Background and objectives The most important feature of B-cells is the production antibodies upon activation; additionally, B-cells produce both pro-inflammatory and anti-inflammatory cytokines in response to certain stimuli. IL-10 producing B10 cells represent a major subset of regulatory B-cells (Bregs). Bregs suppress autoimmune and inflammatory responses by multiple mechanisms. B-cells play crucial role in the development and maintenance of the chronic inflammatory autoimmune disease, Rheumatoid arthritis (RA); however, controversial data are available on B10 population in RA. Our aim was to identify the optimal conditions inducing B10 cells in samples from healthy controls and RA patients; furthermore, to shed light on signalling pathways resulting in the expansion of the B10 subset.
Materials and methods Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and RA patients. B cells were purified by magnetic separation, using negative selection. We assessed IL-10 and TNF expressing B-cells after intracellular staining by flow cytometry, and measured the secreted cytokines with multiplex bead array. Phosphorylation of key signalling molecules was monitored by phospho-flow method.
Results The results show that dual stimulation by CpG and CD40L for 48h was optimal for IL-10 induction, which was synergistically boosted by IL-21. We identified CD19+ CD27+ memory B-cells as the major source of B10 cells. IL-21 increased the ratio of BLIMP1 and IL-10 double positive plasmablasts. In RA patients we detected significantly less CD27+ B10 cells as compared to the controls, while addition of IL-21 to the dual stimuli significantly elevated the number of B10 cells both in the CD19+CD27+ and in the CD19+ CD27- population. Different combinations of stimuli induced preferentially the secretion of pro-inflammatory cytokines (TNF, IFNγ, IL-17) and the suppressor cytokine, IL-10. We assumed that activation of ERK, p38 and CREB are indispensable to induce IL-10, while STAT3 appears to be a co-activator for IL-10 transcription in human Bregs.
Conclusions CD19+CD27+ memory B-cells are the major source of human B10 cells, which may expand and differentiate to IL-10 producing plasmablasts in the presence of IL-21. CREB and the co-activator STAT3 are the key transcription factors responsible for the expansion of the B10 population.
Support: National Research, Development and Innovation Office (OTKA104846), and ELTE TÁMOP 4.2.1./B-09/1/KMR-2010–0003
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