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. 2011 Jun;164(3):337–345. doi: 10.1111/j.1365-2249.2011.04378.x

Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti-cyclic citrullinated peptide (CCP) and anti-VCP1 antibodies

F Pratesi *, C Tommasi *, C Anzilotti *, I Puxeddu *, E Sardano *, G Di Colo *, P Migliorini *,†
PMCID: PMC3087929  PMID: 21413944

Abstract

Anti-citrullinated protein/peptide antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and can be measured using different citrullinated substrates. In this paper we describe a new viral citrullinated peptide – VCP2 – derived from the Epstein–Barr virus-encoded protein EBNA-2 and analyse its potential as substrate for ACPA detection. Analysing sera from 100 RA patients and 306 controls, anti-VCP2 immunoglobulin (Ig)G were found in 66% of RA sera, IgM in 46% and IgA in 39%, compared with less than 3% of control sera. Anti-VCP2 IgG was associated with erosive arthritis, the presence of rheumatoid factor and anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Anti-VCP2 antibodies were detected in 1% and anti-VCP1 antibodies in 4% of CCP-negative RA sera; conversely, 3% of the VCP-negative sera were CCP-positive. Taken together, these data suggest that VCP2 could offer a valuable tool for ACPA detection. Inhibition assays showed that two non-overlapping epitopes – a citrulline–glycine stretch shared between VCP1 and VCP2 and the N-terminal portion of the VCP2 sequence – were targeted by anti-VCP2 antibodies. Moreover, in some RA sera that tested positive in CCP and VCP2 assays, preincubation with VCP2 inhibited binding to CCP, whereas in other sera the binding was unaffected. Thus, the reactivity with more than one ACPA substrate might be due in some RA patients to antibody populations with different specificities, and in others to cross-reactive antibody populations. Finally, affinity-purified anti-VCP2 antibodies immunoprecipitated deiminated Epstein–Barr virus nuclear antigen (EBNA-2) from an EBNA-2-transfected cell line, suggesting that viral sequences may be involved in the generation of the ACPA response.

Keywords: antibody specificity, autoantibodies, cyclic citrullinated peptide, Epstein–Barr virus, rheumatoid arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPA) represent an important tool for the serological diagnosis of RA [1]. ACPA recognize different citrullinated substrates, such as filaggrin, fibrinogen, vimentin, collagen II and enolase [2]. A comparative study of the sequences recognized by anti-filaggrin [3] and anti-fibrin [4] antibodies showed that their main feature is the presence of citrulline flanked by neutral amino acids such as glycine, serine or threonine. Similar amino acid repeats are often found in nucleic acid-binding proteins; some of these are of viral origin, such as the transcription-regulating proteins in herpesviruses [5].

One of the nuclear proteins encoded by Epstein–Barr virus (EBV), EBNA-1, contains in its N-terminal region a sequence (35–58) characterized by six glycine–arginine repeats. This sequence, synthesized as viral citrullinated peptide (VCP1) – a multiple antigen peptide (MAP) containing citrulline in the place of arginine – detected antibodies in 45% of RA sera, but in less than 5% of normal and connective tissue disease controls [6].

A bioinformatic analysis of sequence databases (http://www.uniprot.org) for EBV proteins showed that repeats of arginyl residues surrounded by neutral and small amino acids are also present in the EBNA-2 protein.

In this study we synthesized viral citrullinated peptide 2 (VCP2), a peptide corresponding to the modified 338–358 sequence of the EBNA-2 protein, by substituting each arginine with citrulline, and then analysed its potential as a substrate for the detection of ACPA. By studying the antibodies reactive with VCP2, in terms of their frequency, isotypes and correlations with clinical parameters, we determined that anti-VCP2 antibodies have a high specificity and sensitivity for RA.

Furthermore, in the wake of recent studies employing different citrullinated antigens to investigate the fine specificity of ACPA [7,8], we evaluated by cross-inhibition experiments the relationship of anti-VCP2 antibodies with anti-VCP1 and anti-cyclic citrullinated peptide (CCP), providing further evidence for the heterogeneity of ACPA in RA sera.

