in community cases of very early RA, particularly in some seronegative forms, ACAST-C27 may be useful in discriminating recent-onset RA from the more common non-RA rheumatic diseases, such as spondylarthropathies.

KEY WORDS: Early rheumatoid arthritis, Diagnosis, Anti-calpastatin antibodies.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is the most frequent and most destructive inflammatory rheumatic disease. It is characterized histologically by angiogenesis and hyperplasia of the synovial membrane, which is infiltrated by lymphoid cells and macrophage-like and fibroblast-like synoviocytes [1]. The latter cells are activated by proinflammatory cytokines secreted by macrophages and produce large quantities of proteinases, which are thought to play an important role in joint destruction. In RA, proteolytic enzymes belong to two major families: the matrix metalloproteinases, especially the stromelysins, and the cysteine proteinases, particularly the calpains, which are Ca+-dependent enzymes [2, 3]. In collagen-induced arthritis of mice, it has been demonstrated that calpain induction is correlated with the appearance of arthritis and subsequent cartilage destruction [4]. In RA there is overactivation of calpains, the levels of which are increased in the synovial fluid [5]. The natural inhibitor of calpains is calpastatin. There are two types of calpastatin, viz. the muscle type (110 kDa) and the erythrocyte type (70 kDa). Muscle-type calpastatin contains four repetitive sequences, referred to as domains I–IV, and one non-homologous sequence on the amino-terminal part of the molecule, i.e. domain L, whereas the erythrocyte type lacks domains L and I [6].

Two groups have shown that calpastatin is the target of autoantibodies present in sera of RA patients [7, 8]. Indeed, calpastatin was identified as an autoantigen by the screening of a human placenta or HeLa cell {lambda}gt11 cDNA expression library using selected sera from patients with erosive RA, sequence analysis of positive clones, and subsequent Western blotting against recombinant antigen. This last procedure detects anti-calpastatin antibodies (ACAST) in almost 50% of RA patients, but also in other autoimmune disorders, such as systemic lupus erythematosus (SLE), progressive systemic sclerosis, polymyositis and autoimmune infertility and venous thrombosis, and in patients infected by HTLV-1, a retrovirus that causes RA-like arthropathy [7–11]. However, identification of the immunodominant calpastatin epitopes recognized by RA sera remains controversial, as there are discrepancies between the results obtained by three groups [7, 8, 12, 13] who have analysed the reactivity of RA sera with human calpastatin. All of these groups identified these autoantibodies with an immunoblotting assay that used recombinant calpastatin fusion proteins as the antigen. Lackner et al. [13] studied the reactivity of ACAST with the N-terminal part of the molecule and found that most positive RA sera recognized a fusion protein containing domain L and part of domain I but with a frequency similar to that observed in control sera. All three groups analysed the reactivity of RA sera with the C-terminal part of calpastatin. For two groups [7, 8, 12], ACAST were directed against domains III and IV of the molecule. The most recent study of calpastatin epitope mapping indicated that the 27 C-terminal amino acids of domain IV (CAST-C27) were a major epitope recognized by B-cells. Indeed, Lackner et al. [13] showed that it was the sole epitope of the C-terminal part of the protein recognized by RA sera. This autoantibody population was found to be slightly more common (8.9%) in RA than in healthy control sera (3.4%). Thus, CAST-C27 appears to be a well-defined epitope and could have clinical relevance in RA. This observation led us to develop an enzyme-linked immunosorbent assay (ELISA) using a synthetic CAST-C27 peptide as the substrate, with a procedure comparable to that of Schlosser et al. [14] but with some modifications.

