Glucocorticoid therapy of antigen-induced arthritis depends on the dimerized glucocorticoid receptor in T cells

Abstract

Despite several side effects, glucocorticoids (GCs) have been widely used for 60 y to treat rheumatoid arthritis on the basis of their antiinflammatory effects. However, the cells targeted by GCs and the transcriptional mechanisms underlying their actions through the glucocorticoid receptor (GR) in steroid therapy remain poorly defined. Using cell type-specific GR-deficient mice subjected to antigen-induced arthritis (AIA) as a model of human rheumatoid arthritis, we show that GC action on T cells but not myeloid cells is critical for therapeutic intervention in AIA. Furthermore, the resistance of mice expressing a DNA binding-defective GR (GR(dim)) to GC treatment reveals that dimerization of the GR is indispensable for the antiinflammatory effects. In these mice, the GC-induced suppression of T(H)1 and T(H)17 cell-derived proinflammatory cytokines is impaired. Our finding that IL-17A(-/-) mice are resistant to GC therapy, whereas IFN-γ(-/-) mice respond as efficiently as WT mice implies that IL-17-producing T cells and not IFN-γ-producing T cells are the most important targets for an efficient GC therapy. The present study's identification of the critical cell type and the mode of GR action in steroid therapy of AIA significantly advances our understanding of steroid therapy and should lead to therapies with greater efficiency and fewer side effects.

Fig. 1.

GC treatment suppresses AIA. (A) Treatment scheme of AIA induction and Dex application (Materials and Methods). (B) Effect of Dex treatment on AIA knee joint swelling at indicated time points. (C) Representative H&E stainings of naïve healthy knee joints and of arthritic PBS- and Dex-treated joints at day 1. B, bone of joint; C, connective tissue; SL, synovial layer. (Scale bars, 0.2 mm.) (D) Histological score of PBS- and Dex-treated arthritic knee joints at day 1 according to Tolk and Földi's grading of joint inflammation (Materials and Methods). In B and D, n = 8; **P < 0.01.

Fig. 2.

GC-mediated suppression of AIA requires the GR in T cells. (A–D) Knee joint swelling of (A) GR^LysMCre, (B) GR^CD11cCre, (C) GR^CD19Cre, and (D) GR^LckCre mice and their respective littermate controls (GR^flox) subjected to AIA and PBS or Dex treatment. (E) Histological score of arthritic knee joints of PBS- and Dex-treated GR^flox and GR^LckCre mice at day 1. (F) Representative H&E stainings of arthritic knee joints of PBS- and Dex-treated GR^flox and GR^LckCre mice at day 1 (arrows indicate infiltrations of inflammatory cells) (Scale bars, 0.2 mm.) (G–I) Serum levels of (G) IL-6, (H) IFN-γ, and (I) IL-17 in PBS- and Dex-treated arthritic WT and GR^LckCre mice at day 1. In A–E and G–I, n = 5–6; *P < 0.05, **P < 0.01.

Fig. 3.

GC-mediated suppression of AIA requires dimerization of the GR in T cells. (A) Clinical development of AIA in PBS- and Dex-treated WT mice and mice harboring a dimerization-deficient GR (GR^dim) determined from measurements of knee joint swelling. (B) Histological score of knee joints of PBS- and Dex-treated arthritic WT and GR^dim mice at day 1. (C) Representative H&E stainings of PBS- and Dex-treated arthritic knee joints of WT and GR^dim mice (arrows indicate infiltrations of inflammatory cells) (Scale bars, 0.2 mm.) (D–F) Serum levels of (D) IL-6, (E) IFN-γ, and (F) IL-17 measured in PBS- and Dex-treated arthritic WT and GR^dim mice at day 1. (G) Knee joint swelling of PBS- and Dex-treated arthritic GR^dim/flox and GR^{dim/flox;LckCre} mice, which lack the dimerized function of GR exclusively in T cells. In A, B, and D–G, n = 5–7; *P < 0.05, **P < 0.01.

Fig. 4.

GCs reduce T_H1 and T_H17 cell numbers in WT mice but not in GR^dim mice. (A) Total cell numbers of draining lymph nodes derived from arthritic PBS- or Dex-treated WT and GR^dim mice at day 1. (B) Cells were restimulated ex vivo for 6 h with mBSA, then stained with anti-CD4 and intracellularly with anti-CD154. The number of CD4⁺ cells (Left) and CD154⁺ cells (Right) was calculated using the frequency of CD4⁺ cells and CD154⁺ cells, respectively, from the total number of cells (shown in A). (C) Intracellular FACS staining of restimulated draining lymph node cells from B. Cells were stained with anti-TNFα (Left), anti–IFN-γ (Center), and anti–IL-17 (Right). Subsets of CD4⁺ cells are represented, and P values are indicated to the right of plots. (D) Intracellular FACS analysis of mBSA-restimulated lymph node cells determining the numbers of cytokine-producing cells in the total number of cells. In all four panels, n = 18; *P < 0.05; n.s., not significant.

Fig. 5.

GC response of IFN-γ^−/− and IL-17A^−/− mice. (A) Clinical development of AIA in PBS- and Dex-treated WT mice and mice deficient for IFN-γ determined from measurements of knee joint swelling. (B) Serum level of IL-17 measured in PBS- and Dex-treated arthritic WT and IFN-γ^−/− mice at day 1. (C) Clinical development of AIA in PBS- and Dex-treated WT mice and mice deficient for IL-17 determined from measurements of knee joint swelling. In all three panels, n = 5–7; **P < 0.01.