1. Aryl hydrocarbon receptor controls murine mast cell homeostasis
by Yufeng Zhou, Hui-Ying Tung, Ying-Ming Tsai, Shih-Chang Hsu, Hui-Wen Chang, Hirokazu Kawasaki, Hsiao-Chun Tseng, Beverly Plunkett, Peisong Gao, Chih-Hsing Hung, Becky M. Vonakis, and Shau-Ku Huang
Blood
Volume 121(16):
April 18, 2013
©2013 by American Society of Hematology

2. AhR ligands potentiate IgE-mediated mast cell activation.
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 ), 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.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology

3. FICZ-induced enhancement of mediator release is calcium and ROS dependent.
FICZ-induced enhancement of mediator release is calcium and ROS dependent. BMMCs were sensitized with E-C1 ± 1 nM FICZ or equal amount of vehicle as a control (Con) for 16 hours and then stimulated with 10 μg/mL OVA. The release of (A) Hex and (B) LTC4 in the absence or presence of a calcium blocker (2-APB) and an antioxidant (N-acetyl cysteine) was added 30 minutes before OVA stimulation. Data are representative of 3 independent experiments. (C) FICZ enhances oxidation of a PTP (SHP-2) in mast cells. BMMCs were sensitized with E-C1 ± 1 nM FICZ or an equal amount of vehicle as a control for 16 hours and then stimulated with 10 μg/mL OVA for the indicated time. The cells were lysed, and then SHP-1 and SHP-2 were immunoprecipitated (IP). The immunoprecipitates were subjected to alkylation by IAA, followed by reduction of the reversibly oxidized PTPs with dithiothreitol, and by final oxidation with pervanadate. After size fractionation by SDS-PAGE, the samples were subjected to immunoblotting (IB) with oxPTP mAbs and after stripping, with the respective Abs as loading controls. Data are representative of 3 independent experiments. (D) BMMCs were treated as above. OVA-stimulated cells were lysed and SHP-2 was immunoprecipitated, and the relative activity of PTPs was determined by measuring the levels of FDP fluorescence (RFU). *P < .05. Data are representative of 3 independent experiments.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology

4. Growth defect in AKO and AhRd mast cells in response to IL-3.
Growth defect in AKO and AhRd mast cells in response to IL-3. (A) The percentages of c-Kit+FcεRI+CD49b- mast cells. Bone marrow cells were cultured in 30% WEHI-3-conditioned medium, and at indicated time points, aliquots of cultured cells from WT, AhRd, and AKO mice were stained for c-Kit, FcεRI, and CD49b and analyzed by flow cytometry. *P < .05 (WT vs AKO). Data are representative of 3 independent experiments. (B) The relative level of c-kit analyzed by flow cytometry and measured as mean fluorescence intensity (MFI). Inset: c-kit expression detected by real-time reverse-transcription polymerase chain reaction from D31 mast cells.*P < .05. Data are representative of 3 independent experiments. (C) To determine the cell numbers, media were changed weekly and the numbers of suspension cells excluding trypan blue were recorded over 5 weeks. *P < .05 (WT vs AhRd); #P < .05 (WT vs AKO). Data are representative of 3 independent experiments. (D) Mast cells were negatively selected on day 21 (D21) and day 30 (D30) after the initiation of the culture and stimulated with 5 ng/mL IL-3 for 3 days and the cell proliferation was evaluated with Cell counting kit-8. *P < .05. Data are representative of 3 independent experiments. BMMCs were first synchronized at G0/G1 in media devoid of cytokines for 16 hours and then stimulated with IL-3 (5 ng/mL) and/or SCF (20 ng/mL). The percentages of the cell population at (E) the S+G2/M and (F) G1 phases were determined at the 24-hour time point. *P < .05. Data are representative of 3 independent experiments. (G) Analysis of IL-3–induced signaling. Mature BMMCs from WT or AKO mice were first cultured in the cytokine-free media for 6 hours and then stimulated with 5 ng/mL IL-3 for different time points, followed by western blotting analysis of activation of STATs, AKT, and MAPKs in cell lysates by the use of the respective anti-phospho or total protein antibodies. Data are representative of 3 independent experiments.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology

5. Immunohistochemical staining of mast cells.
Immunohistochemical staining of mast cells. Toluidine blue staining (A) and safranin/alcian blue staining (B) of mast cells (indicated by arrows in red) in the back skin from WT and AKO mice at 12 weeks of age (original magnification ×200). Scale bar represents 50 μm. Data are representative of 3 independent experiments. (C) Quantitative analysis of (A) *P < .05, n = 9. (D) Quantitative analysis of peritoneal mast cells by Toluidine blue staining. *P < .05, n = 10. Data are representative of 3 independent experiments.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology

6. AKO mast cells were refractory to IgE/antigen stimulation.
AKO mast cells were refractory to IgE/antigen stimulation. (A) Ca2+-mobilization and influx. BMMCs of WT and AKO were sensitized with IgE (E-C1) for 16 hours and then loaded with the Ca2+ indicator Fluo-3-AM and Fura red-AM. Ca2+ release was elicited by stimulation with 10 μg/mL OVA (Ag) first in Ca2+-free conditions, followed by shifting to Ca2+ (2 mM)-containing buffer 4 minutes later. The resulting changes in intracellular calcium were monitored over time. Data are representative of 3 independent experiments. (B) BMMCs were sensitized with E-C1 and then stimulated with 10 μg/mL OVA for the indicated time. Whole-cell lysates were analyzed by western blot. Data are representative of 3 independent experiments. (C) The levels of degranulation, IL-13 (D), and LTC4 (E) in WT or AKO mast cells sensitized with IgE (E-C1) for 16 hours and stimulated with OVA for 30 minutes for degranulation and LTC4 or 6 hours for IL-13 measurement. *P < .05. Data are representative of 5 independent experiments.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology

7. AKO mast cells demonstrate mitochondrial damage.
AKO mast cells demonstrate mitochondrial damage. (A) Mitochondrial membrane potential was assessed by JC1 staining. Data are representative of 3 independent experiments. (B) Ratios of JC-1 red versus green fluorescence. (C) Mitochondrial mass was detected by MitoTracker green staining. Data are representative of 3 independent experiments. (D) Numerical data of panel C. Data are representative of 3 independent experiments. (E) Transmission electron microscopy images of WT and AKO mast cells. (F) AKO mast cells showed elevated levels of intracellular ROS. Mast cells were cultured in the cytokine-free medium for 4 hours and then labeled with CM-H2DCFDA, and the levels of ROS were detected by flow cytometry. Data are representative of 3 independent experiments. (G) A heatmap of gene-chip array and gene cluster analyses of WT and AKO mast cells. (H) Analysis of apoptosis in WT and AKO mast cells. Mast cells were cultured in the WEHI-3–conditioned medium for 4 weeks. BMMCs were treated with 100 μM H2O2 overnight or using medium (M) as a control, and cellular apoptosis was detected by the use of annexin V and 7-AAD staining. Data are representative of 3 independent experiments.
Yufeng Zhou et al. Blood 2013;121:
©2013 by American Society of Hematology