Patients and methods

Patients

One hundred consecutive RA patients (74 women and 26 men; mean age 54·3 years, range 21–79; mean disease duration 10 years, range 9–40 months) diagnosed according to the American College of Rheumatology (ACR) classification criteria [9] were enrolled in this study. They were evaluated for systemic involvement (presence of xerostomia, xerophthalmia, peripheral vasculitis, rheumatoid nodules), disease activity [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), active synovitis, morning stiffness lasting more than 30 min], and disease severity (presence of erosions in the hands and/or feet). Self-reported smoking habits were recorded; the patients were classified as ever-smokers and never-smokers using the criteria of Stolt et al. [10], and the correlation between smoking habits and ACPA status was analysed [11].

Controls

Two hundred and six disease controls were studied, comprising patients with: ankylosing spondylitis (AS) (n = 27); psoriatic arthritis (PsA) (n = 29); undifferentiated arthritis (UA) (n = 11); systemic sclerosis (SSc) (n = 19); mixed cryoglobulinaemia (MC) (n = 25); systemic lupus erythematosus (SLE) (n = 23); polymyalgia rheumatica (PMR) (n = 34); Sjögren's syndrome (SjS) (n = 12); and infectious mononucleosis (IM) (n = 26). AS was diagnosed according to the revised New York criteria [12] and PsA according to the criteria of Vasey and Espinoza [13]. The diagnoses of SLE [14] and SSc [15] were made based on ACR criteria; MC was diagnosed in the presence of Meltzer's triad (purpura, weakness and arthralgia) and cryoglobulins in the sera [16]; and SjS was diagnosed according to the criteria of the American–European Consensus Group [17]. The diagnosis of infectious mononucleosis was made on the basis of clinical and serological findings.

One hundred blood donors from the University Hospital of Pisa were used as normal healthy subjects (NHS).

The written consent of all study participants was obtained in accordance with the Declaration of Helsinki and the study was approved by the University of Pisa Ethics Committee.

Serum samples were stored at −20°C until tested.

Peptide synthesis

The peptides for this study were obtained from Espikem s.r.l. (Florence, Italy). Briefly, using the solid-phase synthesis procedure described by Merrifield [18] and modified by Atherton and Sheppard [19], multiple antigen peptides (MAPs) on a 4-mer branching polylysine backbone were produced from 9-fluorenylmethoxycarbonyl-protected amino acids [20]. The peptide sequences are listed in Table 1.

Table 1.

The peptide sequences used in the direct binding and inhibition experiments

Name Sequence
VCP1 (G G D N H G Cit G Cit G Cit G Cit G Cit G G G Cit P G A P G)4 K2 K βA
VCP2 (G Q S Cit G Q S Cit G Cit G Cit G Cit G Cit G Cit G K G)4 K2 K βA
MAP-GC (G Cit G Cit G Cit G Cit G Cit G G G Cit G)4 K2 K βA
MAP-GQSC (G Q S Cit G Q S Cit G)4 K2 K βA
P-RIB (E E E D E D M G F G L F D)4 K2 K βA
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Cit, citrulline; VCP, anti-viral citrullinated peptide; MAP, a multiple antigen peptide; P-RIB, P-Ribosomal Protein derived peptide.

