Inhibitors of mTOR signaling dose-dependently decrease SP-stimulated proinflammatory mediator release from human MCs. (A and B) LAD2 MCs (0.5 × 10⁶ cells) were pretreated with the mTOR inhibitor rapamycin (0.05–0.2 µM, 24 hours) or the ATP-competitive mTOR inhibitor Torin1 (0.05–0.2 µM, 24 hours) prior to stimulation with SP (1 μM) for 24 hours to measure release of TNF (A) and CXCL8 (B) by ELISA. (C) LAD2 MCs (0.5 × 10⁶ cells) were also pretreated with the upstream PI3K inhibitor LY294002 (1–10 µM, 2 hours) prior to stimulation with SP (1 μM) for 24 hours to measure release of TNF and CXCL8 mediators. All inhibitors were dissolved in water or DMSO with a final concentration of <0.1%. All conditions were performed in triplicate for each data set and were repeated three times. Results are presented as means ± S.D. Significance of comparisons was assessed for stimulated cells without any inhibitor (control) and for those with an inhibitor/flavonoid, as denoted by the horizontal lines that indicate significance at P < 0.001 for each comparison made. All inhibitor/flavonoid treatments were also compared among themselves, and the vertical brackets indicate the corresponding levels of significance when present (*P < 0.05; **P < 0.001; ***P < 0.0001).

Human MC proinflammatory mediator release in response to NT or SP is attenuated by PI3K/mTOR inhibitors and the flavonoids luteolin and methoxyluteolin. (A–F) LAD2 MCs (0.5 × 10⁶ cells) were pretreated with the mTOR inhibitors rapamycin (Rap) and Torin1 (0.2 µM, 24 hours), the upstream PI3K inhibitor LY294002 (LY; 10 µM, 2 hours), or the natural flavonoids luteolin (Lut) and methoxyluteolin (Methlut; 50 µM, 30 minutes) and then stimulated with NT (10 μM) (A–C) or the positive control trigger SP (1 μM) (D–F) for 24 hours to measure release of TNF, CXCL8, and VEGF mediators by specific ELISAs. All conditions were performed in triplicate for each data set and were repeated three times, with results presented as means ± S.D. Significance of comparisons was assessed for stimulated cells without any inhibitor (control) and for those with an inhibitor/flavonoid, as denoted by the horizontal lines that indicate significance at P < 0.0001 for each comparison made. All inhibitor/flavonoid treatments were also compared among themselves, and the horizontal brackets indicate the corresponding levels of significance when present (*P < 0.05; **P < 0.001; ***P < 0.0001).

Human LAD2 MC proinflammatory mediator gene expression in response to NT or SP is inhibited by the PI3K/mTOR inhibitors luteolin and methoxyluteolin. (A–F) LAD2 MCs (1 × 10⁶ cells) were pretreated with the mTOR inhibitors rapamycin (Rap) and Torin1 (0.2 µM, 2 or 24 hours), the upstream PI3K inhibitor LY294002 (LY; 10 µM, 2 hours), or the natural flavonoids luteolin (Lut) and methoxyluteolin (Methlut; 50 µM, 30 minutes) prior to stimulation with NT (10 μM) (A–C) or the positive control trigger SP (1 μM) (D–F) for 6 hours to measure gene expression of TNF, CXCL8, and VEGF by quantitative real-time polymerase chain reaction. All inhibitors were dissolved in water or DMSO with a final concentration of <0.1%. All conditions were performed in triplicate for each data set and were repeated three times, with results presented as means ± S.D. Significance of comparisons was assessed for stimulated cells without any inhibitor (control) and for those with inhibitor/flavonoid, as denoted by the horizontal lines that indicate significance at P < 0.0001 for each comparison made. All inhibitor/flavonoid treatments were also compared among themselves, and the horizontal brackets indicate the corresponding levels of significance when present (*P < 0.05; **P < 0.001; ***P < 0.0001).

NT and SP stimulate mTOR activation in human LAD2 MCs. (A–F) LAD2 MCs (1 × 10⁶ cells) were stimulated with NT (10 µM) (A–C) or the positive control trigger SP (1 μM) (D–F) for 0–60 minutes to probe for the total and phosphorylated levels of mTOR and substrates p70S6K and 4EBP1 by Western blot analysis. Peak mTOR activation was denoted at 20 minutes for pmTORSer2448 and pp70S6KThr389 proteins, whereas the levels of p4EBP1Thr37/46 protein remained unchanged. Results were quantified using densitometric analyses for pmTORSer2448 to mTOR levels (B and D) and pp70S6KThr389 to p70S6K levels (B and E) in response to NT (10 µM) or SP (1 μM). All conditions were performed in triplicate for each data set and were repeated three times, with results presented as means ± S.D. Significance of comparisons was assessed for unstimulated cells without NT or SP treatment and for those stimulated by the neuropeptides (*P < 0.05; **P < 0.001; ***P < 0.0001).