Objective Protein citrullination is an important posttranslational modification recognized by rheumatoid arthritis (RA)–specific autoantibodies. One of the citrullinating enzymes, peptidyl arginine deiminase type 4 (PAD‐4), is genetically associated with development of RA in some populations, although the mechanism(s) mediating this effect are not yet clear. There have been descriptions of anti–PAD‐4 autoantibodies in different rheumatic diseases. This study was undertaken to investigate whether anti–PAD‐4 antibodies are specific to RA, are associated with disease phenotype or severity, and whether PAD‐4 polymorphisms influence the anti–PAD‐4 autoantibody response. Methods Sera from patients with established RA, patients with other rheumatic diseases, and healthy adults were assayed for anti–PAD‐4 autoantibodies by immunoprecipitation of in vitro–translated PAD‐4. The epitope(s) recognized by PAD‐4 autoantibodies were mapped using various PAD‐4 truncations. PAD‐4 genotyping was performed on RA patients with the TaqMan assay. Joint erosions were scored from hand and foot radiographs using the Sharp/van der Heijde method. Results PAD‐4 autoantibodies were found in 36–42% of RA patients, and were very infrequent in controls. Recognition by anti–PAD‐4 autoantibodies required the 119 N‐terminal amino acids, which encompass the 3 nonsynonymous polymorphisms associated with disease susceptibility. Strikingly, the anti–PAD‐4 immune response was associated with the RA susceptibility haplotype of PADI4. Anti–PAD‐4 antibodies were associated with more severe joint destruction in RA. Conclusion Our findings indicate that anti–PAD‐4 antibodies are specific markers of RA, independently associated with more severe disease, suggesting that an anti–PAD‐4 immune response may be involved in pathways of joint damage in this disease. Polymorphisms in the PADI4 gene influence the immune response to the PAD‐4 protein, potentially contributing to disease propagation.

Objective. Arthritis in the K/BxN mouse model results from pathogenic immunoglobulins that recognize glucose-6-phosphate isomerase (GPI), a glycolytic enzyme residing in the cytoplasm of all cells. Antibodies directed against GPI can, alone, transfer arthritis to healthy recipients. Previous experiments have revealed significant titers of anti-GPI antibodies in the serum of many patients with rheumatoid arthritis (RA). We evaluated the generality of these observations in cohorts of patients with 12 different arthritic and chronic autoimmune diseases and in population-matched healthy control subjects.Methods. Anti-GPI antibodies were assayed in 811 individual serum samples by enzyme-linked immunosorbent assay with 2 forms of GPI, recombinant and native. Results were confirmed by immunoblotting.Results. Several patients had significantly elevated anti-GPI antibody titers, but without the prevalence or the specificity reported previously. Only 15% of RA patients had anti-GPI antibodies (range 12-29% in different cohorts), with a higher prevalence in patients with active disease. Psoriatic arthritis, undifferentiated arthritis, and spondylarthropathy patients also displayed anti-GPI antibodies at similar frequencies (12-25%). Similar titers were detected in a proportion (5-10%) of control subjects or patients with Crohn's disease or sarcoidosis. Very high titers were found in rare cases of RA and systemic lupus erythematosus.Conclusion. No disease-specific pattern of antibody positivity to GPI was apparent. While the antibody-mediated mechanism at play in the mouse model may exemplify a generic mechanism for some forms of arthritis in humans, GPI itself does not appear to be a target common to the majority of RA patients.

Objective To determine the incidence of inflammatory arthritis and autoantibody prevalence in Indigenous North American people. Methods Unaffected relatives of Indigenous North Americans with rheumatoid arthritis (RA) from central Canada and Alaska were systematically monitored from 2005 to 2017. Rheumatoid factor (RF) and anti–citrullinated protein antibodies (ACPAs) were tested at every visit, and a subset was tested for ACPA fine specificity using a custom multiplex assay. Multistate models based on all available study visits were developed to determine the likelihood of transitioning between autoantibody states, or to inflammatory arthritis. Results Eighteen of 374 relatives (4.8%) developed inflammatory arthritis during follow‐up (after a mean ± SD of 4.7 ± 2.4 years), yielding a transition rate of 9.2 cases/1,000 person‐years. Thirty percent of those who developed inflammatory arthritis were seronegative at baseline, but all were seropositive at inflammatory arthritis onset. Although 30% of ACPA/RF double‐seropositive individuals developed inflammatory arthritis (after 3.2 ± 2.2 years), the majority of these individuals did not develop inflammatory arthritis. Multistate modeling indicated a 71% and 68% likelihood of ACPA and RF seropositive states, respectively, reverting to a seronegative state after 5 years, and a 39% likelihood of an ACPA/RF double‐seropositive state becoming seronegative. Fine specificity testing demonstrated an expansion of the ACPA repertoire prior to the development of inflammatory arthritis. Conclusion Despite a high incidence of inflammatory arthritis in this cohort of at‐risk relatives of Indigenous North Americans with RA, a large proportion of autoantibody‐positive individuals do not develop inflammatory arthritis and revert back to an autoantibody‐negative state.

Objective Anti–citrullinated protein antibodies (ACPAs) are disease‐specific biomarkers in rheumatoid arthritis (RA). More than 90% of IgG ACPAs harbor N‐linked glycans in the antibody variable (V) domain. The corresponding N‐glycosylation sites in ACPA V‐region sequences result from somatic hypermutation, a T cell–dependent process. As ample evidence indicates that T cells drive the maturation of the ACPA response prior to arthritis onset, we undertook this study to investigate whether the presence of glycans in IgG ACPA V domains predicts the transition from predisease autoimmunity to overt RA. Methods We analyzed 2 independent sets of serum samples obtained from 126 ACPA‐positive first‐degree relatives (FDRs) of RA patients. Both sets originated from an Indigenous North American population and comprised cross‐sectional and longitudinal samples of individuals who did or did not develop inflammatory arthritis. Serum IgG ACPAs were affinity‐purified and subjected to ultra high‐performance liquid chromatography–based glycan analysis. Results In both data sets, FDR‐derived IgG ACPA displayed markedly lower levels of V domain glycans (<50%) compared to IgG ACPA from RA patients. Notably, FDRs who later developed RA showed extensive V‐domain glycosylation before the onset of arthritis. Moreover, IgG ACPA V‐domain glycosylation was strongly associated with future development of RA (hazard ratio 6.07 [95% confidence interval 1.46–25.2]; P = 0.013). Conclusion Extensive glycosylation of the IgG ACPA V domain is present in a subset of predisposed FDRs of Indigenous North American RA patients. The presence of this feature substantially increases the risk of RA development. Based on these findings, we propose that glycosylation of the IgG ACPA V domain represents a predictive marker for RA development in ACPA‐positive individuals and may serve to better target prevention measures.