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Antibodies that target carbamylated proteins (anti-CarP antibodies) have been described as a biomarker in rheumatoid arthritis (RA).1 However, little is known about the factors that predispose to the production of anti-CarP antibodies. Carbamylation is a posttranslational modification resulting from the conversion of lysines into homocitrullines that requires the presence of cyanate. There are several conditions in which the concentration of cyanate (and therefore carbamylation) is increased, such as renal failure, chronic inflammation and heavy smoking.2–4 We therefore addressed the question whether conditions of enhanced carbamylation could result in the induction of anti-CarP antibodies.
To investigate this, we determined the presence of anti-CarP antibodies in serum samples from patients with renal failure,5 inflammatory bowel disease (IBD)6 and in heavy smokers with or without chronic obstructive pulmonary disease (COPD)7 (see table 1). The presence of anti-CarP antibodies in healthy controls and patients with RA was used as a comparison.1 ,8 The collection of these cohorts was approved by the Leiden University Medical Center ethics committee and informed consent was obtained from all patients. Anti-CarP IgG and IgA antibodies were determined by ELISA using carbamylated fetal calf serum as antigen.1 Positivity for the presence of anti-CarP antibodies was defined as signal higher than the 97th percentile of the healthy controls. Each cohort was tested separately for this study and control samples (between 120 and 187) were randomly selected from a pool of 209 controls and taken along with each measurement. The anti-CCP2 ELISA (anti-cyclic citrullinated peptide) was carried out as described before.9
The percentage of individuals positive for anti-CarP antibodies was 2.3%, 4.1%, 3.7%, 11.9% and 44.9% for controls, smokers (no differences were observed in anti-CarP antibodies between smokers with and without COPD) and patients with IBD, renal failure and RA, respectively (figure 1A). Pearson χ2 test analyses revealed that a statistically increased frequency of antibody positivity was found in RA (p=<0.001) and in patients suffering from renal failure (p=0.004), when compared with controls.
As a comparison to the presence of anti-CarP antibodies, a second autoantibody, targeting a structurally similar antigen, namely anti-CCP2, was measured. The percentages of individuals positive for anti-CCP2 antibodies were 1.5%, 1.9%, 0.5%, 2.4% and 50.5% for controls, smokers and patients with IBD, renal failure and RA, respectively (figure 1B). These data indicate that anti-CCP2 antibodies are present in patients with RA but hardly in any of the other conditions. While in patients with RA a certain extent of cross-reactivity between anti-CarP and anti-CCP2 antibodies can be observed, there was no overlap between these autoantibodies in renal failure, smoking or IBD (figure 1D). Therefore, the presence of a (low) percentage of anti-CarP IgG antibodies in the individuals analysed is not due to the presence of anti-CCP2 antibodies.
Both IBD and smoking might involve more carbamylation in the lungs or intestine, in which IgA antibodies are thought to be more prevalent than IgG antibodies. Therefore, anti-CarP IgA antibodies were determined as shown in figure 1C (2.7%, 7.8%, 4.6%, 7.9% and 43% for controls, smokers and patients with IBD, renal failure and RA, respectively). Compared with controls, positivity in patients with RA, renal failure and smoking was statistically significant. When correcting for multiple testing, only the difference between controls and patients with RA remained significant.
In summary, we observed an increase in anti-CarP antibodies in patients with RA and to a lesser extent in patients suffering from renal disease. Since an increase in anti-CarP antibodies was not detected in other conditions with enhanced carbamylation, we conclude that increased carbamylation alone is not sufficient for a break of tolerance against carbamylated proteins. Therefore, our data indicate that next to carbamylation, also other (genetic or environmental) factors are required for the induction of anti-CarP antibodies.
Contributors MKV, SJHE, EWNL, MJKM, JS, AEM, REMT and LAT contributed to the planning and design of the study, while MKV and EWNL are responsible for the (technical) execution. Analysis and interpretation were carried out by MKV, SJHE, DW, HWV, REMT, PSH, CK and LAT. All authors contributed to the writing of the manuscript.
Competing interests REMT and LAT are listed as inventors in a patent application regarding the detection of anti-CarP antibodies for RA.
Ethics approval Leiden University Medical Center ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.