The effect of rapamycin on LPS-induced cytokine expression in BMDCs. (A) BMDCs were stimulated with 1 μg/mL LPS in the presence or absence of either 100 nM wortmannin or 10 ng/mL rapamycin for 24 hours and assayed for the production of IL-12p70, IL-10, IL-6, and TNF-α by ELISA. (B) BMDCs were stimulated with 1 μg/mL LPS with or without 10 ng/mL rapamycin along with the indicated concentrations of FK506 for 24 hours and assayed for the production of IL-12p70, IL-12p40, and IL-10 by ELISA. (C) BMDCs were pretreated with or without either 100 nM wortmannin or 10 ng/mL rapamycin for 20 minutes before stimulation with 1 μg/mL LPS. After 4 hours, total RNA was isolated, and IL-12p40, IL-12p35, IL-10, and IFN-β mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. All data are indicated as median plus or minus SD of duplicate samples. Similar results were obtained with 2 to 4 independent experiments.