Figure 5
Figure 5. The effect of rapamycin on LPS-induced IL-12 production depends on the IL-10–STAT3 signaling pathway. (A) BMDCs were pretreated with or without 10 ng/mL rapamycin along with or without 5 μg/mL BFA for 20 minutes before stimulation with 1 μg/mL LPS. After 4 hours, total RNA was isolated, and IL-12p40 and IL-12p35 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. (B) BMDCs were pretreated with or without 10 ng/mL rapamycin for 20 minutes and then stimulated with 1 μg/mL LPS for the indicated times. Total RNA was isolated, and IL-12p40, IL-12p35, and IL-10 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. (C) BMDCs were pretreated with or without 100 ng/mL rapamycin for 20 minutes and then stimulated with 1 μg/mL LPS for the indicated times. The cell lysates were analyzed for phospho-STAT3, STAT3, p70S6K, and ERK2 by Western blotting. The white lines indicate that intervening lanes have been removed. The right panel indicates relative intensities of tyrosine-phosphorylated STAT3 normalized by STAT3 signals. (D) BMDCs were pretreated with or without 100 ng/mL rapamycin for 20 minutes before stimulation with 10 ng/mL IL-10. After 1 hour, total RNA was isolated, and SOCS3 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. In panels A, B, and D, data are indicated as mean plus or minus SD of duplicate samples. Data are representative of 2 (B,C) or 3 (A,D) independent experiments with similar results. (E) BMDCs from WT or IL-10−/− mice were stimulated with 1 μg/mL LPS in the presence or absence of 10 ng/mL rapamycin for 24 hours and assayed for the production of IL-12p70 by ELISA. Absolute IL-12p70 levels in the stimulation of LPS alone: WT, 24.1 plus or minus 3.6 pg/mL; IL-10−/−, 1120 plus or minus 230 pg/mL. Data are indicated as median plus or minus SD of 3 independent experiments. *P < .05 by Mann-Whitney U test comparing WT with IL-10−/− groups. (F) BMDCs from STAT3−/− mice or littermate controls were stimulated with 0.1 μg/mL LPS in the presence or absence of 100 ng/mL rapamycin for 24 hours. Cytokine production was evaluated as in panel E. Absolute IL-12p70 levels in the stimulation of LPS alone: control, 11.8 plus or minus 3.7 pg/mL; STAT3−/−, 299 plus or minus 67 pg/mL. *P < .05 by Mann-Whitney U test comparing control with STAT3−/− groups. Indicated below are the expression levels of STAT3 and ERK2 in BMDCs determined by Western blotting.

The effect of rapamycin on LPS-induced IL-12 production depends on the IL-10–STAT3 signaling pathway. (A) BMDCs were pretreated with or without 10 ng/mL rapamycin along with or without 5 μg/mL BFA for 20 minutes before stimulation with 1 μg/mL LPS. After 4 hours, total RNA was isolated, and IL-12p40 and IL-12p35 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. (B) BMDCs were pretreated with or without 10 ng/mL rapamycin for 20 minutes and then stimulated with 1 μg/mL LPS for the indicated times. Total RNA was isolated, and IL-12p40, IL-12p35, and IL-10 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. (C) BMDCs were pretreated with or without 100 ng/mL rapamycin for 20 minutes and then stimulated with 1 μg/mL LPS for the indicated times. The cell lysates were analyzed for phospho-STAT3, STAT3, p70S6K, and ERK2 by Western blotting. The white lines indicate that intervening lanes have been removed. The right panel indicates relative intensities of tyrosine-phosphorylated STAT3 normalized by STAT3 signals. (D) BMDCs were pretreated with or without 100 ng/mL rapamycin for 20 minutes before stimulation with 10 ng/mL IL-10. After 1 hour, total RNA was isolated, and SOCS3 mRNA levels were assessed by real-time PCR using cyclophilin A mRNA as a reference. In panels A, B, and D, data are indicated as mean plus or minus SD of duplicate samples. Data are representative of 2 (B,C) or 3 (A,D) independent experiments with similar results. (E) BMDCs from WT or IL-10−/− mice were stimulated with 1 μg/mL LPS in the presence or absence of 10 ng/mL rapamycin for 24 hours and assayed for the production of IL-12p70 by ELISA. Absolute IL-12p70 levels in the stimulation of LPS alone: WT, 24.1 plus or minus 3.6 pg/mL; IL-10−/−, 1120 plus or minus 230 pg/mL. Data are indicated as median plus or minus SD of 3 independent experiments. *P < .05 by Mann-Whitney U test comparing WT with IL-10−/− groups. (F) BMDCs from STAT3−/− mice or littermate controls were stimulated with 0.1 μg/mL LPS in the presence or absence of 100 ng/mL rapamycin for 24 hours. Cytokine production was evaluated as in panel E. Absolute IL-12p70 levels in the stimulation of LPS alone: control, 11.8 plus or minus 3.7 pg/mL; STAT3−/−, 299 plus or minus 67 pg/mL. *P < .05 by Mann-Whitney U test comparing control with STAT3−/− groups. Indicated below are the expression levels of STAT3 and ERK2 in BMDCs determined by Western blotting.

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