Methods: Immunofluorescence (IF) was performed on esophageal biopsies from patients with eosinophilic esophagitis or control individuals. Biochemical fractionation, IF, fluorescence recovery after photobleaching (FRAP), and enzyme-linked immunosorbent assay (ELISA) were performed on an esophageal epithelial cell line (TE-7) overexpressing IL-33 via lentiviral transduction.
Results: IF for IL-33 revealed only nuclear staining in esophageal epithelial cells in vitro and during allergic inflammation in vivo. Biochemical fractionation demonstrated IL-33 enrichment in the chromatin fraction compared with heat shock protein 90. Assessment of nuclear mobility by FRAP demonstrated that GFP-tagged full-length IL-33 had a 70% recovery time of 29 seconds compared to 3 seconds for IL-1 alpha. A truncated form of IL-33 (IL-33112-270) which lacks the chromatin binding domain was highly mobile within the nucleus similar to the GFP-alone control. Functionally, IL-33112-270 demonstrated increased extracellular release following induction of necrosis with calcium ionophore or hydrogen peroxide (8 to 20-fold, p < 0.0001) compared with chromatin-bound full-length IL-33.
Conclusions: In summary, herein we have demonstrated that IL-33 binding to chromatin is dynamic and regulates its availability for release during necrosis. We propose that intracellular retention of IL-33 during necrosis may serve as a mechanism to curtail inflammatory responses in allergic disease.