Introduction
Presence of anti-citrullinated protein antibodies (ACPA) is a hallmark of rheumatoid arthritis (RA), and together with rheumatoid factor (RF), part of the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria [
1]. Patients positive for ACPA and/or RF may be referred to as “seropositive” and make up approximately two thirds of the RA population. Seropositive and seronegative RA seem to have disparate mechanisms in predisposition, since HLA-DRB1 shared epitope (SE) alleles, as well as a number of other genes, associate primarily with ACPA-positive RA [
2,
3], while other genes have been linked to ACPA-negative disease [
4], suggesting that these subsets are partly separate disease entities.
While typically considered having a less inflammatory and less destructive form of RA [
5], seronegative patients require more clinical symptoms to be classified as having RA according to the 2010 ACR/EULAR criteria [
1], compared to seropositive patients, and may consequently be diagnosed later [
6]. Since all patients benefit from early treatment, early diagnosis is critical also for seronegative patients [
7‐
9]. A major obstacle to this is the lack of biomarkers.
ACPA status is commonly determined using assays based on cyclic citrullinated peptides (CCP), such as the “second generation” CCP2 test. However, these tests utilise synthetic peptides as surrogate markers for in vivo citrullinated antigens, while recent years have seen a number of studies describing ACPA targets on defined proteins (e.g. citrullinated fibrinogen [
10], vimentin [
11], collagen type II (CII) [
12], α-enolase [
13], histones [
14], and tenascin C [
15]). Since the CCP2 test does not capture all ACPA, multiplex assays capable of detecting multiple ACPA fine-specificities simultaneously may be useful, as more patients could potentially be categorised as ACPA-positive/seropositive [
14,
16‐
19]. Using such a multiplex citrullinated peptide array, we have recently shown that 16% of anti-CCP2-negative RA patients were in fact ACPA-positive [
19].
The other major autoantibody in RA, RF, is most often defined as IgM directed against the constant region of IgG, although other RF isotypes (mainly IgG and IgA) are also present in subsets of patients, but generally not screened for in the clinic [
20]. More recently, antibodies to carbamylated proteins (anti-CarP antibodies) have been described in RA [
21], and additional autoantibodies, including anti-nuclear antibodies, which are primarily linked to other rheumatic/autoimmune conditions, have also been detected in subsets of RA [
22].
In this study, we have used the Swedish population-based RA case-control study EIRA (Epidemiological Investigation of RA) as a basis for the analysis of autoantibodies present in the RA subset conventionally defined as seronegative, and we have investigated whether the presence of such antibodies associates with classical RA risk factors and have predictive value for disease activity.
Discussion
We find autoantibodies in “seronegative” RA, at significantly higher frequencies, levels, and co-occurrence compared to controls, and with a similar pattern of reactivity as in traditionally defined seropositive RA, albeit with a narrower serology in regards to RA-associated autoantibodies. Our data are thus in line with previous studies demonstrating the presence of ACPA in anti-CCP2-negative RA [
16,
17,
19] and a recent report, detecting IgA RF and anti-CCP2 IgA in 5.2% of “seronegative” RA [
30]. A novel finding in our study is that the extended ACPA/RF serology defines a group of RA patients with a more severe prognosis, and since additional screening of ACPA fine-specificities and IgA/IgG RF was able to identify 35% of the patients in the conventionally defined seronegative RA subset, our data suggest that the use of such extended serology may be clinically useful.
While the presence of ACPA fine-specificities and RF in “seronegative” RA associated with higher disease activity during follow-up, the presence of anti-CarP antibodies associated with lower disease activity in our study. This observation is somewhat contradictory to what has been reported previously, where presence of anti-CarP antibodies was shown to associate with higher disease activity and worse clinical outcome [
21,
31,
32]. However, the other studies have either only analysed baseline DAS28, and at baseline, we also found a non-significant trend with higher disease activity in the anti-CarP-positive group, or the other studies have analysed radiological progression over time, while we have analysed DAS28 over time, with no access to radiological data. Moreover, the other studies did not investigate presence of ACPA fine-specificities. Based on our data, nearly 40% of anti-CarP-positive patients within the “seronegative” RA population would also be ACPA-positive. Therefore, it cannot be ruled out that the observed joint destruction in the other studies is primarily associated with ACPA, rather than anti-CarP antibodies.
