Background Regulatory B cells (Breg) are newly defined B cell subsets that downregulate the immune response. The pleiotrophic cytokine, IL-10 seemed to be responsible for this function. B cells play a crucial role in the development and maintenance of Rheumatoid arthritis (RA). Therefore this study was undertaken to identify the optimal stimuli (BCR, CpG, CD40L) that induce Breg cells, furthermore, our aim was to compare the number of Breg in RA patients and healthy controls. We also aimed to examine, which inflammatory cytokines are produced by B cells in response to the same stimuli.
Materials and methods Blood samples were collected from healthy donors and RA patients. Intracellular IL-6, IL-10 and TNF were measured in purified B cells and in PBMC. Cytokines were detected in B cells prior or after stimulation by intracellular fluorescent staining using specific antibodies. IL-10, IL-6, TNF, IL-1b and INFg were measured in the supernatants of purified B cell with FlowCytomix multiplex bead array.
Results At our experimental conditions, dual stimulation by CpG and CD40L for 48 h was found to be optimal for IL-10 induction in B cells. We identified CD19+ CD27+ memory B cells as the main source of IL-10. The dual stimuli induced significantly lower number of IL-10 producing B cells from RA patients as compared to healthy controls, while we didn’t find a difference in the inflammatory cytokine (IL-6 and TNF) production. However, in unstimulated samples the frequency of IL-6 producing B cells was lower in healthy controls, than in RA patients.
Furthermore, we observed that in the supernatant of purified B cells, beside IL-10, other inflammatory cytokines (IL-6, TNF, IL-1b and INFg) were also present. Therefore we double stained B cells with anti-IL-10 and anti-TNF antibodies and found that these cytokines were produced by different subsets.
Conclusion We detected a significant difference between the number of IL-10 producing CD27+Breg cells of RA patients and healthy controls. The lower frequency of activation-induced IL-10 producing Bregs and on the other hand, the increased ratio of spontaneously IL-6 secreting B cells in RA patients may contribute to the exacerbation of the disease.
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