Figure 5
COX-2 expression in MLN-DC regulates mucosal Tr-cell conversion and suppresses IL-4-producing T-cell differentiation. (a) Mice were fed with 5 mg OVA labeled with Alexa Fluor-488 succinimydyl ester in 200 μl saline. At 24 h after antigen administration, MLNs were isolated and single-cell suspensions were stained for CD11c. The percentage of DC that had ingested OVA were determined by gating on the CD11c and Alexa Fluor-488 double-positive population and compared with the overall CD11c-positive population. (b) MLN, spleen, and peripheral LN of naive BALB/c mice were isolated, and DCs, T cells, B cells, and CD45- cells were purified by flow cytometric cell sorting and were compared for COX-2 expression relative to cyclophillin by quantitative PCR. (c) DC subsets were purified from MLN of naive BALB/c mice by flow cytometric cell sorting and were compared for COX-2 expression relative to cyclophillin by quantitative PCR. (d and e) MLN-DCs (2 × 104) were preincubated with 0.2 μg ml−1 OVA peptide with or without retinoic acid and with or without TGF-β for 6 h. After washing, the cells were incubated with 5 × 105 CFSE-labeled CD4+KJ1.26+ T cells in the presence of TGF-β and retinoic acid for 96 h. To assess the role of COX-2, NS-398 75 μM or DMSO control was added to the co-culture. At 96 h, CD4+ T cells were isolated from the culture by eliminating DCs with mAbs and anti-rat magnetic beads and analyzed for Foxp3 expression (d), Th2 cytokine profile IL-4 (e), and transcription factor GATA-3 (e) mRNA expression by quantitative PCR. In parallel, CD4+KJ1.26+ cells were analyzed for Foxp3 by flow cytometry (d) (n=3). *Statistically significant (P<0.05). CFSE, 5,6-carboxy-succinimidyl-fluoresceine ester; COX-2, cyclooxygenase-2; DCs, dendritic cells; DMSO, dimethyl sulfoxide; RA, retinoic acid; TGF-β, transforming growth factor-β.