In accordance with data published by Wagner et al. [
17], as well as our recent report [
19], we found an association of SE with the presence of ACPA in anti-CCP2-negative RA, suggesting that these patients belong to the same disease entity as anti-CCP2-positive patients. A more detailed analysis of the ACPA fine-specificity response showed that SE associates primarily with some ACPA fine-specificities, not all. These ACPA fine-specificities co-occurred to a large extent, which could possibly indicate cross-reactivity rather than co-occurrence, something that has been shown on a monoclonal level for different ACPA [
33‐
35]. Other ACPA did not co-occur to the same extent, which may suggest different triggering mechanisms for different ACPA fine-specificities. This, we have recently explored in a separate study, demonstrating unique genetic characteristics for different ACPA fine-specificities [
36]. We did not find an association between HLA-DRB1 SE and RF in anti-CCP2-negative RA, supporting data generated from single-cell RNA sequencing of RF-positive and ACPA-positive B cells, which suggest a T cell-dependent affinity maturation for the generation of ACPA, but innate immune pathways for the generation of RF [
37].
We could not detect an association between smoking and presence of ACPA in anti-CCP2-negative RA, but a significant association with RF, in particular IgA RF. We also observed significantly elevated RF (but not anti-CCP2 IgG) levels in current smokers compared to former smokers. These data are thus in line with recent reports, demonstrating a lack of association between ACPA and smoking in RF-negative RA [
38] and that the association between smoking and RA-associated antibodies is likely dependent on RF/RF levels [
39]. The association between smoking and IgA RF is well known [
40,
41] and points to the putative role of mucosal inflammation in the generation of IgA RF. Notably, RF has been reported in healthy smokers [
41,
42].
Contrary to ACPA and RF, the other autoantibodies—not primarily associated with RA—were low in frequency, equally distributed among anti-CCP2-positive and anti-CCP2-negative RA patients, and showed no associations with genetic and environmental risk factors. Most common were anti-R060/SSA and anti-Ro52/SSA antibodies, each with a frequency of around 5%, similar to previous reports [
43]. Their presence may indicate clinical features associated with other systemic inflammatory diseases, in addition to classical RA symptoms.
Although the PTPN22 polymorphism (rs2476601) was more frequent among anti-CCP2-positive patients compared to anti-CCP2-negative, we found a significant association with both subsets, similar to previous reports [
44,
45]. Presence/absence of ACPA fine-specificities, RF, and other autoantibodies did not affect this positive association. PTPN22 polymorphism has been described as a risk factor for several autoimmune diseases [
46], and healthy carriers have been reported to have more autoreactive B cells compared to non-carriers [
47]. Hence, the association of PTPN22 with autoantibody-negative RA was a bit surprising and potentially suggests the presence of yet other autoimmune reactions.
Our study is by no means complete when it comes to the analysis of RA-associated autoantibodies. We did not investigate the presence of anti-CCP2 IgA [
48] nor did we analyse presence of ACPA using other commercial clinical tests, like the CCP3/CCP3.1 assays or the mutated citrullinated vimentin (MCV) assay. The antibody response against human PAD enzymes [
49] and acetylated proteins [
50] was not investigated either, and anti-CarP antibodies were analysed using carbamylated fibrinogen as an antigen [
28], while other studies have used carbamylated foetal calf serum [
21,
39].
Another point to be considered is the loss of specificity when analysing multiple autoantibody reactivities. Hence, for a diagnostic setting, highly specific cutoff values should be strictly applied to each individual antibody and be based not only on population controls but on disease controls with other rheumatic and autoimmune conditions. Still, our data suggest that the number of ACPA fine-specificities, as well as co-occurrence of ACPA and IgA/IgG RF, could be useful diagnostic markers.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.