The frequency of ACAST-C27 and their clinical relevance for RA remain imprecise. Therefore, the objectives of the present study were (i) to determine the frequencies of these autoantibodies in different autoimmune and inflammatory rheumatisms, (ii) to determine their frequency in community cases of very early RA, and (iii) to attempt to correlate in established RA the presence of these autoantibodies with clinical, biological and radiological features.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Populations studied to determine the sensitivity and specificity of ACAST-C27 autoantibodies in RA
From 1989 to 1998, sera from patients with classified rheumatic diseases referred to the departments of rheumatology and immunology were collected and stored at -80°C. A total of 500 disease-associated and control sera were tested. This group of patients was divided into three subgroups: (i) 102 patients with RA fulfilling the 1987 American College of Rheumatology (ACR) criteria [15] (group 1a); (ii) 289 patients with non-RA rheumatic diseases (Table 1Go) defined according to international criteria [16–21] (group 1b); and (iii) 109 blood donors as the healthy control group (group 1c).


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  		TABLE 1. Frequencies of antibodies directed against ACAST-C27 in patients with various rheumatic diseases and in healthy controls

 
From 1998 to 2000, a prospective longitudinal study was conducted in out-patients with very early peripheral inflammatory arthritis (group 2).

Recruitment and characteristics of patients with RA (group 1a)
One hundred and two patients who had RA that had been evolving for <5 yr and who fulfilled at least four of the seven ACR criteria [15] were enrolled prospectively in 1996 in the Normandy region of France. This recruitment was made possible by the major contribution of rheumatologists in private practice (80% of the cases were community cases). The evaluation was conducted at the patient's home by two investigators from the Department of Rheumatology of the Centre Hospitalier Universitaire de Rouen. During the evaluation, the following data were recorded: clinical characteristics, i.e. demographic data, the duration of RA (defined as the time between onset of the first symptoms and the time of the evaluation), the presence or absence of extra-articular manifestations, the Ritchie articular index, the validated French version of the Health Assessment Questionnaire (HAQ) [22]; biological analyses, i.e. C-reactive protein (CRP; normally 10 mg/l) measured nephelometrically, the latex fixation test, ACAST-C27, anti-keratin antibodies (AKA), anti-perinuclear factor (APF), and HLA-DR typing by a standard polymerase chain reaction procedure; radiological assessment, including radiographs of the wrists and hands (read separately by two trained rheumatologists) and the modified Sharp score [23] (to evaluate joint destruction); and drugs prescribed to patients at the time of evaluation and during the preceding year. The characteristics of this RA population have been described elsewhere [24] and are reported in Table 2Go. Briefly, the cohort was composed mostly of women (female:male ratio=2.9). RA was mainly of recent onset (mean disease duration 2.2 yr); it had been evolving for <3 yr in 71% of the patients and <1 yr in 19%. The disease was mildly active, mildly invalidating and mildly destructive. One-quarter of the patients had at least one rheumatoid nodule and some of them had other extra-articular manifestations, primarily serous inflammation (one case of pleurisy and one of pericarditis). Rheumatoid vasculitis was not observed. Half the patients were positive for rheumatoid factor (RF) in the latex fixation test. Almost half the patients were positive for AKA and/or APF, and among them 70% were positive for RF detected by the agglutination test. Almost all patients (96%) were being treated at the time of the evaluation. Oral prednisone was prescribed at a mean dose of 6.5 mg/day. One or several disease-modifying anti-rheumatic drugs (DMARDs) were prescribed to 95% of the patients; more than half of them were taking methotrexate at a mean dose of 10 mg/week (range 5–20 mg/week).


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  		TABLE 2. Demographic, clinical and biological characteristics of the RA population (group 1a)

 

Recruitment of community cases of very early arthritis (group 2)
Eighty-eight patients were recruited by rheumatologists in private practice in three French regions: the entire province of Upper Normandy and the metropolitan areas of Amiens and Tours. Patients were required to have swelling of at least two joints that had persisted for >=4 weeks, disease that had evolved for <6 months (median disease duration 4 months), and no local or systemic corticoid therapy or DMARD treatment before inclusion. Exclusion criteria were inflammatory back pain, pregnancy and suckling. The follow-up period was 1 yr after the first symptoms. At the end of the study, patients were classified as having RA (n=58) according to ACR criteria [15] or as having other rheumatic diseases [n=30, comprising 10 patients with spondylarthropathies (including four patients with psoriatic arthritides), four patients with osteoarthritides, two patients with crystal-induced arthritides, one patient with primary Sjögren's syndrome, one patient with viral arthritis due to parvovirus B19 and 12 patients with undifferentiated arthritides]. At entry to the study, we performed the latex fixation test and tested the patients for AKA, APF and ACAST-C27. The characteristics of this cohort are summarized in Table 3Go. The Ethics Committee (Centre Hospitalier Universitaire de Rouen) gave its approval for the studies on this group and group 1a. Before entry into the study, each patient was informed of the nature, duration and purpose of the study and gave his or her written consent.


