Figure 3: Free cysteines control the conformational switch in IL-33.

(a) SDS–PAGE of human N-terminal His-Avi tagged IL-33^112–270 following overnight treatment as indicated. (b) NMR analysis with overlay of the ¹H–¹⁵N HMQC spectra for ¹⁵N-labelled human IL-33 following 0 and 10 h incubation in cell culture media without air (left panel) or with air (right panel). Media was degassed before addition of IL-33. No air was added (left panel) or air was pumped through the sample for 5 min (right panel), representative of two separate experiments. (c) Signalling in human umbilical vein endothelial cells (HUVEC) stimulated by untreated or media treated human IL-33^112–270 WT or complete Cys→Ser mutant. NFκB p65/RelA translocation was measured at 30 min post stimulation. Data points are mean±s.e.m. of duplicate determinations, representative of three independent experiments. (d) Stimulation of IL-6 production from human cord blood derived mast cells with human IL-33^112–270 WT or Cys→Ser mutant. Data points are mean±s.e.m. of duplicate determinations, representative of two independent experiments. (e,f) Concentration of BALF IL-13 (e) or eosinophil counts (f) following 3 consecutive daily intranasal challenges of BALB/c mice with PBS (n=4 per group), human IL-33 WT or Cys→Ser mutant (n=6 per group). Endpoint was 24 h after final challenge. Statistical comparisons were made using one way ANOVA with Bonferroni multiple comparisons test (*P<0.05, **P<0.01, ***P<0.001). (g) NFκB p65/RelA translocation in human umbilical vein endothelial cells (HUVEC) stimulated by L-cystine treated human IL-33^112–270 WT or Cys→Ser mutants. Data points are mean±s.e.m. of duplicate determinations, representative of two independent experiments.