Figure 2
COX-2-dependent arachidonic acids inhibit the differentiation of IL-4-producing T cells in MLN. Donor BALB/c mice were injected with 1 × 107 CD4+ KJ1.26+ cells i.v., Starting the next day, mice were treated with NS-398 (black symbols) as described in Figure 1. Control groups were injected with vehicle consisting of a 1% DMSO solution in saline (white symbols). Tolerization was induced by a single i.g. dose of 70 mg OVA. At 48 h after OVA administration, MLN and PP (not shown) were isolated and single-cell suspensions were stained for the presence of CD4+ KJ1.26+ cells. CFSE profiles of CD4+ KJ1.26+ T cells were determined in MLN by flow cytometry. (a) Representative division plot at 48 h after OVA feed. (b) The percentages of CD4+ KJ1.26+ T cells in each peak of division were calculated and are represented as the mean for at least three mice±s.d. (c–e) At 48 h after oral OVA administration, MLN cells were re-stimulated overnight with 0.5 mg ml−1 OVA or medium at a concentration of 5 × 106 cells per ml. (c) Single cell suspensions were stained for KJ1.26+ cells and the percentage of IL-4-secreting cells per peak of division was determined with a cytokine secretion assay. Data are represented as mean percentage of OVA-induced cytokine-secreting antigen-specific T cells per peak of division of at least three separate experiments, with s.d. (d) Cytokine concentrations in the supernatants of overnight cultures were determined by cytometric bead array. Data are represented as the mean cytokine concentration of at least three separate mice, with s.d. (e) At 48 h after oral OVA administration, dividing OVA-specific T cells were isolated from MLN of three mice and the expression of transcription factors GATA-3 and T-bet was performed by quantitative PCR. *Statistically significant (P<0.01). CFSE, 5,6-carboxy-succinimidyl-fluoresceine ester; COX-2, cyclooxygenase-2; DMSO, dimethyl sulfoxide; i.g., intragastric; IL, interleukin; i.v. intravenous; MLN, mesenteric lymph node; OVA, ovalbumin; PP, Peyer's Patch.