Detection of anti-VCP2 antibodies

Anti-viral citrullinated peptide antibodies were detected in the sera by enzyme-linked immunosorbent assay (ELISA) as described previously [6,21]. Briefly, 10 µg/ml VCP2 was added to polystyrene plates (Nunc MaxiSorp F96; Nunc, Roskilde, Denmark) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Saturation was carried out with PBS containing 3% bovine serum albumin (BSA) for 45 min at room temperature. Sera diluted 1:200 in PBS containing 1% BSA and 0·05% Tween 20 were then incubated in duplicate on the plates for 3 h at room temperature. After washing with PBS 1% Tween and PBS, alkaline phosphatase-conjugated anti-human IgG, IgA or IgM (Sigma-Aldrich, St Louis, MO, USA) was added to the wells, and the plates were incubated for 2 h at room temperature. Alkaline phosphatase activity was revealed with p-nitrophenyl phosphate in 50 mm Na2CO3 (pH 9·6). The results are expressed as the percentage of an internal positive sample; the positivity threshold adopted for the test (>97·5 percentile for healthy controls) was 13% for IgG, 20% for IgM and 19% for IgA.

The anti-VCP1 assay was conducted using the same procedure and the positivity threshold adopted for IgG was 20%. Anti-CCP IgG were detected using a commercial kit (anti-CCP second generation; Euro-Diagnostica AB, Malmö, Sweden), according to the manufacturer's instructions. Rheumatoid factor (RF) was measured by nephelometry.

Purification of antibodies specific for VCP2 from RA patient sera

VCP2 was conjugated to CNBr-activated Sepharose (Sigma) following standard procedures.

Seven RA sera containing anti-CCP, anti-VCP1 and anti-VCP2 antibodies and, for comparison, five ACPA-negative NHS sera were evaluated. Total immunoglobulins were precipitated with 50% saturated ammonium sulphate from each sample, and the precipitates were dissolved in phosphate buffer (pH 7·4) and dialysed overnight against phosphate-buffered saline (PBS). IgG immunoglobulins were then extracted from each sample using a protein G-Sepharose column (Sigma) according to standard procedures.

The IgG preparation from each serum sample was applied to a VCP2 column, and the flow-through was collected for analysis. The column was washed extensively with 20 mm Na2HPO4 and 150 mm NaCl (pH 7·2) and the antibodies binding to the column were eluted with 0·1 m glycine buffer (pH 2·8) (0·5 ml/fraction), neutralized immediately with 50 µl Tris 1 m (pH 8·0), and dialysed overnight against PBS. The anti-VCP antibody content in the eluates and flow-through was measured by ELISA.

Competition assays

For the competition assays on CCP, nine RA sera that had tested positive in the VCP1, VCP2 and CCP assays were selected and assayed at doubling dilutions (1/100–1/12 800). The serum concentration corresponding to 50% of maximum binding to the solid phase was determined. This serum dilution was preincubated in the liquid phase with VCP1, VCP2 or P-RIB for 30 min at 37°C before being transferred to CCP-coated plates. ELISA was carried out subsequently according to the manufacturer's instructions.

For the competition assays on VCP2, the amount of affinity-purified anti-VCP2 antibodies that yielded 50% of maximum binding to the solid phase was determined. This quantity of antibodies was diluted in PBS containing 1% BSA and 0·05% Tween 20 and then preincubated in the liquid phase with synthetic peptides for 30 min at 37°C before being transferred to the VCP2-coated plates. Thereafter, ELISA assays were carried out as for the direct binding tests.

Preparation of the cell lysate and in vitro deimination

Burkitt's lymphoma cell line DG75 transfected with EBNA-2 (a kind gift from Dr G. Klein, Karolinska Institute, Stockolm, Sweden) was lysed at a rate of 2·5 × 106 cells per 150 µl of lysis buffer [50 mm Tris-HCl, pH 8·0, 150 mm NaCl, 0·1% sodium dodecyl sulphate (SDS), 1% Nonidet P40, protease inhibitors]. Protein concentrations were measured by the Pierce bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA).

Deimination was performed by in vitro treatment with rabbit muscle peptidylarginine deiminase (PAD), as described previously [22].

Immunoprecipitation

Deiminated and non-deiminated cell lysates (250 µg/sample immunoprecipitation) were incubated overnight at 4°C with 100 µg affinity-purified anti-VCP2 antibodies or control IgG from NHS diluted in Tris-buffered saline (TBS) containing 0·1% Tween-20. Preformed antibody–antigen complexes incubated with PureProteome™ Protein G Magnetic Beads (Millipore, Billerica, MA, USA), according to the manufacturer's instructions, were isolated using a MagnaGrIP™ Magnetic Rack (Millipore).

The immunoprecipitates were eluted in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, subjected to SDS-PAGE under non-reducing conditions, and blotted onto a nitrocellulose membrane (Millipore). The membrane strips were saturated for 30 min at room temperature in TBS containing 3% BSA and incubated overnight at 4°C with a rat monoclonal antibody specific for EBNA-2 (rat monoclonal antibody anti–EBNA-2 R3; Ascenion GmbH, Berlin, Germany) diluted 10 µg/ml in TBS containing 5% BSA and Tween-20 0·05%. After washings with TBS containing 0·1% Tween 20 (TBST), anti-rat IgG–horseradish peroxidase (Sigma) diluted at 1:800 in TBST was added and the strips were incubated at room temperature for 2 h. The anti-citrulline (modified) detection kit (Millipore) was used, following the manufacturer's instructions, to detect deiminated proteins. Peroxidase activity was visualized by means of enhanced chemiluminescence (ECL) using Luminata™ Western HRP substrate (Millipore). ECL images were acquired and analysed with the VersaDoc Imaging System and QuantityOne analysis software (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Quantitative variables were compared between groups using Kruskal–Wallis one-way analysis of variance or the Mann–Whitney U-test as appropriate. Clinical parameters were compared between sera that tested positive or negative for anti-VCP2 antibodies using the χ2 or Fisher's exact test as appropriate. Correlations were determined using Spearman's rank correlation coefficient. P < 0·05 was considered as significant; Prism 4 for Windows (GraphPad Software Inc., San Diego, CA, USA) and Stat View for Windows (SAS Institute Inc., Cary, NC, USA) were used for the analyses.

Results

Anti-VCP2 antibodies in RA patients and controls

The deiminated viral peptide VCP2 was synthesized as a MAP and used in an ELISA assay to test the sera from 100 RA patients, 100 healthy subjects and 206 disease controls. Setting the threshold value at the 97·5 percentile of the normal healthy population, we detected anti-VCP2 antibodies of the IgG isotype in 66%, of the IgM isotype in 46% and of the IgA isotype in 39% of RA sera, compared with fewer than 3% of the disease and healthy controls (Fig. 1). Therefore, anti-VCP2 antibodies of the IgG, IgM and IgA isotypes differentiated RA patients clearly from healthy subjects and disease controls (P < 0·0001).

Fig. 1.

Fig. 1

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Levels of anti-viral citrullinated peptide 2 (VCP2) immunoglobulin (Ig)G, IgM and IgA in rheumatoid arthritis (RA) and disease control patients. Anti-VCP2 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in normal healthy subjects (NHS) and in patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS, undifferentiated arthritides (UA), polymyalgia rheumatica (PMR), systemic lupus erythematosus (SLE), mixed cryoglobulinaemia (MC), systemic sclerosis (SSc), Sjögren's syndrome (SjS), infectious mononucleosis (IM) or RA. Results are expressed as the percentage (%) of an internal positive control. Horizontal bars represent the mean values for each group. The dotted lines represent cut-off values: 13% for anti-VCP2 IgG, 20% for anti-VCP2 IgM and 19% for anti-VCP2 IgA.

We then tested individual RA serum samples to determine which anti-VCP2 isotypes were present, and found that 22 sera were single-positive (20 for IgG, one for IgA and one for IgM); eight sera were double-positive for IgG and IgM; and one was double-positive for IgG and IgA. All three anti-VCP2 antibody isotypes were detected in 37 RA sera (Fig. 2).

Fig. 2.

Fig. 2

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Anti-viral citrullinated peptide 2 (VCP2) isotype distribution in rheumatoid arthritis (RA) patients. The distributions of anti-VCP2 IgG, IgM and IgA in 100 RA patients. The number above each bar indicates the frequency (%) of positive patients.

