Antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies.

Evan Reed,A Jimmy Ytterberg,Xia Jiang,Monika Hansson,Lars Alfredsson,Nastya Kharlamova,Lena Israelsson,Johan Rönnelid,Linda Mathsson-Alm,Karin Lundberg,Anca I Catrina

doi:10.1186/s13075-016-1001-6

Abstract

BackgroundIn addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e., homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). However, the extent to which anti-CarP antibodies are truly distinct from ACPA remains unclear, and few studies have focused on specific autoantigens. Here, we examine cross-reactivity between ACPA and anti-CarP antibodies, in the context of the candidate autoantigen α-enolase.MethodsCross-reactivity was examined by immunoblotting of citrullinated and carbamylated proteins using purified ACPA; and by peptide absorption experiments, using the citrullinated α-enolase peptide CEP-1 and a homocitrulline-containing version (carb-CEP-1) in ELISA. The population-based case-control cohort EIRA (n = 2836 RA; 373 controls) was screened for reactivity with CEP-1 and carb-CEP-1, using the ISAC multiplex array. Associations between anti-CarP antibodies, smoking and genetic risk factors were analysed using unconditional logistic regression models. Differences in antibody levels were investigated using the Mann-Whitney U test.ResultsAffinity-purified ACPA was found to bind carbamylated proteins and homocitrulline-containing peptides, demonstrating definitive cross-reactivity between ACPA and anti-CarP antibodies. Anti-carb-CEP-1 reactivity in EIRA was almost exclusively confined to the CEP-1-positive subset, and this group of RA patients (21 %) displayed a particularly strong ACPA response with marked epitope spreading. The small RA subset (3 %) with homocitrulline reactivity in the absence of citrulline reactivity did not associate with smoking or risk genes, and importantly had significantly lower anti-carb-CEP-1 antibody levels.ConclusionOur data presented herein cast doubt on the specificity of anti-CarP antibodies in RA, which we posit may be a subset of cross-reactive ACPA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1001-6) contains supplementary material, which is available to authorized users.

Highlights

In addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated proteins have been described in rheumatoid arthritis (RA)
Purified ACPA immunoglobulin G (IgG) bind carbamylated proteins Using a pool of affinity-purified anti-anti-cyclic citrullinated peptide (CCP2) IgG, previously described to bind both citrullinated α-enolase and fibrinogen [36], we could demonstrate cross-reactivity of human ACPA with carbamylated α-enolase for the first time, and in line with previous reports [20, 24], we could show cross-reactivity with carbamylated fibrinogen (Fig. 1a)
Purified anti-the immunodominant peptide epitope of citrullinated alpha-enolase (CEP-1) IgG displays cross-reactivity with a homocitulline-containing version of CEP-1 To further investigate the specificity and extent of crossreactivity between citrullinated and carbamylated epitopes, we focused on α-enolase and the immunodominant CEP-1 epitope

Summary

Introduction

In addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e., homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). Autoantibodies to citrullinated proteins (ACPA) are today a well-known and accepted feature of rheumatoid arthritis (RA) [1, 2]. These autoantibodies have been linked to RA risk factors, most notably HLA-DRB1 shared epitope (SE) alleles and cigarette smoking, and their presence predicts a more destructive disease process [3,4,5,6,7]. Antibodies to carbamylated proteins containing homocitrulline (anti-CarP antibodies) were described in RA [13]. There was no specific association between HLA-DRB1 SE or smoking and anti-CarP antibodies, when the analyses were adjusted for the presence of ACPA [21]

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Discussion

Conclusion