Mutated citrullinated vimentin antibodies in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is the most common inflammatory systemic autoimmune disease, primarily affecting the peripheral joints. The past decade has been marked with revolutionary changes both in the therapeutic and diagnostic perspectives of RA. The discovery of an RA-specific citrullination-driven immune reaction gave a substantial contribution in the diagnostic approach to RA. Efforts directed towards the identification of the antigenic target specifically recognized by these autoantibodies resulted in the identification of vimentin in citrullinated form as the potential native antigen, among other proteins. Furthermore, it was found that the mutation of vimentin represents an independent trigger of antigenic properties, in addition to citrullination. As a result of this discovery, a commercial ELISA using mutated citrullinated vimentin (MCV) was developed. Increasingly, data now support the use of anti-MCV in RA diagnosis and prognosis for errosion. This review summarizes the research to date on the use of anti-MCV in RA diagnosis and prognosis and its potential use as a therapeutic marker. The pathologic role of these antibodies in RA disease is also discussed.

Introduction

Rheumatoid arthritis (RA) is the most common chronic, inflammatory autoimmune disease with a frequency of 0.5–1.0% in the adult population of developed countries. It is marked by chronic synovial inflammation and associated damage to articular cartilage and underlying bone, leading to substantial disability. Due to the systemic nature of the disease, different comorbidities develop throughout the course of the disease, contributing to the overall impaired quality of life. The past decade has brought several revolutionary changes in the therapeutic approach to RA, based on the improved understanding of the joint inflammation pathogenic mechanism, the hallmark of RA. The new approach promotes early aggressive therapy with synthetic disease-modifying anti-rheumatic drugs (DMARDs) and also the introduction of biological agents that target specific molecules and pathways in the pathogenesis of RA [1], [2], [3]. Bearing in mind the potential toxic effects vs. the benefit of such aggressive therapy, reliable early diagnosis of RA is of crucial importance. The disease course can follow two distinct clinical phenotypes characterized by mild disease activity, less joint destruction and fewer comorbidities or high disease activity, more joint destruction and higher comorbidities. Therefore, it is important to select patients who would benefit most from aggressive treatment in the early phase of disease [1], [4]. In light of the advanced demands of RA management, the necessity for a reliable early diagnostic and prognostic marker is more pronounced than ever. Historically, rheumatoid factor (RF), an antibody directed to the Fc fragment of human immunoglobulin G, has been used as an RA serological marker. RF is present in 60–80% of RA patients but has inferior specificity due to its presence in other autoimmune diseases (in up to 70% in primary Sjögren syndrome), and a number of other non-autoimmune conditions, such as infectious diseases. It is also present in 3–5% of the healthy population, with a rising trend among the elderly [5], [6], [7]. A true breakthrough in the RA serologic profile was the identification of amino acid citrulline as the main compound of the antigenic determinant recognized by RA specific autoantibodies [8]. Some members of the family of anti-citrullinated peptide/protein antibodies (ACPA) have long been identified in the serum of RA patients, including antikeratin antibodies (AKA) and antiperinuclear factor (APF) or anti-Sa antibodies, though insufficient knowledge of the structure of the antigenic determinants resulted in tests with unsatisfactory diagnostic characteristics [9], [10], [11]. The use of synthetic cyclic citrullinated peptides (CCP) that mimic RA-specific epitopes improved the diagnostic performances of immunological tests for ACPA detection. Currently, the second generation anti-cyclic citrullinated peptide test (anti-CCP2) has proven to be the most sensitive assay (about 80%, depending on the disease phase), which is comparable to the sensitivity of RF, but with significantly higher specificity (> 95%) [12], [13], [14]. Numerous studies have confirmed the correlation of the presence of this antibody with erosive progression of RA, indicating the high prognostic value of anti-CCP2 test for RA development [15], [16], [17], [18]. Due to the high clinical potential of ACPA, this biomarker was deservedly included in the new RA classification criteria released based on collaborative efforts between the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) [19].

Despite the obvious improvement in RA diagnosis and management stemming from the discovery of a specific immunological response to citrullinated proteins, the pathophysiological significance of anticitrulline immunity and identity of the native citrullinated protein as the target antigen remain largely unknown. The local ACPA production demonstrated in inflamed RA joints strongly suggests that citrullinated proteins present in the inflamed synovium are involved in anticitrulline immunity [20]. Among candidate proteins abundantly present in the inflamed synovium, studies have been conducted on fibrin, fibrinogen, alpha-enolase (originated from monocyte and granulocyte) collagen types I and II, vimentin, fibronectin and Epstein-Barr nuclear antigen 1 (EBNA-1) [20], [21], [22], [23], [24], [25], [26]. ACPA targeting these citrullinated proteins is also detectable in early RA and can predict radiological progression.

This review focuses on the antibodies targeting citrullinated vimentin, and addresses their usefulness for RA diagnosis and prognosis in comparison to the established anti-CCP2 test.