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  		TABLE 3. Characteristics of the patients with very early arthritis (group 2)

 

ELISA for detection of ACAST-C27
An ELISA in which the antigen was a synthetic peptide corresponding to the 27 C-terminal amino acids of calpastatin (SSKAPKNGGKAKDSAKTTEETSKPKDD) was set up according to the procedure described by Schlosser et al. [14] with some modifications. Half of the wells of 96-well plastic microtitre plates (Nunc Maxisorp; Polylabo, Strasbourg, France) were coated overnight at room temperature with the synthetic peptide (Neosystem, Strasbourg, France) dissolved in phosphate-buffered saline (PBS) at 0.1 µg/ml. The purity of this peptide was >75%; the preparation may have contained smaller fragments of the peptide sequence but no exogenous contaminant. The remaining wells received 100 µl of PBS. After four washes with PBS containing 0.1% Tween 20 (PBST), the wells were saturated for 2 h at room temperature with 200 µl of PBST solution containing 50 g/l of sucrose and 3% milk powder. After removal of excess buffer, test sera diluted 1:200 in 1% bovine serum albumin–PBST were incubated for 30 min at room temperature. After washing with PBST, F(ab')2 fragments of goat anti-human immunoglobulin G (IgG) conjugated to alkaline phosphatase (Caltag Laboratories, San Francisco, CA, USA), diluted 1:2000 in PBST, were added and the preparations were incubated for 30 min at room temperature. Plates were then washed four times and 100 µl of a solution containing 1 mg/ml of p-nitrophenyl phosphate (Sigma, St Louis, MO, USA) dissolved in 0.1 M Tris–HCl (pH 9.8) and 1.35 M NaCl was added. After 15 min at room temperature, the optical densities (OD) of the different wells were determined with a microplate spectrophotometer (Labosystems, Les Ulis, France) set to an absorbance of 405 nm. The OD was calculated by subtracting the OD measured in uncoated wells from that measured in coated wells. A calibration curve was generated with an ACAST-C27-positive reference serum at dilutions ranging from 1:200 to 1:4800. This reference serum, which was obtained from one RA patient followed in the Department of Rheumatology, had a high OD at a dilution of 1:200. Serum autoantibody titres were expressed in arbitrary units (AU) relative to the reference serum, whose OD at a dilution of 1:200 was equal to 240 AU. The threshold of positivity was two standard deviations (S.D.) above the mean value calculated for the sera of 70 healthy blood donors whose OD values were normally distributed, i.e. 20 AU. The specificity of the assay was confirmed by inhibition studies using the synthetic CAST-C27 peptide as a soluble inhibitor (data not shown). The intra- and interassay coefficients of variation were 1 and 10% respectively. To confirm that reactivity with the synthetic peptide was attributable to ACAST, sera were tested in assays using human erythrocyte calpastatin as the antigen. All ACAST-C27-positive sera were positive in ELISA and/or Western blotting using purified human erythrocyte calpastatin as the substrate (see below for description of the methods).

