Background and objectives Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA).
Materials and methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation method. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR. Cell viability was monitored by impendance-based cell analyzer (CASY-TT).
Results Although the anti-CD3/CD28 activation increased the IL-17 and IL-22 production; it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc and T-bet without stimulation or cytokine treatment and also higher amount of IL-17 compared to the CD45RO- cells. By contrast, there was no difference between the RORc expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. The production of IL-17 and IL-22 of the differentiated CD45RO- cells from RA patients were both increased by IL-23 treatment.
Conclusions The IL-17 and IL-22 production are differently regulated during the human Th17 differentiation. Furthermore, our present data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA.
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