Differing specificities and isotypes of anti-citrullinated peptide/protein antibodies in palindromic rheumatism and rheumatoid arthritis

This cross-sectional study included patients diagnosed with PR according to the criteria of Guerne and Weisman [1] attending our Arthritis Unit from June 2012 to June 2013, with no evolution to RA or other rheumatic diseases at the time of clinical and laboratory assessments; other causes of intermittent arthritis were excluded. As controls, we included patients with established RA (1987 ACR criteria) controlled by gender, age (±5 years), disease duration (±3 years), and CCP2 positivity. Written consent was obtained from all participants and the study was approved by the Hospital Clinic Ethics Committee.

IgG antiCCP2 was measured in serum samples by a second-generation enzyme-linked immunosorbent assay (ELISA; Immunoscan, Eurodiagnostica,; NV <50 UI). The citrullinated peptides used in this study, whose primary sequences are detailed below, are derived from the main protein targets of RA-specific autoantibodies: fibrin [13], vimentin [14], and alpha-enolase [15].

ACPA fine specificities were determined by home-made ELISA tests using synthetic citrullinated peptides previously studied by our group [16, 17] as antigens: fibrin ([Cit⁶³⁰]α-Fibrin(617-631)), vimentin ([Cit⁷¹]Vim(47-72) and [Cit^64,69,71]Vim(47-72)), and α-enolase [Cys^4,22,Cit^9,15]α-Enolase(5-21); namely p18, p48, p55 and CEP-1, respectively. Uncitrullinated peptides were also synthesized and used as a control for citrulline specificity of the anti-citrulline peptide antibodies in both RA and PR patients. The ACPA isotypes IgG, IgA, and IgM of fine specificities were measured using the corresponding secondary antibodies (Jackson Immunoresearch) according to the previously described ELISA procedure [16, 17]. Cut-off values for each ELISA test were determined using receiver operating characteristic (ROC) curves, with a specificity of 98% compared with a healthy population (blood donors, n = 64).

Citrullinated peptides [Cit⁶³⁰]α-fibrin(617-631): HSTKRGHAKSRPVCitG (p18), [Cit⁷¹]Vim(47-72): STSRSLYASSPGGVYATRSSAVRLCitS (p48), [Cit^64,69,71]Vim(47-72): STSRSLYASSPGGVYATCitSSAVCitLCitS (p55), and [Cys^4,22,Cit^9,15]Enolase(5-21): CKIHACitEIFDSCitGNPTVEC (CEP-1), and uncitrullinated versions α-fibrin(617-631): HSTKRGHAKSRPVRG, Vim(47-72): STSRSLYASSPGGVYATRSSAVRLRS, and Enolase(5-21): CKIHAREIFDSRGNPTVEC were synthesized by solid-phase peptide synthesis as C-terminal carboxamides on a Novasyn TGR resin (Novabiochem Merck, Germany) (0.22 meq/g) and following a 9-fluorenylmethoxycarbonyl (Fmoc) strategy. Couplings were performed by 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIEA) activation, with three-fold molar excesses of amino acids. The Fmoc deprotection step was performed twice with 20% piperidine in dimethylformamide (DMF) for 10 min. The peptides were concomitantly side chain-deprotected and cleaved from the resin by treatment with a mixture of TFA in the presence of triisopropylsilane (TIS) and water as scavengers (TFA:TIS:H₂O, 9.5:2.5:2.5) for 3 h with occasional agitation at room temperature. The solvent was removed in vacuum and the crude peptides were precipitated with diethyl ether. The solids were dissolved in 30% acetic acid in water and lyophilized.

Crude peptides were purified by semipreparative HPLC (1260 Infinity, Agilent Technologies) in an XBridge™ Prep BEH130 C18 column (Waters, 5 μm, 10 × 250 mm) at a flow rate of 3.5 mL/min. Final peptides were characterized by analytical HPLC on a 1260 Infinity chromatograph (Agilent Technologies) with an Eclipse Plus C18 column (Agilent, 3.5 μm, 4.6 × 100 mm). The peptides were 95% pure by analytical HPLC at 220 nm. Their identity was confirmed by electrospray ionization mass spectrometry (ESI-MS) performed with a liquid chromatograph–time of flight (LC-TOF) detector, LCT Premier XE (Micromass Waters) coupled to Analytical Ultra Performance Liquid Chromatography apparatus (UPLC, Waters).

For cyclization of CEP-1, its linear version was dissolved in 0.1 M ammonium bicarbonate (0.3 mg/mL). The solution was left to stand open to the atmosphere and stirred for 24 h. The formation of cyclic disulfide was checked by the Ellman test.

