Objective: To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA).Methods: The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis–knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α‐enolase by P gingivalis was studied by incubating live wild‐type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry.Results: Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild‐type P gingivalis with fibrinogen or α‐enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy‐terminal arginine residues, which were identified by mass spectrometry.Conclusion: Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg‐X peptide bonds by arginine gingipains, followed by citrullination of carboxy‐terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis–mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.

Objective: To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated α‐enolase, in rheumatoid arthritis (RA).Methods: Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, α‐enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD‐2) and PAD‐4. Proteins from an RA synovial fluid sample were separated by 2‐dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated α‐enolase peptide 1 (CEP‐1) and cyclic citrullinated peptide 2 were measured by enzyme‐linked immunosorbent assay.Results: Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha‐enolase was detected in all of the samples, with mean levels of 6.4 ng/μl in RA samples, 4.3 ng/μl in SpA samples, and <0.9 ng/μl in OA samples. Two‐dimensional electrophoresis provided evidence that the α‐enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD‐4 was detected in samples from all 3 disease groups. PAD‐2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP‐1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample.Conclusion: These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Objective To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles. Methods Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE–transgenic mice and control mice (class II major histocompatibility complex–deficient [class II MHC^−/−] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA. Results Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE–transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC^−/− control mice. Conclusion This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti–CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA.

Mixed connective tissue disease (MCTD) was first described in 1972 as a disease syndrome with overlapping features of systemic sclerosis, systemic lupus erythematosus (SLE) and polymyositis associated with antibodies to RNAse sensitive extractable nuclear antigen. When the antigen was subsequently characterized as polypeptides on the U1 ribonuclear protein component of the splicesosome (U1RNP), MCTD became the first rheumatic disease syndrome to be defined by a serologic test. Clinical features include a high frequency of Raynaud’s syndrome, swollen hands, sclerodactyly, arthritis, polymyositis and interstitial lung disease. Over the last 30 years there has been a continuing debate as to whether MCTD constitutes a ‘distinct clinical entity’. Here, I will review the pathological, immunogenetic and clinical features of MCTD and conclude that the debate remains unresolved. The early misconception that it has a relatively good prognosis has not stood the test of time with long-term follow-up studies. These have identified a tendency for MCTD to evolve into SLE or systemic sclerosis and highlighted pulmonary hypertension and scleroderma renal crisis as important causes of death. Providing it is realized that our appreciation of the clinical features associated with anti-U1RNP have evolved over time, MCTD remains a useful concept in clinical practice. Whether it can be credited with the term ‘disease’ awaits the demonstration of common etiopathological events underlying the development of antibodies to U1 RNP and their associated clinical features.