We also investigated whether antibody levels varied with the presence/absence of rheumatoid factor. When sera from RF-positive (n = 72) and RF-negative (n = 28) RA patients were compared, it was found that anti-VCP2 levels were higher in the RF-positive patients (P = 0·0006 for IgG, P = 0·006 for IgM, P = 0·02 for IgA). No association was found with increased CRP levels or the presence of active arthritis or extra-articular manifestations of the disease.

The presence of anti-VCP2 IgG was associated with erosive arthritis (P = 0·012); this association was higher in the patient who was positive for both IgG and IgA (P = 0·009) and in the 37 patients with all three antibody isotypes (P = 0·003).

An association was also found between smoking and double-positivity for anti-VCP2 IgG and IgA (n = 1) (P = 0·043) and between smoking and triple-positivity for anti-VCP2 IgG, IgM and IgA (n = 37) (P = 0·003).

VCP2 and other assays for ACPA detection

The panel of RA sera was also tested on VCP1 and CCP-coated plates. The levels of antibodies detected by the three assays were correlated strongly (P < 0·0001) (Fig. 3a–c), although there were some RA sera that recognized only one citrullinated peptide (Fig. 3d). Among the sera tested, 62% were triple-positive for anti-CCP, anti-VCP1 and anti-VCP2 antibodies, 6% were double-positive for varying combinations of antibodies, 3% were single-positive for CCP, 4% were single-positive for VCP1, 1% were single-positive for VCP2 antibodies and 24% were negative for all the ACPAs investigated.

Fig. 3.

Fig. 3

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Correlation between anti-viral citrullinated peptide (VCP) and anti-cyclic citrullinated peptide (CCP) antibodies. (a,b,c) The levels of anti-VCP1, anti-VCP2 and anti-CCP IgG in rheumatoid arthritis (RA) patients measured by enzyme-linked immunosorbent assay are shown along the x- and y-axes. Spearman's rank correlation coefficients and P-values are given. The dotted lines represent cut-off values. (d) The pie chart shows the clustering of different anti-citrullinated protein/peptide antibodies (ACPAs) in the RA patients.

Serum binding to different antigens could be attributable to antibody cross-reactivity or to the simultaneous presence of distinct antibody populations, each reactive to a single antigen. To investigate antibody cross-reactivity, we selected nine triple-positive RA patients and performed liquid phase inhibition assays preincubating sera with VCP1 and VCP2 before testing on CCP-coated plates. High inhibition was seen in seven sera, whereas in two of nine preincubation with VCP1 or VCP2 did not affect binding to CCP. Representative assays are shown in Fig. 4.

Fig. 4.

Fig. 4

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Cross-reactivity of anti-viral citrullinated peptide 2 (VCP2), anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Sera from three representative rheumatoid arthritis (RA) patients containing anti-VCP1, anti-VCP2 and anti-CCP IgG antibodies were preincubated with different amounts of VCP1, VCP2 or a control peptide (P-RIB) before being transferred to CCP-coated plates. The results are expressed as the percentage inhibition of binding to CCP: 100 − [100 × OD (antibody + peptide)/optical density (antibody + buffer)]. (a.b) Inhibition of serum binding by VCP1 and VCP2; in (c) no inhibition is observed.

Fine specificity of anti-VCP2 antibodies

Anti-VCP2 antibodies were purified by affinity chromatography from seven different RA sera; these were found to bind exclusively to the deiminated peptide VCP2, and not to the arginine-containing sequence or to other peptides synthesized on a polylysine core (data not shown).

To explore further the fine specificity of anti-VCP2, affinity-purified antibodies were analysed by a liquid phase inhibition assay using VCP1, VCP2 and two shorter peptides comprised within these sequences: MAP-GC (a run of five consecutive Cit–Gly residues shared by VCP1 and VCP2) and MAP-GQSC (the N-terminal portion of VCP2).