Peptide sequences were coupled covalently to ELISA microplates (Costar Corp., DNA-bind N-oxysuccinimide surface, Cambridge, MA, USA). Peptides were diluted to 10 μg/mL in 0.05 M carbonate/bicarbonate (pH 9.6) buffer; 100 μL of peptide solution was added to each well of microplates and incubated overnight at 4 °C. Each plate contained control wells that included all reagents except the serum sample in order to estimate the background reading and control wells that included all reagents except the peptide to evaluate nonspecific reactions of sera. For blank controls, wells were coupled with 2 μg bovine serum albuin (BSA)/well. After incubation, the plates were blocked with 2% BSA in 0.05 M carbonate/bicarbonate (pH 9.6) buffer for 1 h at room temperature. Sera were diluted 50-fold in RIA buffer (1% BSA, 350 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1% vol/vol Triton X-100, 0.5% wt/vol Na-deoxycholate, 0.1% SDS) supplemented with 10% fetal bovine serum; 100 μL/well were added and incubated for 1.5 h at room temperature. After washing six times with phosphate-buffered saline (PBS)/0.05% Tween-20, 100 μL/well of the anti-human secondary antibodies conjugated to peroxidase at different dilutions in RIA buffer (IgG 1:1000, IgA 1:2000, and IgM 1:40,000) were added. After incubation for 1 h at room temperature, the plates were washed six times with PBS/0.05% Tween-20 and bound antibodies were detected with o-phenylenediamine dihydrochloride (OPD; Sigma Chemical Company) and 0.8 μL/mL 30% hydrogen peroxide. The plates were incubated at room temperature for 30 min. The reaction was stopped with 50 μL of 2 N H₂SO₄ per well and absorbance values were measured at a wavelength of 492 nm.

All sera were tested in duplicate. Control sera were also included to monitor inter- and intra-assay variations.

Microtiter plates coated with uncitrullinated peptides were used as a control for citrulline specificity of the anti-citrulline peptide antibodies. The OD values on the arginine variants were clearly bellow the citrulline respective peptide cut-off values. The number of patients who recognized both the citrullinated and the arginine-containing peptide with similar OD values, and thus are considered not to bind to the respective peptide in a citrulline-specific way, was very small. In this sense, only one serum of RA (4.7% of the 21 IgG anti-CEP-1-positive) and another one RA serum (3.6% of the 28 IgA anti-p18-positive sera) reacted against the respective arginine control peptides of p18 and CEP-1. Besides, for the PR sera included in this study, only one serum (5.9% of the 17 IgG anti-CEP-1-positive sera) reacted against the uncitrullinated CEP-1 peptide. These three patients were considered negative for the presence of IgG and IgA anti-CEP-1 and anti-p18 antibodies because no specific response was detectable.

Continuous data are presented as means and standard deviation and categorical variables as absolute frequencies and percentages. Differences in demographic, clinical, and ACPA fine specificities and isotypes were compared between PR and RA patients. For continuous variables, we used the Student’s or Mann-Whitney tests. For binary variables, we used the Chi-square or Fisher’s exact tests, when appropriate. Differences in reactivity of OD values between PR and RA patients were analyzed using the Mann-Whitney test. We analyzed anti-CCP2 reactivity levels according to the number of IgG specificities using Spearman’s correlation test. The level of statistical significance was established as ≤0.05. The analysis was made using R version 3.2.0 (copyright ©2015, The R Foundation for Statistical Computing).

We included 54 PR patients and 54 RA patients (62.9% female, 66.7% CCP2-positive in both groups). There were no significant between-group differences in mean age, disease duration, and RF positivity (Table 1). The demographic and clinical characteristics of patients with PR are described elsewhere [18].

PR patients had a lower frequency of some IgG ACPA fine specificities than RA patients, which was significant for the two vimentin-derived peptides, p48 vimentin (1.9% RP vs. 14.8% AR, p = 0.03) and, especially, p55 vimentin (PR 24.1% vs. 59.3% RA; p < 0.001) (Table 2). The mean number of additional ACPA specificities (p48 and p55 were grouped into one) was lower in PR patients (0.91 ± 0.96 vs. 1.43 ± 1.03; p = 0.008). Only 25.9% of PR patients recognized ≥2 additional IgG ACPA specificities compared with 46.3% of RA patients (p = 0.028) (Table 3).

Significantly higher levels of IgG ACPA fine specificities were found in RA patients compared with PR patients, except for the enolase peptide, when all samples were considered; however, no significant differences were found when negative samples were excluded except for IgG enolase, which was higher in PR (Fig. 1a, b, c, and d). Comparison of the 18 CCP2-negative patients in each group showed that p48/p55 vimentin was lower in PR patients (5.6% vs. 38.9%; p = 0.04) and that PR patients had a lower mean number of positive specificities (0.17 ± 0.5 PR vs. 0.56 ± 0.6 RA; p = 0.04).

CCP2 antibody levels correlated with the number of citrullinated epitopes recognized by ACPA in RA, but to a lesser extent in PR (Fig. 2).

PR patients had a lower frequency of IgA and IgM ACPA isotypes, which was significant in the case of IgA and IgM against fibrin p18 and IgA against vimentin p55 (Table 2). Mean levels of fibrin p18 IgA and IgM isotypes, enolase CEP1 IgM, and p55 vimentin IgA were lower in PR (Fig. 1e, f, g, h, i, and j). The mean number of IgA (0.37 ± 0.7 vs. 0.96 ± 0.93; p < 0.001) and IgM (0.22 ± 0.46 vs. 0.44 ± 0.6; p = 0.04) ACPA isotypes was lower in PR than in RA patients.