(A.) HuMCs were cultured with or without IL-33 for 7–9 wk and cytospins were stained with toluidine blue and analyzed by light microscopy. The scale bar is 10 μm. (B.) FcεRI and KIT expression in cells from A. was analyzed by flow cytometry. (C.) Naïve HuMCs were sensitized overnight with biotinylated IgE (100 ng/ml) and challenged with various stimulants individually, or in the indicated combinations: SA (streptavidin, 100 ng/ml), IL-33 (10 ng/ml), or SCF (30 ng/ml), and the release of β-hexosaminidase was determined. (D., E.) Cells in A. were sensitized with biotinylated Hu IgE overnight, then stimulated with antigen (streptavidin) alone (D.) or in the presence of SCF (E.) and degranulation after 30 min (β-hex release) (F.) Cells were sensitized and treated as in C. and the calcium signal was determined. (G.) The calcium signal in the cells sensitized and triggered as in D. and E. was similarly assessed. Data in A., B., F., and G. are representative of 3 experiments conducted on separate cell preparations. In C., the results are means ± S.E. of two separate experiments conducted in duplicate or triplicate. Data in D. and E. are represented as the means ± S.E. (n=6 to 14; *: P<0.05, Student’s t-test).

(A.) Developing HuMC cultures were exposed to IL-33 (10 ng/ml) for indicated times prior to examining FcεRI-dependent degranulation in mature (8 wk) HuMCs. (B.) Mature HuMCs were incubated with IL-33 (10 ng/ml) for 1, 4, 12, 24, 48, 72 h and 120 h, then degranulation (β-hex release) of IgE-biotin sensitized cells was determined. (C.) Data from B. re-plotted as a time course using the 10 ng/ml antigen concentration. (D., E.) HuMCs were cultured with IL-33 for 7 weeks, IL-33 removed for an additional 2 weeks, then degranulation (β-hex release) of IgE-biotin sensitized cells determined. Data are represented as the means ± S.E. (n=3; *: P<0.05 for comparison with cells cultured in the absence of IL-33, Student’s t-test).

(A.) BMMCs were cultured with or without IL-33 during the 4–6 wk development, sensitized with DNP-specific mouse IgE overnight, then stimulated with the indicated concentrations of DNP-HSA (antigen) (B.) BMMCs were cultured with or without IL-33 for 4–6 wk and FcεRI and KIT expression was analyzed by flow cytometry. (C.) Cells were sensitized and cultured as in A. then stimulated with DNP-HSA (Ag) or SCF alone or in combination and calcium response determined. (D.) IgE-sensitized mice were injected with of IL-33 (2 μg), DNP-HSA (200 μg) or PBS retro-orbitally, and the change in core body temperature was monitored. (E., F.) Peritoneal cells from PBS treated (E.) and from mice treated for 12 days with IL-33 (6 doses of 1 μg on alternative days) and sensitized with DNP-specific mouse IgE (2 doses of 1 μg on alternative days prior to stimulation) (F.) were labeled with a KIT-specific antibody and Fluo-4 AM. The calcium responses of the KIT-positive population of these cells to vehicle alone (Ctrl) or antigen (Ag, DNP-HSA; 100 ng/ml) were determined by acquisition of their Fluo-4 AM fluorescence using flow cytometry and two-second averaged geometric mean fluorescence (MFI) was analyzed. (G.) Delta MFI values (see methods) for PBS- (E.) and IL-33- (F.) treated cells were calculated and normalized responses evaluated. Data in A. are the means ± S.E. (n=14) and in B. and C. are representative of 3 experiments conducted on separate cell preparations. In D., data are presented as the means ± S.E. of n=4 and the difference among the groups of mice is indicated (*: P<0.05, two-way ANOVA with Bonferroni’s posttest). Data in G. are presented as the means ± S.E. of n=4. The arrows in E. and F. indicate the time of cell challenge. *: P<0.05 for comparison with cells not exposed to IL-33, Student’s t-test)

(A.) BMMCs, developed from wild type (WT) or ST2^−/− mice, cultured in the absence or presence of IL-33 (10 ng/ml) for 4 wk were sensitized then challenged with DNP-HSA (Ag) for 30 min and degranulation (β-hex release) determined. (B., C.) BMMCs, developed from wild type (WT) or MyD88^−/− mice, cultured in the absence or presence of IL-33 (10 ng/ml) for 4 wk, were sensitized then challenged with DNP-HSA (Ag) (B.) or Ag with SCF (C.) for 30 min and degranulation (β-hex release) determined. (D.) The calcium signal in WT and MyD88^−/− BMMCs, sensitized and treated as described in B. and C. was determined by Fura-2. (E.) Conditioned media from WT cells treated or not with IL-33 was added to the MyD88^−/− BMMCs for 2 wk then degranulation (β-hex release) determined. Data are represented as the means ± S.E. of n=3 (A., E.) or (n=6 (B., C.) or are representative of n=3 (D.); *: P<0.05, Paired Student’s t-test.

(A.) The Hck mRNA and protein expression in BMMCs cultured with or without IL-33 for 2 wk were evaluated by real-time PCR and immunoblotting. (B.) Sensitized BMMCs, cultured in the absence or presence of IL-33 (10 ng/ml) for the duration of the culture were challenged with DNP-HSA (Ag; 10 ng/ml) in the presence or absence of SCF (10 ng/ml) for 2 min then phospho- and total proteins detected by immunoblotting. (C.) Immunoblots from B. were normalized to loading control (Syk; see ) and evaluated. (D.) PLCγ₁ expression in MyD88^−/− BMMCs, cultured in the absence or presence of IL-33 (10 ng/ml) as described for B. In A., B., and D., the immunoblots are representative of 3 experiments conducted on separate cell preparations. Data in A. and C. are represented as the means ± S.E. (n=3–8; *: P<0.05, Student’s t-test).

Sensitized BMMCs, incubated or not in IL-33 (10 ng/ml) for the duration of the culture, were challenged with DNP-HSA (10 ng/ml) for 2 min or 10 min. Cells were stained for F-actin (green) and nuclei (blue). The degree of polymerization was evaluated from the normalized fluorescence intensities. The scale bar is 20 μm and the data are represented as the means ± S.E. (n=3; *: P<0.05 for comparison with cells cultured in the absence of IL-33 by Student’s t-test).