The highest inhibition was observed when anti-VCP2 antibodies were preincubated with VCP2. Two patterns were observed for the other inhibitors: in some antibody preparations VCP1, GC and GQSC inhibited binding to VCP2 to more or less the same extent; in others, GQSC and VCP2 inhibited binding to a similar degree, whereas VCP1 and GC were less efficient inhibitors. Representative examples are given in Fig. 5. These data suggest the existence of two epitopes within VCP2, one residing in the Cit–Gly stretch shared with VCP1 and the other corresponding to the N-terminal portion of the sequence.

Fig. 5.

Fig. 5

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Fine specificity of anti-viral citrullinated peptide 2 (VCP2) antibodies. Affinity-purified anti-VCP2 immunoglobulin (Ig)G antibodies from two representative rheumatoid arthritis (RA) patients were preincubated with different amounts of VCP1, VCP2, multiple antigen peptide (MAP)-GC or MAP-GQSC peptides before being transferred to VCP2-coated plates. The results are expressed as the percentage inhibition of VCP2: 100 − [100 × optical density (antibody + peptide)/optical density (antibody + buffer)]. (a) Inhibition of antibody binding by VCP2 and MAP-GQSC; (b) Inhibition of antibody binding by VCP1, VCP2 and MAP-GC.

Anti-VCP2 antibodies immunoprecipitate citrullinated EBNA-2

To investigate whether affinity-purified anti-VCP2 antibodies react with cells containing citrullinated EBNA-2, immunoprecipitation experiments were performed. Cell lysate from a DG75 cell line transfected with EBNA-2 was citrullinated in vitro by treatment with PAD, and incubated with IgG from normal subjects or anti-VCP2 antibodies from RA patients. The immunoprecipitate was run on SDS-PAGE, transferred to nitrocellulose and probed with a monoclonal anti-EBNA-2 antibody and with polyclonal rabbit anti-modified citrulline antibodies (anti-MC).

Bands comprised between 90 and 60 kDa were detected by the anti-EBNA-2 antibody on the immunoprecipitates of anti-VCP2 antibodies and in lower amounts on the immunoprecipitates of normal IgG (Fig. 6). A similar heterogeneity in molecular weight has been reported previously for EBNA-2 [23,24]. In contrast, anti-MC antibodies reacted with the 70–75 kDa polypeptides present exclusively in the immunoprecipitates of anti-VCP2 antibodies (Fig. 6).

Fig. 6.

Fig. 6

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Immunoprecipitation of citrullinated Epstein–Barr virus nuclear antigen (EBNA)-2 by affinity-purified anti-viral citrullinated peptide 2 (VCP2) antibodies. Immunoprecipitation of deiminated EBNA-2 from the in vitro deiminated lysate of DG75 transfected with EBNA-2. Deiminated DG75 lysate was incubated with anti-VCP2 antibodies from two rheumatoid arthritis (RA) patients (RA1 and RA2) or with control immunoglobulins (NH IgG). Immunoprecipitated proteins were blotted with a monoclonal anti-EBNA-2 antibody or an anti-modified citrulline antibody. Bands comprised between 90 and 60 kDa were detected by anti-EBNA-2 antibody on the immunoprecipitate of anti-VCP2 antibodies and in lower amounts on the immunoprecipitate of normal IgG. Anti-citrulline antibody reacted with 70–75 kDa polypeptides present exclusively in the immunoprecipitate of anti-VCP2 antibodies.

These results suggest that both normal sera and RA sera contain antibodies that react with EBNA-2, but only affinity-purified antibodies from RA sera can immunoprecipitate citrullinated EBNA-2-related polypeptides.

Moreover, anti-VCP2 antibodies immunoprecipitate citrullinated protein/proteins with a molecular weight comprised between 37 and 50 kDa, not recognized by anti-EBNA-2 antibody, that may represent either fragments or alternatively spliced versions of EBNA-2 or unrelated deiminated proteins.