IgG subclass and light-chain expression analysis of ACAST-C27
IgG subclasses and light-chain expression were identified by ELISA. The method was the same as that used to determine ACAST-C27 of the IgG class. For the detection of IgG subclasses, subclass-specific mouse monoclonal autoantibodies (mAb) to IgG1 (clone HP-6001), IgG2 (clone HP-6014), IgG3 (clone HP-6050) and IgG4 (clone 6025) (Sigma) were used at a dilution of 1:1000 in PBST and were incubated for 1 h at room temperature. After four washes, 100 µl of goat anti-mouse IgG coupled to biotin (Caltag), diluted 1:1000 in PBST, was added for 1 h at 37°C. For the determination of the light chain, biotin-coupled goat anti-human {kappa} or {lambda} chain (Southern Biotechnology Association, Birmingham, AL, USA), diluted 1:2000 in PBST, was incubated for 1 h at 37°C. For both tests, streptavidin (diluted 1:10 000 in PBST) was added and the preparation was incubated for 15 min at 37°C. After washing the wells, enzyme reaction detection and OD determination were performed as described above.

ELISA on purified human erythrocyte calpastatin
The procedure was similar to that used for the detection of ACAST-C27 with some modifications. Purified human erythrocyte calpastatin (Research Diagnostics, Flanders, NJ, USA) was coated at 2 µg/ml under the same conditions. Test sera were diluted 1:50 in the same buffer and were incubated for 2 h at room temperature.

Immunoblotting on purified human erythrocyte calpastatin
Fifteen nanograms of purified human erythrocyte calpastatin (Research Diagnostics) was deposited on each lane of an 8% Tris–glycine–polyacrylamide minigel (Novex, San Diego, CA, USA). The erythrocyte calpastatin isoform contained domains II, III and IV of calpastatin and had a molecular mass of 70 kDa. After electrophoresis, the proteins were transferred onto nitrocellulose filters (Hybond-C Extra; Amersham Life Sciences, Buckingham, UK) for 2 h at 35 V in Tris–glycine–20% methanol. The nitrocellulose bands were saturated with PBST containing 5% milk powder (PBSTM). After washing, they were incubated for 2 h at room temperature with individual RA patients' sera that had been shown to have ACAST-C27 activity in the peptide ELISA or normal control sera diluted 1:100 in PBSTM. A mouse mAb (CSF 3-3) directed against domain IV of human calpastatin (Takara Shuzo, Kyoto, Japan) was used as the positive control at a concentration of 0.1 µg/ml. After three washes with PBST, the bands were incubated with F(ab')2 fragments of alkaline phosphatase-conjugated goat anti-human IgG (Caltag), diluted 1:1000 in PBSTM, and incubated for 1 h at room temperature. After washing, the reactivity was revealed with an indoxyl phosphate-staining solution (BCIP/NBT alkaline phosphatase substrate; Sigma). Sera recognizing a 70-kDa protein were considered to have ACAST reactivity.

Latex fixation test
This test was performed using the Rapitex RF agglutination kit (Behring Diagnostics, Westwood, NJ, USA). An RF titre >=80 IU/ml was considered to be positive.

Detection of AKA and APF by immunofluorescence
AKA and APF were detected as described previously [25, 26].

Statistical analysis
Fisher's exact test was used for frequency comparisons. Correlations between the presence of ACAST-C27 and/or their titres and the different criteria investigated in the cohort of 102 RA patients were assessed using Fisher's exact test, Spearman's rank correlation coefficient and the Mann–Whitney U-test when appropriate. A P value of <0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Frequencies of ACAST-C27 in established RA (group 1a), autoimmune and inflammatory rheumatic diseases (group 1b) and controls (group 1c)
ACAST-C27 of isotype IgG were detected in RA, primary Sjögren's syndrome and SLE with frequencies of 19.5, 18.5 and 15.5% respectively (Table 1Go). They were also detected in healthy controls, but significantly less frequently than in RA patients. ACAST-C27 were not detected in other connective tissue diseases (CTD) or inflammatory rheumatisms. ACAST-C27 were found in ankylosing spondylitis patients' sera at a rate (5.7%) similar to that observed in healthy donors (5.5%).

