Figure 4.

Impaired Th17 differentiation in cyclooxygenase (COX)-2^−/− naive CD4⁺ T cells in vitro. (A) Naive CD4⁺ T cells isolated from spleens of COX-2^+/+ and COX-2^−/− mice were treated with or without anti-CD3 (3 μg/ml), anti-CD28 (3 μg/ml), anti–INF-γ (3 μg/ml), transforming growth factor (TGF)-β (10 ng/ml), and IL-6 (10 ng/ml) for 4 days. The cells were then stimulated for 4 hours with 12–0-tetradecanoyl-phorbol-13-acetate and inomycin (500 ng/ml each) in the presence of brefeldin A (1 μg/ml) before flow cytometry analysis. Data are representative of three independent experiments. (B) The percentage of naive CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice that differentiated to CD4⁺ IL-17A⁺ (Th17) cells is shown. (C) Inhibition of COX-2 with NS-398 during Th17 cell differentiation from naive CD4⁺ T cells was examined. Naive CD4⁺ T cells were cultured with anti-CD3 (3 μg/ml), anti-CD28 (3 μg/ml), anti–INF-γ (3 μg/ml), TGF-β (10 ng/ml), and IL-6 (10 ng/ml) in the presence or absence of NS-398 (20 μM) for 4 days. The cells were then stimulated for 4 hours with 12–0-tetradecanoyl-phorbol-13-acetate and inomycin (500 ng/ml each) in the presence of brefeldin A (1 μg/ml) before flow cytometry analysis. (D) The percentage of IL-17A⁺ CD4⁺ cells was examined by flow cytometry at different time points (Days 1–7) after treatment with anti-CD3, anti-CD28, anti–INF-γ, TGF-β, and IL-6. (E) Cell culture supernatants from D were collected at different time points and IL-17A levels were assayed by ELISA. (F) Naive CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice were differentiated to Th17 cells in the presence of different amounts of IL-6 (0, 5, 10, and 20 ng/ml) for 4 days and the percent of CD4⁺ IL-17A⁺ cells was analyzed by flow cytometry. (G) IL-17A levels in cell culture supernatants from F were assayed by ELISA. For panels C–F, data are representative of three independent experiments; closed circles = COX-2^+/+; open circles = COX-2^−/−; * P < 0.05 versus COX-2^+/+. (H) Total number of CD4⁺ T cells were enumerated in spleen, lymph nodes, and lung from COX-2^+/+ and COX-2^−/− mice (n = 10 per group). (I) Splenocytes from COX-2^+/+ and COX-2^−/− mice were analyzed for CD62L⁺ and CD4⁺ expression. Representative scattergrams are illustrated and the percentages of CD62L⁺ CD4⁺ cells (mean ± SE; n = 10 per group) are shown. (J) Naive CD4⁺ CD62L⁺ T cells from spleens of COX-2^+/+ and COX-2^−/− mice were first sorted by flow cytometry. These highly purified naive CD4⁺ CD62L⁺ T cells were then differentiated into Th17 cells as described in A. On Day 4, the cells were collected and the percentage of IL-17A⁺ CD4⁺ cells was analyzed by flow cytometry. (K) Quantification of Th17 cell differentiation of COX-2^+/+ and COX-2^−/− naive CD4⁺ CD62L⁺ T cells is shown.