Fig. 3.

Ciclesonide inhibits MCP-1 mRNA production and MCP-1 promoter and enhancer activity. A and B: human ASM cells were preincubated for 30 min with 10⁻⁷ M ciclesonide and then stimulated for 8 h with TNFα (0.1 ng/ml) or IL-1β (0.1 ng/ml). Cells were lysed, RNA was extracted and reverse transcribed, and cDNA levels were determined by quantitative PCR. Values are means ± SE (n = 3). C and D: 50–60% confluent human ASM cells were transfected for 16 h with MCP-1 promoter plasmid (0.4 μg/well), pretreated for 30 min with ciclesonide (10⁻⁷ M), and then incubated for 6 h with TNFα (10 ng/ml) or IL-1β (10 ng/ml). Firefly and Renilla luciferase activity was assayed, and the ratio of firefly to Renilla luciferase activity was calculated to represent activity of the reporter. Values are means ± SE (n = 3). E and F: 50–60% confluent human ASM cells were transfected for 16 h with MCP-1 enhancer plasmid (0.4 μg/well), pretreated for 30 min with ciclesonide (10⁻⁷ M), and then incubated for 6 h with TNFα (10 ng/ml) or IL-1β (10 ng/ml). Firefly and Renilla luciferase activity was assayed, and the ratio of firefly to Renilla luciferase activity was calculated to represent activity of the reporter. Values are means ± SE (n = 3). +P < 0.05, +++P < 0.001 vs. control. *P < 0.05, **P < 0.01 vs. stimulated alone.