Among the 102 community cases of RA (group 1a), 20 (19.5%) were positive (>=20 AU) and the mean titre was 49.2±60 AU. Only 4/20 sera had high titres (>70 AU) (Fig. 1Go). All ACAST-C27-positive sera also reacted with purified human erythrocyte calpastatin in a solid-phase ELISA. Only sera with high titres (>70 AU) reacted strongly by immunoblotting with the 70 kDa protein (Fig. 2Go), while sera with moderate titres bound only weakly. All ACAST-C27 were of the IgG isotype, either IgG1 (20%) or IgG3 (80%), and 100% bore {lambda} light chains.



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  		FIG. 1. Titres of autoantibodies directed against the 27 C-terminal amino-acids of calpastatin as a function of RA duration in the cohort of 102 RA patients. Threshold of positivity is 20 AU.

 


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  		FIG. 2. Western blot analysis of calpastatin-specific monoclonal antibodies and RA sera using purified human erythrocyte calpastatin as the substrate. Molecular mass (expressed in kDa) is indicated on the left. Lane 1: mouse monoclonal antibody [CSF 1-2] directed against domain I of human calpastatin (negative control); lane 2: mouse monoclonal antibody [CSF 3-3] that reacts with domain IV of calpastatin (positive control); lanes 3 and 4: RA patients' sera with high ACAST-C27 Ab titres; lanes 5 and 6: RA patients' sera without ACAST-C27 Ab; lane 7: RA sera with a moderate ACAST-C27 Ab titre.

 

Specificity of ACAST-C27 for RA
Compared with normal controls (group 1c) and patients with non-RA rheumatic diseases (group 1b), the specificity of ACAST-C27 for RA was 94.5 and 93.1% respectively. All tested sera from CTD patients were positive for at least one autoantibody characteristic of the disease, i.e. anti-double-stranded DNA (anti-dsDNA) for SLE patients (n=84) and anti-SSA/Ro and/or anti-SSB/La autoantibodies for patients with primary Sjögren's syndrome (n=27). In contrast, only two RA patients' sera (group 1a), which were negative for ACAST-C27, recognized the SSA/Ro antigen. Therefore, the autoantibody patterns observed in patients positive for ACAST-C27 differed according to the type of autoimmune disease. Thus, in the absence of autoantibodies usually associated with SLE and/or Sjögren's syndrome, the presence of ACAST-C27 in patients with a rheumatic disorder was suggestive of RA.

Among the 88 community cases of very early arthritis (group 2), ACAST-C27 of the IgG isotype were detected in 6/58 (10.3%) RA patients but not in patients with other rheumatic diseases or undifferentiated rheumatism (Table 1Go). In group 2, therefore, the specificity of ACAST-C27 for RA was 100%.

Correlations between ACAST-C27 and clinical and/or biological and/or radiological findings in RA
In group 1a, no statistically significant relationship was found between ACAST-C27 and the other autoantibody populations. Indeed, 8/20 of ACAST-C27-positive RA sera were negative for both RF and AKA/APF and 3/20 were negative for RF or AKA/APF (Table 4Go). It should be noted that patients who had the highest titres of ACAST-C27 had RA of significantly shorter duration (P=0.01) (Fig. 1Go). However, the presence of ACAST-C27 was not associated with parameters of disease activity (Ritchie articular index, CRP) or severity (HAQ index, Sharp score). Furthermore, ACAST-C27-positive patients had significantly fewer nodules than patients who were ACAST-C27-negative (P=0.02).


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  		TABLE 4. Relationship between ACAST-C27 and RF and/or AKA/APF in the cohort of 102 community cases of RA (group 1a)

 
In group 2, all six ACAST-C27-positive RA patients were negative for both AKA and APF and four of them were also negative for RF. Thus, ACAST-C27 can be detected in very early seronegative RA.


