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

Abstract

Objective: The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts.

Methods: Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis.

Results: We identified elevated anti-CarP antibodies 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 the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; 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 anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and α enolase.

Conclusion: In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP 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.

Keywords: AUTOANTIBODIES; CYCLIC CITRULLINATED PEPTIDE; RHEUMATOID ARTHRITIS; VIMENTIN.

Figure 1

Relationships of anti-carbamylated protein antibody (ACarP Ab) titer to seropositivity and anti-cyclic citrullinated peptide antibody (anti-CCP). Panels (A) and (B) represent the Dartmouth established RA cohort, and panels (C) and (D) represent the Sherbrooke EUPA cohort. A, C. Log-transformed ACarP Ab is higher in seropositive than in seronegative RA patients in both the Dartmouth cohort (seronegative: n = 45, mean (SEM) = 0.66 (0.08); seropositive: n = 167, mean (SEM) = 1.23 (0.05); p<0.0001) and the Sherbrooke cohort patients (seronegative: n = 165, mean (SEM) = 0.40 (0.05); seropositive: n = 171, mean (SEM) = 0.97 (0.05); p< 0.0001). B, D. Among seropositive samples, a positive correlation is observed between ACarP Ab titer and anti-CCP in both cohorts (p< 0.0001, p= 0.01, respectively). Diagonal lines represent lines of best fit.

Figure 2

ACarP antibody titer robustly correlates with anti-Sa Ab levels among seropositive RA patients in both the Dartmouth and Sherbrooke cohorts. A. Anti-Sa(+) among seropositive patients in the Dartmouth cohort also exhibit a significantly higher mean ACarP Ab level than anti-Sa(−) among seropositive patients (anti-Sa positive: n = 91, mean (SEM) = 1.51 (0.08); anti-Sa negative: n = 67, mean (SEM) = 0.93 (0.07); p < 0.0001). B. Anti-Sa(+) among seropositive patients in the Sherbrooke cohort have a significantly higher mean ACarP Ab level than anti-Sa(−) among seropositive patients (anti-Sa positive: n = 72, mean (95% CI) = 1.22 (1.15-1.29); anti-Sa negative: n=71, mean (95% CI) = 0.82 (0.74-0.90); p = 0.0002). C. Within the Dartmouth cohort, ACarP Ab is significantly correlated with anti-Sa antibody titer (n=158; p<0.0001). D. A similar relationship is seen with seropositive RA patients from the Sherbrooke cohort (n=169; p=0.0002).

Figure 3

Competition for anti-Sa reactivity. A. Immunoblot analysis of native FCS and carbamylated FCS using rabbit anti-carbamyl lysine antibody confirming extensive carbamylation. B. Five sera were tested for anti-Sa reactivity or (C) CarP reactivity in the absence or presence of varying (1:100 and 1:10) dilutions of carbamylated FCS (0.32-3.2 mg/ml).

Figure 4

Variability in reactivity to CarP, Sa antigen, citrullinated (cit-) fibrinogen, and cit-peptides in the Dartmouth Cohort. A. ACarP(+) samples, have a stronger relationship to cit-fibrinogen reactivity than in (B) AcarP(−) samples (p< 0.0001). There is no relationship between anti-Sa and cit-fibrinogen values either in the ACarP(+) samples (R= -0.08; p= 0.79; n=13) or in the ACarP(−) samples (R= −0.028; p= 0.91; n=18). C. Using a multiplex fluorescent bead array, antibody reactivity to citrullinated (cit-) vimentin 59-78, cit-fibrinogen-α 616-635, cit-fibrinogen-α 39-50, cit-fibrinogen-β 36-52, and cit-α-enolase 5-21 was measured in ACarP Ab(+) (n=14) and ACarP Ab(−) (n=30) samples. Values displayed represent AU/mL for CarP reactivity while the values for the peptides represent mean MFI (median fluorescence intensity) of reactivity to the citrullinated peptide minus the value obtained from native peptide (corrected value).