Discussion

The data obtained in the present study indicate that different citrullinated sequences derived from EBV-encoded proteins are recognized by antibodies of the ACPA family. Therefore, an ELISA assay using VCP2 could offer a sensitive and specific tool for the diagnosis of RA: anti-VCP2 of all three isotypes (IgA, IgM and IgG) were detected almost exclusively in RA sera. While anti-VCP2 antibodies are not a marker of active disease or extra-articular involvement, they are associated with erosive arthritis.

The association of ACPA with severe erosive arthritis has been reported in several studies using different substrates to detect ACPA [25–27]. In two recent studies different ACPA assays were applied to the same cohort of patients. In the first study, anti-modified citrullinated vimentin (MCV) and anti-CCP antibodies showed similar correlations with the rate of joint destruction [28]. In a 10-year prospective study, anti-MCV antibodies were found to be better predictors of the radiographic progression of RA than anti-CCP antibodies [29]. Using viral peptides, we demonstrated an association of erosive arthritis with VCP2, but not VCP1 [21]. The simultaneous presence of different anti-VCP2 isotypes thus constitutes an additive risk, increasing the strength of the association with bone erosions.

When a panel of sera were tested on VCP2, VCP1 and CCP, a high correlation overall in the reactivity of the antibodies was found. However, some sera reacted with only one or two antigens, suggesting the presence of non-cross-reactive anti-VCP1 and anti-VCP2 antibodies. The results of our inhibition assays support this hypothesis, and showed a considerable variability between individual sera. In some sera, different antibody populations that reacted independently with a single substrate were detected, whereas in other sera the reactivity on different ACPA substrates was due to the presence of cross-reactive antibodies. It is worth noting that after preincubation with a single peptide (VCP1 or VCP2) some sera inhibited up to 60% of the binding to CCP2-coated plates, which employ different citrullinated peptides [30], thus indicating the broad cross-reactivity of these members of the ACPA family.

When we analysed the fine specificity of anti-VCP2 antibodies on polyclonal antibodies affinity-purified from individual sera, we found direct evidence of non-overlapping epitopes within the VCP2 sequence. The glycine–citrulline stretch represents an epitope shared by VCP1 and VCP2, while other sera contain antibodies that react mainly with the N-terminal portion of the VCP2 molecule.

Studies of the citrullinated sequences contained in filaggrin [5] or fibrin [4] have led to similar conclusions. Sebbag et al. [4] measured anti-citrullinated fibrinogen antibodies using a panel of overlapping deiminated peptides that cover a large portion of the fibrin sequence. Although fibrin contains two major epitopes that are recognized by many RA sera, the study found 12 different reactivity profiles. Liquid phase inhibition assays using fibrin-derived [31] and filaggrin-derived peptides [5] suggest the presence of cross-reactive as well as non-cross-reactive ACPA. In a study of antibodies reacting with enolase or collagen type II-derived peptides, Snir et al. reported a more limited cross-reactivity of ACPA [31]; however, absorption on antigen-coated plates was used instead of liquid phase inhibition and this approach may have limited the ability to detect cross-reactive antibodies. When Van de Stadt et al. analysed five sera from CCP+ patients with arthralgia and three sera from RA patients, they detected no cross-reactivity among antibodies that were reactive with citrullinated peptides from fibrinogen, enolase and vimentin [7]. At this point, a study involving a larger number of sera from RA patients in different stages of the disease and using a panel of peptides that includes EBV-derived sequences could help us to unravel the complexity of the ACPA response in RA and shed light on how anti-citrulline immune response is incited in RA.

Given the broad heterogeneity of ACPA, a more sensitive, multiplex assay based on the combination of different citrullinated antigens could be envisaged. In this respect, citrullinated proteins such as fibrinogen, vimentin and enolase are of interest because they contain a variety of citrullinated epitopes. There are some major drawbacks, however, to the use of citrullinated proteins; first of all, they contain non-citrullinated epitopes that are recognized by non-RA sera and would thus lower the specificity of the test; and secondly, it is difficult to prepare sufficient amounts of high-quality antigen with a defined degree of citrullination. Conversely, synthetic citrullinated peptides are obtained easily in pure form with a well-defined chemical structure; moreover, an exact orientation of the epitopes in the plate can be obtained by covalent binding of the peptides via their carboxy- or amino-terminal.

