Modulation of cytokine production by β-agonist and PGE₂. (A,B) PBLs were cultured 14 days with IL-2 + PHA + CD28mAb ± IL-4 (IL-4 was not used in cultures for experiments testing IFN-γ production). After culture, the cells were treated 15 minutes with vehicle, PGE₂ (1 μM), or ISO (10 μM), and then stimulated either 5 hours with PMA + calcimycin + IL-2 or 18 hours with CD3 + CD28 mAb + IL-2 (+monensin). Cytokine production was analyzed by flow cytometry (filled region, staining for IL-13 expression; open region, background from isotype control mAb—same for vehicle and PGE₂ treated). To allow comparison of results among subjects (“Statistical analyses” section in “Materials and methods”), MFI of cytokine staining (background subtracted) was normalized as the percent change relative to MFI of the respective stimulus only (vehicle treated = 0-change). Histograms of cytokine production by T cells for a representative experiment for PGE₂ are shown in panel A. (B) Reported are mean ± SD, n = 9 for IL-13 and IL-2, n = 6 for IFN-γ. ^*P < .05 for difference from 0 to change. (C) The experiments described in panel B were performed replacing PGE₂/ISO with 200 μM db-cAMP (n = 5-7). (D) PBLs were treated 15 minutes with vehicle, PGE₂, ISO, or db-cAMP at the indicated concentrations, then stimulated 18 hours with CD3 + CD28 + IL-2 for cytokine production. IL-13 production was analyzed, and MFI normalized to vehicle-treated cells as described in Figure 5B. Data represent mean ± SD from 6 independent experiments using PBLs freshly isolated (n = 3 for PGE₂- and ISO treated; n = 2 for db-cAMP treated) or after culture for 14 days with IL-2 + IL-4 ± PHA + CD28 mAb (n = 3 for PGE₂ and ISO treated; n = 1 for db-cAMP treated). Because effects were nearly identical for both populations, data were combined and fitted to a sigmoidal dose-response curve. Increased IL-13 production with 1 nM PGE₂, 1 to 10 nM ISO, and 5 μM db-cAMP was observed in all 6 of the independent experiments performed. (E) PBLs were cultured 14 days with IL-2 + PHA + CD28 mAb + IL-4. After culture, the cells were treated 15 minutes with vehicle, PGE₂, or ISO at the indicated concentrations, and then stimulated 18 hours with CD3 + CD28 mAb + IL-2 (without monensin). Culture supernatants were then analyzed by flow cytometry. The levels of IL-13 are reported as ng/mL (limit of detection: 20 pg/mL) for 1 representative of 2 independent experiments. (F) After culture of PBLs as in panel A, cells were incubated 5 minutes with ICI-188551 or CGP20712A, treated 15 minutes with subsaturating concentration of ISO (100 nM), then stimulated 18 hours with CD3 + CD28 mAb, IL-2, and monensin. After stimulation, IL-13 production was determined by flow cytometry. Reported is the net MFI for IL-13 production (MFI from isotype control staining subtracted) for 1 representative of 2 independent experiments. (G) PBLs were cultured as described in panels A and B, treated 15 minutes with vehicle or 100 nM ISO, then stimulated 18 hours with CD3 + CD28 mAb, IL-2, and monensin. IL-13 production and expression of CD3 and CD8 were determined by flow cytometry. MFI for IL-13 levels (isotype-control MFI subtracted) is reported for CD8⁺ and CD4⁺ (= CD8^-) T cells for 1 representative of 3 independent experiments.