Background and objectives Th17 cells produce several inflammatory cytokines, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α and play an important role in the regulation of inflammation. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA).
Materials and methods CD4+ T cells were negatively separated by magnetic method from peripheral blood mononuclear cells (PBMC) then CD45RO+ and CD45RO- cells were separated. The cells were treated for 5–10 days with anti-CD3 and anti-CD28 antibodies and with TGFβ (2.5 ng/ml) and IL-6 (25 ng/ml) or with IL-6 (25 ng/ml), IL-1 (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) and anti-IFNγ (10 μg/ml) blocking antibodies. The IL-17 and IL-22 production were measured by ELISPOT and ELISA, the RORγt expression was measured by real-time PCR and by Western blot methods. Cell viability was monitored by Trypan blue staining, by Annexin V binding and by an impendance-based cell analyser.
Results Anti-CD3/CD28 activation increased the IL-17 production, but did not alter the RORγt expression. The anti-CD3/CD28 activation and TGFβ, IL-6, and IL-1 cytokine induced RORγt expression of the CD4+ T cells was further increased by the anti-IL-4 and anti-IFNγ antibody treatment. The IL-22 production of CD4+ T cells was significantly reduced in the RORγt producing CD4+ cells. The CD45RO+ cells expressed high level of RORγt and produced high amount of IL-17 without stimulation or cytokine treatment. The RORγt expression of the CD4+RO- T cells increased severalfold upon stimulation and cytokine treatment. The differentiated Th17 cells of patients with RA produced more IL-17 than those of the healthy donors’. The applied treatments did not change the viability of the cells.
Conclusions Our results suggest that the IL-17 and IL-22 production are differently regulated during Th17 differentiation. The Th17 cell differentiation is most effective from CD45RO- naive T cells.