Methods. Highly purified B cells were obtained from tonsils or peripheral blood by using a multiple-step separation procedure which included rosette depletion, adherence depletion, CD3+ cell magnetic-activated depletion and CD19+ magnetic-activated positive cell selection. CD20+ purity was verified by flow cytometry. In these cells fractions, the percentage of double-labeled CD3+CD4+ cells (T lymphocytes) was negligible (0.2%); CD14+ cells (macrophages/monocytes) were also very low (<0.2%). By Q-RTPCR, CD8 (a cytotoxic T cell marker) and CD161 (a NK cell marker) mRNAs were undetectable in these CD19+ B cell fractions. The CD19+CD20+ B cells were stimulated with IL-4, IFN-gamma, IL-6, IL-23, and TGF-beta and analyzed the expression of both IL-17A and F in response to stimulation by real-time RT-PCR, western blot, immunocytochemistry and by ELISA.
Results. Freshly purified B cells from tonsils and from blood express detectable amounts of the mRNA and protein of IL-17A and F. When B cells were stimulated with IL-6, IL-23 and TGF-beta, the mRNA and protein expression of both IL-17A and F was significantly increased (n=3; P≤0.001). In contrast, stimulation with IL-4 alone or in combination with anti-CD40 antibody, decreased the expression of IL-17A and F in cultured B cells (n=3; P≤0.001).
Conclusions. These novel findings provide evidence that B lymphocytes could be a significant source of IL-17A and IL-17F cytokines and support the notion that B lymphocytes actively participate in immune responses via a mechanism in addition to the classic release of antibodies. Moreover, our results also set the stage for investigating the role of the B cells-IL-17 interaction in autoimmune diseases.