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
Confronted with a peripheral inflammatory rheumatic disorder having evolved for only a few weeks, it is difficult for the clinician to diagnose RA and predict its outcome. However, early recognition of RA and prediction of its prognosis are determinants of the choice of therapy. At the onset of the disease, clinical and radiological findings are often insufficient for the diagnosis of RA. Several biological markers that may contribute to the diagnosis and prognosis of RA have been identified. RF, whose presence is generally considered predictive of a severe form, is often absent at the onset of RA and lacks disease specificity. Other autoantibody populations, such as AKA/APF and anti-Sa autoantibodies, which are more specific to RA, might be present at the early stages of the disease and, according to several authors, may help predict its outcome [26–30]. However, the assays used to detect these autoantibodies, i.e. indirect immunofluorescence analysis for AKA/APF and immunoblotting for anti-Sa, have not yet been validated as they are highly dependent on the laboratory running the test. Moreover, as shown by a recent study, only one-third of RF-negative patients with early RA were positive for anti-filaggrin and/or anti-Sa autoantibodies [31]. The cumulative sensitivity of the three autoantibodies populations having clinical relevance to RA is therefore not sufficient for the diagnosis of all very early cases of RA. Thus, new biological markers are clearly needed. Recently, ACAST have been detected in the sera of RA patients [7, 8], but their frequencies and clinical significance in a well-documented RA population has not been determined.

To evaluate the clinical relevance of ACAST, we set up a solid-phase ELISA using a synthetic peptide corresponding to the 27 C-terminal amino acids of calpastatin as the antigen. This assay has several advantages: (i) it is rapid, easy to perform and reproducible with low intra- and interassay coefficients of variation, as demonstrated in our study and that of Schlosser et al. [14]; (ii) in contrast to the interpretation of immunoblots, reading of the result is not prone to investigator bias; and (iii) the antigen used in the assay is pure, without exogenous contaminant(s). In addition, the reactivity against the synthetic peptide was shown to correspond to true ACAST reactivity, as all ACAST-C27-positive sera also reacted with native human erythrocyte calpastatin by ELISA or immunoblotting.

Therefore, the peptide-based ELISA was used to determine the sensitivity and specificity of ACAST-C27 in various CTD and several inflammatory rheumatic disorders and to study their clinical significance in a well-documented cohort of RA from a population-based group of 102 patients. This procedure allowed us to detect ACAST-C27 in almost 20% of the RA patients' sera. This frequency is lower than that observed by two other groups (approximately 50%), who used immunoblot analysis and a recombinant fusion protein corresponding to the 178 C-terminal amino acids of human calpastatin [8] or heated HeLa cell extracts [32] as the antigen. Thus, one might conclude that the peptide-based ELISA is less sensitive than these immunoblotting procedures. However, in the light of the results obtained by those groups, it is likely that the use of a large protein as the antigen led to lower specificity of ACAST for RA; indeed, ACAST were identified in several CTD, i.e. SLE, progressive systemic sclerosis, polymyositis–dermatomyositis and overlap syndrome, with sensitivities of 20–27, 11–38, 13–24 and 29% respectively. Similarly, in a cohort of 238 patients with persistent synovitis (>6 weeks) that had been evolving for <1 yr, autoantibodies directed towards domain 3 and 4 of calpastatin were detected in 49/132 (37%) non-RA patients [31]. Moreover, those authors did not test sera from patients with various inflammatory rheumatic diseases. In contrast, the peptide-based ELISA enabled us to detect ACAST exclusively in SLE and Sjögren's syndrome among non-RA sera. In clinical practice, French rheumatologists are rarely confronted with CTD such as SLE and, to a lesser degree, primary Sjögren's syndrome, the incidences of which are lower than that of RA and the spondylarthropathies. In this respect, a recent study has shown that the prevalences of RA and spondylarthropathies are nearly similar in Brittany, a region of France close to ours [33]. Moreover, CTD are usually easy to diagnose because of the availability of several serological markers. In our study, all sera from patients with SLE or Sjögren's syndrome contained the autoantibodies usually associated with these diseases, i.e. anti-dsDNA and/or anti-extractable nuclear antigen autoantibodies. Thus, the autoantibody patterns of SLE and Sjögren's syndrome patients differ from that of RA patients, whose sera only rarely react with these proteins. The major difficulty is in distinguishing RA from inflammatory rheumatic disorders that share clinical features, particularly spondylarthropathies with peripheral manifestations, such as psoriatic arthritis. This is why we studied a cohort of 88 community cases of arthritis that had been evolving for a median of 4 months. Because of the type of recruitment, this population was probably highly representative of very early arthritis in our region of France. In this group, one-third of the non-RA patients had peripheral spondylarthropathy; in contrast, only one non-RA patient had a CTD, which was primary Sjögren's syndrome. Such a profile of rheumatic diseases is not surprising, and has already been observed in other cohorts of patients with very early arthritis [34, 35]. Pertinently, in this cohort, ACAST-C27 were not detected in the more common non-RA rheumatic diseases, i.e. crystal-induced arthritides and osteoarthritides in the elderly, and spondylarthropathies, in particular with peripheral manifestations, in younger patients. These results are concordant with those obtained in our study of stored sera. Thus, ACAST-C27 of isotype IgG were detected only in RA patients and a restricted set of those with CTD and, in the absence of autoantibodies classically found in sera of patients with the latter, could be useful for diagnosing very early RA.

