Figure 3.

Cyclooxygenase (COX)-2–derived prostaglandins (PGs) regulate T helper cell type 9 (Th9) differentiation in vitro. (A) Eicosanoid levels in supernatants of naive CD4⁺ T cells and differentiated Th9 cells from COX-2^+/+ and COX-2^−/− mice were measured by liquid chromatography–tandem mass spectrometry (n = 5 per group; *P < 0.05). (B) Naive CD4⁺ T cells were cultured with anti-CD3, anti-CD28, anti–INF-γ, IL-4, and transforming growth factor (TGF)-β in the presence or absence of PGD₂, PGE₂, PGF_2α, or PGI₂ (1 μM each). Th9 differentiation from naive CD4⁺ T cells was examined by RT-PCR. Results are representative of at least three independent experiments. *P < 0.05 versus vehicle. (C) Th9 differentiation, as above, was measured in the presence of increasing concentrations of PGD₂ and PGE₂, *P < 0.05 versus vehicle. (D) Schematic of in vivo minipump experiment. Alzet minipumps filled with either PGD₂, PGE₂, or the combination of PGD₂ and PGE₂ were implanted into sensitized COX-2^−/− mice from Day 13 to Day 20. (E) Sensitized mice were exposed to ovalbumin (OVA) daily for 4 days. At 48 hours after the last OVA exposure, mice were killed and the percentages of IL-9⁺ CD4⁺ T cells in lung, bronchoalveolar lavage fluid (BALF), lymph nodes, and blood were determined by flow cytometry (n = 4–5; *P < 0.05 versus saline containing minipump). Lines indicate the mean, and each symbol represents an individual mouse.