IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells

Abstract

CD4(+) T cells producing IL-17 [T helper (Th)17], as distinct from Th1 or Th2 cells, have recently been shown to be associated with autoimmunity, but it is not entirely clear how Th17 cells are generated from naïve T cells. We demonstrate here that IL-6, but not TNF-alpha or IL-1beta, can, in combination with TGF-beta, induce Th17 cell generation from naïve T cells and inhibit TGF-beta-induced Foxp3 expression. Moreover, conditioned medium from lipopolysaccharide-stimulated bone marrow-derived dendritic cells (DCCM) can induce IL-17 production in naïve T cells. Interestingly, IL-17 was produced by DCCM even with the addition of anti-gp130 antibody or DCCM from IL-6 KO mice. The combination of IL-6 and TGF-beta could maintain activation of signal transducer and activator of transcription (Stat)3, but not of Stat1. IL-27 or IFN-gamma suppressed the induction of Th17 cells by TGF-beta plus IL-6 and maintained Stat1 activation under these conditions. In contrast, both Stat1 and Stat3 remained to be activated in naïve T cells cultured with DCCM. These findings represent a different basis for Th17 differentiation from naïve T cells.

Fig. 1.

The combination of IL-6 and TGF-β induces the development of Th17 cells. Isolated naïve T cells were cultured with anti-CD3/CD28 beads and the indicated cytokines for 3 days. (A and B) Naïve T cells were stimulated with the indicated cytokines in the presence or absence of anti-mouse gp130 antibody or anti-mIL-6R monoclonal antibody MR16-1. After stimulation, cells were restimulated with PMA and ionomycin for 5 h and with GolgiStop (last 2 h) and were then subjected to intracellular cytokine staining. Dot plots show intracellular staining for IFN-γ and IL-17. (C) Foxp3 expression was determined by staining with anti-mouse Foxp3 antibody. These results are representative of three independent experiments.

Fig. 2.

TNF-α or IL-1β in combination with TGF-β has no effect on the differentiation of Th17 cells. (A) MACS-sorted naïve T cells were cultured with anti-CD3/CD28 beads. Cells were stimulated with IL-6 or TGF-β alone and with IL-6, TNF-α, or IL-1β combined with TGF-β. After 3 days, cells were restimulated with PMA and ionomycin before intracellular cytokine staining for IFN-γ and IL-17 and analysis by flow cytometry. Dot plots show intracellular staining for IFN-γ and IL-17. (B) Isolated naïve T cells were activated with anti-CD3/CD28 beads and cultured with TGF-β alone and with IL-6, TNF-α, or IL-1β combined with TGF-β. Foxp3 expression was determined by staining with anti-mouse Foxp3 antibody. These results are representative of three independent experiments.

Fig. 3.

RORγt mRNA expression by IL-6 and TGF-β with or without IL-27 or IFN-γ. Isolated naïve T cells were stimulated with anti-CD3/CD28 beads and the indicated cytokines, followed by total RNA and cDNA prepared as described in Materials and Methods. RORγt induction was examined by using RT-PCR.

Fig. 4.

IL-17 is produced by DCCM without IL-6-mediated activity in naïve T cells. (A) Purified naïve T cells were cultured with anti-CD3/CD28 beads and the indicated cytokines, LPS, or CM from DCs stimulated with LPS. (B) Naïve T cells were stimulated with TGF-β plus IL-6, DCCM, or DCCM from IL-6 KO mice in the presence or absence of IL-27 or neutralizing antibodies for IL-6. Supernatants were collected 2 days after stimulation, and IL-17 production was measured by means of ELISA. Data show means ± SE of three independent experiments. (C) MACS-sorted naïve T cells were activated with anti-CD3/CD28 beads. Cells were cultured with the indicated stimulators. After 3 days, cells were restimulated with PMA and ionomycin before intracellular cytokine staining for IFN-γ and IL-17 and analysis by flow cytometry. Dot plots show intracellular staining for IFN-γ and IL-17.

Fig. 5.

Activation of Stat1 and Stat3 by stimulation to produce IL-17 in naïve T cells. MACS-sorted naïve T cells were cultured with anti-CD3/CD28 beads. (A) Naïve T cells were stimulated with TGF-β alone or TGF-β plus IL-6 for 30 min or 24 h. (B) Naïve T cells were stimulated with TGF-β alone or TGF-β plus IL-6 in the presence or absence of IL-27 or IFN-γ for 24 h. (C) Naïve T cells were cultured with DCCM with or without IL-27. Cells were fixed and permeabilized in 90% methanol, followed by staining with Alexa Fluor 488-conjugated phospho-Stat1 and phycoerythrin-conjugated phospho-Stat3. Intracellular phospho-Stat1 and -Stat3 were measured by flow cytometry. These results are representative of three independent experiments.