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. 2011 Apr 7;184(1):37–49. doi: 10.1164/rccm.201010-1637OC

Figure 7.

Figure 7.

Expression of prostaglandin (PG) receptors on CD4+ T cells in vivo and in vitro differentiated Th17 cells from cyclooxygenase (COX)-2+/+ and COX-2−/− mice. (A) The percentage of CD4+ T cells isolated from spleen, lymph nodes, lung, blood, and bronchoalveolar lavage fluid (BALF) of allergic COX-2+/+ and COX-2−/− mice was determined by flow cytometry. Antibodies to each of the PG receptors were conjugated with AlexaFlur-488 or AlexaFlur-594 using a Therm Scientific DyLightTM 488/594 microscale antibody labeling kit. Cell suspensions were prepared and stained with the conjugated receptor antibodies. EP1-EP4, DP1, DP2, FP, and IP receptor-positive CD4+ T cells were quantified by flow cytometry by gating on the IL-17+ CD4+ T cell population. Lines indicate the mean, and each symbol (open squares, COX-2+/+; solid squares, COX-2−/−) represents an individual mouse. (B) Levels of EP1-EP4, DP, FP, and IP receptor mRNAs in naive CD4+ T cells and in in vitro differentiated Th17 cells from COX-2+/+ and COX-2−/− mice. Naive CD4+ T cells isolated from COX-2+/+ and COX-2−/− mice were stimulated with or without anti-CD3, anti-CD28, anti–INF-γ, IL-6, and transforming growth factor (TGF)-β for 4 days. Total RNA was then extracted and reverse transcribed to cDNA for detection of EP1-EP4, DP, FP, and IP receptor expression by real-time polymerase chain reaction. (C) The effects of FP and IP receptor antagonists on Th17 cell differentiation of naive CD4+ T cells were investigated. Naive CD4+ T cells from wild-type mice were differentiated to Th17 cells in the presence or absence of the FP receptor antagonist, AL8810, the IP receptor antagonist, CAY10441, or a combination of AL8810 and CAY10441. The percentage of Th17 cells was analyzed by flow cytometry after 5 days. n = 3; * P < 0.05. (D) The effects of FP and IP receptor knockdown on Th17 differentiation of naive CD4+ T cells were investigated. Naive CD4+ T cells from wild-type mice were transfected with IP receptor siRNAs, FP receptor siRNAs, a combination of IP receptor and FP receptor siRNAs, or control siRNA. Transfected cells were then differentiated in the presence of 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) for 5 days and the percentage of Th17 cells was analyzed by flow cytometry. n = 3; * P < 0.05 versus control siRNA.