Figure 1. IL-33 induces granzyme c expression in BMMCs.

(A) The average copy numbers of proteins in the mouse chymase locus detected in BMMCs, NK cells, and CD8 cytotoxic T (CTL) cells proteomes in the ImmPres database (http://immpres.co.uk/). Gene abbreviations are: Cma1, Chymase; Mcpt, Mast Cell protease; Ctsg, Cathepsin G; Gzm, Granzyme. (B) BMMCs were stimulated with 10 ng/ml IL-33 for the indicated times. Cells were then lysed and total RNA isolated. The levels of granzyme B and granzyme C were determined by qPCR and normalised to the level of GAPDH mRNA in the same sample. Data show mean and 95% confidence interval of four stimulations. For comparisons to the 0 h time point, a P<0.05 is indicated by * and P<0.001 by *** (Brown-Forsythe and Welch ANOVA and Dunnett’s post hoc testing). (C–E) BMMCs were stimulated with 10 ng/ml IL-33 for 48 h and lysates generated for proteomics analysis as described in the Methods section and the estimated copy numbers of granzyme B (C), granzyme C (D), and chymase (E) determined. Graphs show mean and standard deviation of four independent cultures. A P<0.05 is indicated by * and P<0.01 by ** (two-tailed unpaired ttest with Welch’s correction). (F–H) BMMCs were incubated with 10 ng/ml IL-33 for the indicated times or maintained in the absence of IL-33 (unstimulated) as indicated. Cells were then fixed, stained for granzyme C, and analysed by flow cytometry as described in the Methods section. A short time course of IL-33 stimulation is shown in (F), while a separate experiment looking at IL-33 treatment for 1–3 days is shown in (G), with representative flow cytometry histograms in (H). The graphs show the mean and standard deviation of three separate stimulations. For comparisons to the unstimulated cells, a P<0.01 is indicated by ** and P<0.001 by ***. Data in (F) were analysed by one-way ANOVA and Dunnett’s post hoc testing and (G) was analysed by two-way ANOVA with Sidak’s post hoc testing between unstimulated and IL-33-stimulated conditions.