Fig. 8

IL-33 and histones synergistically activate ST2 signaling. a Acid-purified histones were run by electrophoresis on an SDS-PAGE gel and subjected to Coommassie blue staining; the right lane depicts the indicated molecular weight markers. b Supernatants from HMC-1 mast cells treated overnight with 6.7 µg/mL acid-purified histones or 23.4 nM recombinant wild-type (WT) IL-33 as indicated were subjected to analysis by a 42-cytokine multiplex array. Heat map shows fold change (log10) compared to untreated samples. Bolded cytokines have a ≥2-fold change between samples treated with IL-33 plus histones compared to IL-33 alone. c HMC-1 mast cells were treated with anti-ST2 or control IgG for 1 h with subsequent overnight treatment with 6.7 μg/mL of acid-purified histones and 11.7 nM IL-33 as indicated. IL-8 levels in the supernatants were determined by ELISA. d–f IL-8 levels in supernatants of HMC-1 mast cells treated overnight with indicated amounts of wild-type IL-33 (d), truncated IL-33 (e), or IL-1α (f) protein in the presence of 6.7 µg/mL of acid-purified histones as indicated. g–i Normalization of data from d–f depicting fold change as a function of the amount of histones and IL-33 (g–h) or IL-1α (i) compared to IL-33 or IL-1α treatment alone. Untreated samples and samples receiving only histone are excluded from the graphs. j Representative western blot analysis of phospho-NFκB and total NFκB expression in cell lysates of HMC-1 mast cells treated for 20 min with 4.0 µg/mL acid-purified histones or 3.5 nM IL-33 with quantification of proportion of NFκB protein that was phosphorylated in k. The data in (c) are representative of five independent experiments, d–i are representative of 2–3 independent experiments, and (k) are cumulative data of three independent experiments. All graphs depict mean ± standard error of the mean. ****, p < 0.0001 by Student’s t-test; *, p < 0.05 by one-way ANOVA with Holm–Sidak correction for multiple comparisons. ELISA: enzyme-linked immunosorbent assay, Ig: immunoglobulin, IL interleukin, NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells, SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis, ST2: suppressor of tumorigenicity 2