Background/Purpose:

Rheumatoid arthritis (RA) is a complex disease with both genetic- and environmental risk factors. By studying first degree relatives (FDR) of RA patients which shares some of the genetic and environmental risk factors, may shed light on the pathogenic mechanisms of RA. A subset of B-cells, identified by their IL-10 producing abilities, when stimulated with CD-40 the B-regulatory cells (CD19⁺CD20⁺CD24^highCD38^high) has been suggested to be less functional in expressing IL-10 in patients with Systemic lupus erythematosus compared with cells from healthy individuals. As a downstream protein of CD-40 stimulation, signal transducer and activator of transcription 3 (Stat-3) expression can be used as a pseudo- marker for IL-10 expression and response to CD-40 stimulation.

In FDR, regarded as high risk population we aim to analyze B-regulatory cells to investigate their Stat-3 response with and without CD-40 stimulation and compare it with healthy controls and RA- patients.

Methods: Mononuclear cell isolation was done with blood samples from 23 FDR and 26 RA patients and 11 control individuals from northern Sweden. Two million cells, either with or without anti-CD40-antibody stimulation was analyzed with LSRII flow cytometer (Becton-Dickinson, San Jose, CA) for Stat-3 expression in CD19⁺CD20⁺CD24^highCD38^high-cells (B-regulatory cells). Samples with a robust coefficient of variation in flow cytometry variables >150 was excluded for subsequent analyses. Anti-CCP2 antibody analysis was performed by ELISA (Euro-Diagnostica). HLA-DRB1 alleles were genotyped using polymerase chain reaction sequence-specific primers. Genotyping of the PTPN22 1858C/T polymorphism was performed using a Taqman instrument. Continuous data were compared by non-parametric analysis using the Mann-Whitney U-test for two groups and the Kruskal-Wallis test comparing several groups. For within-patient comparisons the Wilcoxon signed-rank test was used.

Results: Comparing the Stat-3 levels with and without CD-40 stimulation in B-regulatory cells on individual level, a significant increase was found in controls (p=0.018) while no change of significance was found in the FDR nor in the RA patients. No significant differences could be found in Stat-3 expression in B-regulatory cells when stratified for HLA-SE or PTPN22 T-carrier.  No significant difference could be found between the three studied populations (Controls/FDR/Patients) regarding; Stat-3 expression, relative cell number to parent (CD19⁺CD20⁺) or relative cell number to grandparent population, in neither CD40-stimulated nor unstimulated B-regulatory cells, respectively.

Conclusion: We have demonstrated that the B-regulatory cells of RA-patients and their FDR could be functionally impaired when measuring the Stat-3 response on CD40 stimulation, compared with healthy controls. HLA-SE and PTPN22 1858T genotype did not affect Stat-3 expression. This could indicate that defective B-regulatory cells play a role in the predisposition for RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-regulatory-cd19cd20cd24highcd38high-cells-are-functionally-impaired-in-patients-with-rheumatoid-arthritis-and-healthy-first-degree-relatives-compared-with-controls/