AhR ligands potentiate IgE-mediated mast cell activation. (A) A low dose of AhR ligands enhances IgE-mediated mast cell degranulation. BMMCs from C57BL/6 mice were sensitized with 1 μg/mL anti-OVA IgE (E-C1) ± FICZ or an equal amount of vehicle as a control (Con) for 16 hours and then stimulated with 10 μg/mL OVA for 30 minutes. Degranulation was monitored by the release of Hex. *P < .05. Data are representative of 3 independent experiments. (B) BMMCs were sensitized with E-C1 ± 1 nM FICZ or an equal amount of vehicle as control (Con) for 16 hours, then washed and stimulated with 10 μg/mL OVA for 30 minutes. *P < .05. Data are representative of 5 independent experiments. (C) AhR ligands enhance IgE-mediated IL-13 production. BMMCs were sensitized with E-C1 ± FICZ or equal amount of vehicle as control (Con) for 16 hours, then washed and stimulated with 10 μg/mL OVA for 6 hours. *P < .05. Data are representative of 3 independent experiments. (D) Analysis of PCA. Mice were injected intradermally with phosphate-buffered saline or 200 ng E-C1 IgE mAbs ± 10 nM or 100 nM FICZ. After 24 hours, 1 mg OVA was administered intravenously together with Evans blue dye, followed by measurement of the extravasation of Evans blue into the skin. In each group, n = 6. *P < .05. Data are representative of 3 independent experiments. (E) Intracellular Ca²⁺. BMMCs were sensitized with E-C1 ± 1 nM FICZ or equal amount of vehicle as control for 16 hours, loaded with the Ca²⁺ indicator Fluo-3-AM and Fura red-AM, and resuspended in 2 mM Ca²⁺ buffer stimulated with 10 μg/mL OVA for 5 minutes. Data are representative of 3 independent experiments. (F) Immunoblotting analysis of whole-cell lysates. BMMCs were sensitized as above, and then the cells were stimulated with 10 μg/mL OVA for the indicated time periods. For detection of phosphorylated and total proteins, 2 equal samples (1 for each) were loaded on the same Criterion XT Bis-Tris Gel (26 wells; Bio-Rad, catalog number 345-0125), so that all the samples were done on 1 gel and transferred to 1 membrane simultaneously. After the transfer to the same membrane, the membrane was cut and each group of samples was detected for phosphorylated and total proteins in parallel. Data are representative of 3 independent experiments. (G) Increased ROS levels in FICZ-exposed, IgE-activated mast cells. BMMCs were sensitized and treated as above, loaded with 5 μM CM-H2DCFDA, and stimulated with 10 μg/mL OVA. The fluorescence was monitored at 30-second intervals using a microplate fluorometer. An inhibitor of Ca²⁺ signaling (2-APB) was added 30 minutes before OVA stimulation. Data are representative of 3 independent experiments.