The history of citrullinated vimentin as an RA specific antigenic target began nearly two decades ago, when Despres et al.[27] described new antibodies in the serum of RA patients, which they named Sa antibodies after the surname of the French Canadian patient (Savoie) in whom it was first identified. It was shown that these antibodies recognise protein bands of approximately 50 kDa on the western blots of extracts from human spleen, placenta and, mostly important, rheumatoid synovial tissue. Using an immunoblotting method with purified Sa antigen from human placenta extracts, Sa antibodies were detected in 20–25% of early RA cohorts and in up to 47% patients with the established disease. The specificity of this antibody was exceptional, ranging from 92 to 100%. It was shown that they appear early in the course of the disease and identify patients designated to have the aggressive, destructive form of RA [28], [29]. Since the anti-Sa antibodies were shown to be present in the rheumatoid synovium, the molecular nature of the Sa antigen and its potential pathogenic role in articular inflammation has become a challenging goal.

In 2004, Vossenaar et al.[30]. identified citrullinated forms of vimentin as the major antigen recognized by sera reacting to Sa. Firstly, they obtained several peptide sequences from highly purified preparations of the Sa antigen that were unique to the intermediate filament vimetin. Intermediate filaments are a large and structurally diverse group of cellular filaments that form the major structural component of the cytoskeleton of the eukaryotic cell. Along with actin microfilaments and microtubules, they form a supportive network responsible for the mechanical integrity of the cell. This network is also a dynamic structure involved in various processes, such as division and motility [31]. Vimentin is the elementary building compound of the intermediate filaments contained in the cells of mesenchymal origin. Within the synovial tissue, two types of cells rich in vimentin intermediate filaments, fibroblast-like and macrophage-like synoviocytes, play an active role in the main RA-pathophysiological features: inflammation, hyperplasia and joint destruction [32]. It is also known that intermediate filaments containing vimentin are important for the regulation of mechanical stress between chondrocytes and the surrounding matrix tissue [33]. Therefore, vimentin is an abundantly present protein in the joint microenvironment. In the next step of their study, Vossenaar et al [30]. used recombinant human vimentin to obtain IgG antivimentin antibodies in a subset of RA patients, irrespective of their anti-Sa status, and found a strong concordance between their anti-Sa and anti-CCP status. These results suggested that anti-Sa antibodies might be reactive with citrullinated epitopes. As it is known that autoantibodies can be directed towards proteins that are modified during cell death and in particular during apoptosis, the question arose as to whether citrullinated vimentin is the true nature of the Sa antigen. Citrullination of proteins results from the calcium dependent post-translational deimination of arginine residues catalysed by the enzyme peptidylarginine deiminase (PAD), which exists in several isotypes. Isotypes PAD2 and PAD4 are present in macrophages, while monocytes contain only PAD2 . Under normal physiological conditions, the cytosolic and nucleoplasmic calcium concentrations are too low for activation of this enzyme. During apoptosis (and necrosis) of the cell, the plasma membrane calcium pump is cleaved by caspases and the influx of calcium can no longer be efficiently cleared. It was shown that vimentin is citrullinated in macrophages during apoptosis, most likely by PAD2 located in the cytosol. Citrullination of the amino terminal domain of vimentin induces the disassembly of vimentin filaments, and causes the disruption and collapse of the filament network into perinuclear agregates [34], [35]. In RA, 80–100% of the synovial lining cells are macrophage-like synoviocytes (compared to 20–30% in healthy synovium) with an activated phenotype due to the pro-inflammatory microenvironment. Activated macrophages are very prone to cell death. The abundance of macrophages in the inflamed synovium is further supported by constant recruitment of circulating monocytes into the synovium, where they differentiate into macrophages. Therefore, the likely presence of citrullinated vimentin in the rheumatoid synovium makes it a potential target for anti-Sa antibodies. This was the leading idea in the further investigation of Vossenaar et al. in identifying the Sa antigen. Through immunoblotting and immunoprecipitation experiments, they succeeded in providing evidence that citrulline is essential for vimentin antigenicity toward anti-Sa antibodies and confirmed that the Sa antigen is indeed citrullinated vimentin ^[30].

Despite the high RA-specificity of this new member of the growing ACPA family, the prevalence of anti-Sa antibodies was too low (40%) to improve the diagnosis of RA. In order to determine whether there are additional vimentin modifications that can influence its antigenicity, Bang et al [33]. investigated the expression of vimentin in synovial sites under pathological conditions. Their assumption of the existence of different isoforms of vimentin was based on the previous evidence of abundant microsatellite instability and suppression of the DNA repair mechanism in the RA synovium. As the representative pathological condition that could induce DNA damage, persistent oxidative stress in an RA typical synovial microinvironment was proposed. Using mass spectroscopy analysis of purified vimentin from a human fibroblast cell line and synovial fluid from RA patients, mutations of glycine to arginine residues within vimentin DNA were identified. Enhanced antibody reactivity was observed in the sera of RA patients against the mutated citrullinated vimentin, as compared to the citrullinated wild-type vimentin. Therefore, mutation of vimentin represents an independent trigger of antigenic properties, in addition to citrullination.

Encouraged by this discovery, the same authors used mutated and in vitro citrullinated recombinant human vimentin antigen (MCV) in the ELISA assay for quantitative measurement of IgG isotype autoantibodies against MCV in serum/plasma [33].