Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359–369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising β-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.

Little is known about the genetic diversity of pale flax (Linum bienne Mill.), the wild progenitor of cultivated flax (L. usitatissimum L.), and ex situ germplasm of pale flax was scarce. Effort was made to collect 34 pale flax accessions and five landrace accessions of cultivated flax in Turkey. The inter simple sequence repeat (ISSR) technique was applied to characterize this set of flax germplasm, along with one Turkish cultivar, one Russian cultivar, five winter and four dehiscent type accessions of cultivated flax. Twenty-four ISSR primer pairs detected a total of 311 DNA fragments, of which 298 bands were polymorphic across 493 flax samples (roughly 10 samples per accession). These polymorphic bands had frequencies ranging from 0.002 to 0.998 and averaging 0.38. Accession-specific ISSR variation (Fst values) ranged from 0.469 to 0.514 and averaged 0.493. There was 49.3% ISSR variation resided among these 50 accessions, 35.9% harbored among landrace, winter, dehiscent types of cultivated flax and pale flax, and 38.2% present among 34 pale flax accessions. Pale flax displayed more ISSR variation than landraces and dehiscent type, but less than winter type, of cultivated flax. Clustering 493 individual plants revealed that these assayed plants were largely grouped according to their plant types and that pale flax was genetically more close to the dehiscent type, followed by the winter type and landrace, of cultivated flax. Pale flax collected within the geographic range of 180 km displayed a significant spatial genetic autocorrelation. Genetic distances among the pale flax accessions were significantly associated with their geographic distances and elevation differences. These findings are significant for understanding flax domestication and its primary gene pool.

The discovery of antibodies specific for citrullinated protein epitopes [anti-citrullinated protein antibodies (ACPAs)] is a hallmark for the diagnosis and prognosis of rheumatoid arthritis (RA) and will also be a useful tool for understanding the fundamental pathologic processes. There are several essential questions pertaining to ACPA that remain to be explored, such as understanding the early specificity of the underlying T-cell recognition, whether the production of ACPA is a primary or secondary process, and in the event of such antibodies being arthritogenic, whether they could possibly regulate the disease development. To answer these questions, animal models are needed, but unfortunately ACPA is not a prominent feature of any of the classical animal models of RA. However, we showed recently that ACPA can be isolated from animals susceptible to collagen-induced arthritis that are specific for citrullinated type II collagen (CII). The citrulline specificity could be visualized, and the specificity is determined primarily by a direct interaction with citrulline. We also demonstrated that these antibodies are specific for the citrullinated epitopes and are pathogenic in vivo. A new hypothesis to explain how inflammation in RA can be directed to cartilaginous joints and be self-perpetuating is suggested, which involves recognition of post-translational modifications (glycosylation and citrullination) on CII by T and B cells that can have both arthritogenic and regulatory consequences.

IntroductionAntibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA.MethodsAnalysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice.ResultsMany CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group.ConclusionCII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation.