In rheumatoid arthritis patients, total IgA1 and IgA2 levels are elevated: implications for the mucosal origin hypothesis

Abstract

Objective: Mucosal initiated immune responses may be involved in the pathophysiology of RA. The most abundant immunoglobulin at mucosal surfaces is IgA, of which two subclasses exist: IgA1 and IgA2. IgA2 is mainly present at mucosal sites and has been ascribed pro-inflammatory properties. As IgA subclasses might provide insights into mucosal involvement and pro-inflammatory mechanisms, we investigated IgA responses in sera of RA patients.

Methods: In two cohorts of RA patients, the EAC and IMPROVED, total IgA1 and IgA2 were measured by ELISA. Furthermore, IgA subclass levels of RF and anti-citrullinated protein antibodies (anti-CCP2) were determined. The association of these IgA subclass levels with CRP and smoking was investigated.

Results: Total IgA1 and IgA2 were increased in RA patients compared with healthy donors in both cohorts. This increase was more pronounced in seropositive RA vs seronegative RA. For RF and anti-CCP2, both IgA1 and IgA2 could be detected. No strong associations were found between IgA subclasses (total, RF and anti-CCP2) and CRP. In smoking RA patients, a trend towards a selective increase in total IgA2 and RF IgA1 and IgA2 was observed.

Conclusion: RA patients have raised IgA1 and IgA2 levels. No shift towards IgA2 was observed, indicating that the increase in total IgA is not due to translocation of mucosal IgA into the bloodstream. However, mucosal inflammation might play a role, given the association between smoking and total IgA2 levels. Despite its pro-inflammatory properties, IgA2 does not associate strongly with pro-inflammatory markers in RA patients.

Keywords: ACPA; IgA subclasses; RA; mucosal immunity; rheumatoid factor.

Fig. 1

Total IgA subclasses Total IgA1, total IgA2 and percentage IgA2 of total IgA in seropositive and seronegative RA patients compared to healthy controls. Mann–Whitney U tests to compare IgA levels between seropositive or seronegative RA patients and healthy donors. Red bars: median and interquartile range. *P < 0.05, **P < 0.01, ***P < 0.001, no brackets shown when P > 0.05. Not all samples were measured in the same experiment. Colour version available online.

Fig. 2

RF IgA subclasses (A) RF IgA1 levels and (B) RF IgA2 levels in IMPROVED seropositive RA patients, (C) RF IgA1 levels and (D) RF IgA2 levels in EAC seropositive RA patients. Gray dashed line represents cut-off. Red bars: median and interquartile range. Arbitrary units cannot be directly compared between subclasses. Colour version is available online.

Fig. 3

Anti-CCP2 IgA subclasses (A) Anti-CCP2 IgA1 and IgA2 subclass measurements in seropositive IMPROVED RA patients and healthy controls. Number and percentage of patients above the cut-off is indicated. Gray dashed line represents cut-off. Red bars: median and interquartile range. ^#Results might be influenced by RF IgA2 binding. (B) Anti-CCP2 IgA1 in seropositive EAC RA patients. (C–D) Mixing experiment for anti-CCP2 IgA1 and IgA2 ELISA to investigate whether RF IgA binding could influence the read-out. For anti-CCP2 IgA1, no RF interference is observed, for the anti-CCP2 IgA2 ELISA results could be influenced by RF IgA2 binding, as the combination of anti-CCP2 IgG+ IgA- RF IgA- serum with anti-CCP2- RF IgA+ serum can give high OD values. (E–H) Measurements before and after IgG depletion in a selection of IMRPOVED seropositive RA patients. After IgG depletion, the anti-CCP2 IgA2 signal remains clearly visible in part of the samples, while all anti-CCP2 IgG is depleted. The procedure led to some specific loss of total IgA1 and total IgA2. Colour version is available online

Fig. 4

Correlation between IgA subclasses and CRP in seropositive RA patients Correlation between IgA subclass levels and CRP levels in (A) IMPROVED seropositive RA patients and in (B) EAC seropositive patients, calculated using Spearman’s rank correlation coefficient (r_s). In RF IgA subclass analyses, only patients positive for both RF IgA1 and RF IgA2 are included. For anti-CCP2 IgA1 analysis, only anti-CCP2 IgA1 positive patients are included. Of note, RF and anti-CCP2 IgA subclass levels were not titrated.

Fig. 5

Association between IgA subclasses and smoking in seropositive RA patients IgA subclass levels in ever- versus never-smoking seropositive RA patients in (A) IMPROVED and (B) EAC, analysed using Mann–Whitney U tests. Red bars: median and interquartile range. *P < 0.05, **P < 0.01, ***P < 0.001, ^#not significant after correction for multiple testing. In RF IgA subclass analyses, only patients positive for both RF IgA1 and RF IgA2 are included. For anti-CCP2 IgA1 analysis, only anti-CCP2 IgA1 positive patients are included. Of note, RF and anti-CCP2 IgA subclass levels were not titrated. Colour version available online.