Anti-carbamylated Protein Antibody Levels Correlate with Anti-Sa (Citrullinated Vimentin) Antibody Levels in Rheumatoid Arthritis

Abstract

Objective The presence of anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein antibodies (ACarP Ab). We analyzed the clinical and serologic relationships of ACarP in two RA cohorts. Methods Circulating levels of IgG ACarP Ab were determined by ELISA in established (Dartmouth) and early (Sherbrooke) cohorts and evaluated for anti-CCP, specific ACPA, and rheumatoid factor (RF) levels using Student's t-test and correlation analysis. Results We identified elevated ACarP Ab titers in 47.0% of seropositive patients (Dartmouth, n=164), with relationships to anti-CCP (p<0.0001) and IgM-RF (p=0.001). Similarly, 38.2% of seropositive patients from Sherbrooke (n=171) had elevated ACarP Ab; titers correlated to anti-CCP (p=0.01) but not IgM-RF (p=0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa(+) patients were ACarP Ab(+) versus only 25.4% anti-Sa(−) (Sherbrooke; p=0.0002), and 62.6% anti-Sa(+) patients versus 26.9% anti-Sa(−) were ACarP Ab(+) (Dartmouth; p<0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and alpha enolase. Conclusion In two North American RA cohorts we observed a high prevalence of ACarP Ab positivity. We also describe a surprising and unexpected association of ACarP with anti-Sa Ab that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between ACarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

“…In a panel of RA sera enriched for ACPA and ACarPA seropositivity, we observed a lower frequency of anti-BPI autoantibodies in RA patients than previously reported in a serologically undefined RA patient population (53). In RA, there is a strong association of ACarPA with ACPA (24). In contrast, despite seeing ACarPA in CF patients, we observed a poor correlation between ACarPA and anti-BPI autoantibodies.…”

“…In contrast to anti-BPI responses, the lack of association between ACarPa and anti-Cif IgG levels suggests that ACarPA arise independently of infection and are most likely a marker of neutrophil-driven bronchial inflammation reported in other chronic diseases (24,68,69). Alternatively, it may be that ACarPA influence clinical phenotype by impairing the antibacterial activity on NETs.…”

“…Nickel-coated ELISA plates (ThermoFisher Scientific) were used to bind the 6X-His tagged CTBPI (10 μg/ ml), and ELISA was performed as described for nBPI. Anti-carbamylated protein IgG titers (ACarPA) in sera were evaluated by commercial ACarPA ELISA assay (Inova Diagnostics Inc.) as well by a previously described in-house ACarPA ELISA (24). Positive cutoff for the BPI ELISA assays was defined as mean + 2 SDs of the healthy donor cohort.…”

“…neutrophil enzymes that localize to NETs induce posttranslational modifications, such as citrullination and carbamylation, thus creating neoantigens that lead to anti-citrullinated protein autoantibodies (ACPA) and anti-carbamylated protein autoantibodies (ACarPA) (22)(23)(24)(25)(26). Given the abundance of nucleic acids in NETs, the induction of autoantibodies by NETosis is likely to be facilitated by TLR 7/9-mediated B cell activation (27).…”

Carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis

“…This is in agreement with a recently described association of anti-carbP and anti-citrVim that could not be explained by cross-reactivity. 33 Of note, a treatment-related reduction of antibody titres was less obvious for anti-carbamylated antibodies compared with all other tested antibodies. This indicates that anti-carbamylated antibodies are produced by a distinct B-cell subset, which appears to be more treatment-resistant.…”