Figure 3

The failure of IL-33 to induce mast cell degranulation is associated with lack of intracellular mobilisation of calcium. PDMCs (a,c) and BMMCs (b,d) were sensitized with murine anti-DNP IgE (0.5 μg/ml) or not (a,b) and then stimulated with DNP-HSA (0.5 µg/ml) to induce cross-linking of FcεRI (XL), IL-33 (10 ng/ml) or PMA plus Ionomycin (both 1 μM) for 30 mins at 37 °C. Degranulation was determined as the % β-hexosaminidase released relative to the total activity of the cells and data are presented from a single experiment (a,b) or mean ± SEM values from the combined results of five (c) or three (d; IL-33, n = 2) independent experiments where statistical analysis was by one-way ANOVA with Bonferroni’s post test and *p < 0.05, **p < 0.01 and ***p < 0.001. WT or ST2 ^−/− PDMCs (e–g) and BMMCs (h,i) loaded with Fura-2/AM and sensitised or not (e) with murine anti-DNP IgE (0.5 μg/ml) were stimulated at 50 s in serum-free HBSS with 100 ng/ml IL-33. Calcium mobilisation was recorded in real-time and for the analysis of intracellular mobilisation alone, the cells were stimulated in calcium-free buffer supplemented with 100 µM EGTA to remove all extracellular calcium (−Ca²⁺; g, i). Data are presented as the mean (baseline subtracted) values ± SEM from replicate biological analyses collated from at least two independent experiments as follows: for PDMCs, e: IL-33, n = 3, IL-33 + IgE, n = 4; f: WT n = 6, ST2^−/− n = 5; g: IL-33 n = 7, IL-33-Ca²⁺ n = 7; for BMMCs, h: WT n = 6, ST2^−/− n = 5; i: IL-33 n = 10, IL-33-Ca²⁺ n = 9. Statistical analysis was by two-way ANOVA where ***p < 0.001.