Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins.

Xiaobo Meng,Charles N Bernstein,Irene Smolik,Hani S El-Gabalawy,Carol Ann Hitchon,Peyman Ezzati,Salvatore V Pizzo

doi:10.1371/journal.pone.0165501

Abstract

Anti-citrullinated protein antibodies (ACPA) have become an integral part of the clinical definition of rheumatoid arthritis, and are hypothesized to be important in the immunopathogenesis of this autoimmune disease. Several citrullinated proteins have been demonstrated to serve as candidate autoantigens for the ACPA, based on in vitro immune reactions between citrullinated peptides/proteins and RA sera. Yet it remains unclear whether the autoantigens identified in vitro are indeed directly and specifically targeted by the ACPA in vivo. Moreover, it is unclear whether ACPA present in RA sera are directed towards the same spectrum of autoantigens as the ACPA present within the synovial compartment. In this study, we isolated ACPA immune complexes from RA synovial fluids (SF) and sera by using immobilized cyclic citrullinated peptides (CCP3) based immune affinity, and characterized the proteins that are directly and specifically associated with them by mass spectrometry. The results demonstrate that four histone proteins are prominent ACPA autoantigens, with the frequency of detection being histone H4 (89%), H2B (63%), H3 (63%), and H2A (58%) in ACPA positive RA SF. We further demonstrate that a histone 4 peptide containing citrulline at position Cit39 was recognized by 100% of ACPA positive RA SF. An adjacent citrulline residue at Cit40 was recognized by 34% of ACPA positive RA SF. An H4 peptide containing Cit39-40 was recognized in the serum of 94% ACPA positive RA, 77% ACPA positive first-degree relatives (FDR) of RA patients, and 2.5% of healthy controls. The Cit39-40 peptide substantially blocked the ACPA reactivity in both SF and serum. Although the spectrum of ACPA we identified was limited to those isolated using immobilized CCP3 peptides, the findings indicate that H4 is a widely recognized RA autoantigen in both the synovial and serum compartments. The identification of this immunodominant ACPA epitope may be valuable in designing approaches to immune tolerance induction in RA.

Highlights

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease in which autoimmunity is felt to play an important role in its initiation and pathogenesis [1, 2]
In analyzing the IC that were isolated from Anti-citrullinated protein antibodies (ACPA) negative synovial fluids (SF) samples using either CCP3 ELISA or protein-G, the proteins identified by mass spectrometry were considered to be non-antigenic (S3 Table and S4 Table, respectively)
We used proteomic techniques in an attempt to identify in vivo citrullinated autoantigens that were binding directly and to ACPA in RA synovial fluids, and in serum

Summary

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease in which autoimmunity is felt to play an important role in its initiation and pathogenesis [1, 2]. Seminal studies performed almost 20 years ago demonstrated that autoantibodies directed towards citrullinated proteins and peptides are detectable in the majority of RA patients, there appeared to be considerable heterogeneity in the antigens recognized [3]. These autoantibodies, referred to as ACPA, have been shown to have a high degree of specificity and sensitivity in multiple patient cohorts worldwide, and are valuable biomarkers for RA diagnosis and prognosis. To optimize the CCP based ACPA assay, a second (CCP2) and third (CCP3) generation of proprietary CCP were designed and mass produced by the bio-industry These substantially increased the sensitivity and specificity of the assay for RA diagnosis. The CCP based assays are considered the gold standard for detecting ACPA, and have been incorporated into 2010 ACR/EULAR RA classification criteria [13, 14]

Methods

Results

Conclusion