ACPA may vary in terms of their isotype usage as well as their fine specificity. Most RA sera contain anti-VCP1 [21] and anti-VCP2 antibodies of the IgG, IgA and IgM isotypes, indicating a polyclonal response to citrullinated peptides of viral origin. However, sera positive for IgM and/or IgA, but not IgG anti-VCP antibodies, do exist, suggesting that each isotype may be produced independently.

Similar data have been obtained in studies measuring ACPA on other substrates. Verpoort et al. [32] measured anti-CCP antibodies of the IgA and IgM isotypes in CCP-IgG-positive sera from patients with established RA or undifferentiated arthritis, and reported that a high proportion of the RA sera also contained anti-CCP IgA (62%) and anti-CCP IgM (61%). In undifferentiated arthritis, the simultaneous presence of different anti-CCP antibody isotypes was strongly predictive of an evolution towards RA. IgA ACPAs that are reactive with citrullinated enolase or collagen II-derived peptides have been described recently [31]; these were present mainly in IgG-positive sera, which is consistent with the findings in the present study. The authors suggested that the mechanism leading to the production of IgA ACPA could be immune stimulation from citrullinated antigens at the mucosal surfaces. In this regard, it is worth noting that in our patients there was a statistically significant correlation between smoking and the combined presence of IgA and IgG ACPA, but only a trend towards an association between smoking and the presence of IgG ACPA alone. Therefore, IgA ACPA could play a role in the pathogenesis and aetiology of RA, as has been suggested in studies on other patient cohorts [33], and their measurement might increase the sensitivity of ACPA assays.

In a previous study we tested RA sera on a viral citrullinated peptide derived from EBNA-1 [21] and found an analogous polyclonal response, as well as evidence of the persistent production of anti-VCP IgM many years after disease onset. Taken together, these data suggest that B cells which produce IgM ACPA are persistently expanded in RA.

Finally, this discovery of a new EBV sequence that could be used as a substrate for ACPA detection raises once again the question of the role of EBV in RA [34]. EBV establishes a persistent infection in more than 90% of adults and represents a constant source of antigens that in an inflammatory environment might be deiminated and possibly induce or perpetuate the immune response to citrullinated proteins. Alternatively, EBV-derived citrullinated peptides could be targeted by ACPA simply because of a similarity in sequence or conformation with other self- or non-self antigens.

We demonstrated previously that anti-VCP1 antibodies bind in vitro deiminated recombinant EBNA1 and that they immunoprecipitate in vivo deiminated EBNA-1 from EBV-infected cell lines [35]. In this study we have shown that anti-VCP2 antibodies immunoprecipitate deiminated EBNA-2. However, more experiments will be needed to demonstrate that EBNAs act as inciting antigens in the production of ACPA. EBNA-2 is expressed in newly infected B cells and in naive B cells from the tonsillary lymph nodes of healthy EBV carriers [36]. No data are available on the EBNA-2 load in RA patients, which are characterized by a defect in the killing of autologous EBV-infected lymphoblastoid B lines and by an increased frequency of EBV-infected peripheral B lymphocytes. Experiments are now in progress to detect citrullinated EBNA-2 in cells and tissues from RA patients and to identify the stimuli that could lead to the in vivo deimination of EBV antigens. These results should help to shed light on the role that EBV, acting in the appropriate genetic background, may play in the induction of ACPA.

Acknowledgments

We would like to thank Dr G. Klein (Karolinska Institute, Stockolm, Sweden) for the kind gift of EBNA 2-transfected DG75 cells and Ms Lisa Chien for revising the manuscript.

Disclosure

The authors declare no conflict of interest.

References

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