Correlations between ACAST-C27 and clinical, biological and radiological findings were studied in our cohort of 102 RA patients. The presence of ACAST-C27 was not correlated with more active or severe forms of RA, especially parameters of structural joint damage. However, because it is well established that the detection of certain autoantibodies can precede the occurrence of clinical or radiological manifestations [36], we intend to continue monitoring ACAST-C27 in our two cohorts. Furthermore, only two-thirds of the RA sera were positive for RF and/or AKA/APF, two biological markers useful for the diagnosis of RA [37]. In our cohort of 88 patients with very early arthritis, only half of the RA sera were positive for these markers. This finding emphasizes the need for additional biological markers of RA. Although the frequency of ACAST-C27 in our two cohorts was rather low (10.3 and 19.5%), our analysis provided an interesting finding. Indeed, ACAST-C27 may constitute an independent immunological marker, as eight out of 20 (RA cohort) and four out of six (very early arthritis cohort) ACAST-C27-positive RA patients were negative for both RF and AKA/APF.

Analysis of the light-chain and IgG subclass distributions of ACAST-C27 in our RA cohort showed a predominance of IgG3 and the exclusive expression of a {lambda} light chain. The latter observation suggests that the ACAST-C27 response is antigen-driven and, therefore, that the B-cells producing these autoantibodies are expanded selectively during the course of RA. In this regard, the demonstration that RF-negative B-cells from RA synovial tissue used {lambda} light chains [38, 39] should be noted. In addition, IgG1 and IgG3 can activate the classical complement pathway, leading to perpetuation of the inflammatory process and contributing to tissue damage. Finally, it has been demonstrated that ACAST are able to inhibit calpastatin activity, leading to overexpression of calpains, which are proteases implicated in joint injury [8]. Taken together, these findings raise the possibility of a pathogenic role for ACAST-C27 in RA, particularly in the early course of the disease.

In conclusion, ACAST-C27 autoantibodies are detected only in a restricted set of CTD and so appear to be specific to RA when autoantibodies usually associated with SLE or primary Sjögren's syndrome are not detected. Because of their presence in community cases of very early RA, particularly in some seronegative forms, ACAST-C27 may help physicians to distinguish recent-onset RA from the more common non-RA rheumatic diseases, such as the spondylarthropathies. ACAST-C27 were not associated with the more destructive forms of RA, but long-term follow-up of the patients will enable us to determine whether they might also constitute a predictive marker of severity.


   Acknowledgments
 
The authors thank the group ‘Polyarthrite rhumatoïde et consommation de soins’ for its contribution and Janet Jacobson for correcting the manuscript. This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and grants from the Association de Recherche sur la Polyarthrite, the Société Française de Rhumatologie, the Fondation de la Recherche Médicale and Pharmacia France.


   Notes
 
Correspondence to: O. Vittecoq, Service de Rhumatologie, Centre Hospitalier Universitaire de Rouen, 76031 Rouen Cedex, France. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 18 October 2000; Accepted 20 April 2001


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