Fig. 2.
Effect of IL-38 on cytokine/chemokine release in cocultures of human primary bronchial epithelial cells (HBE) and eosinophils (EOS) upon stimulation by poly (I:C)/LyoVec or TNF-α. Primary bronchial epithelial cells (1 × 105) and purified eosinophils (3 × 105) were cocultured with or without human IL-38 pretreatment for 10 min, followed by poly (I:C)/LyoVec (2 μg/ml) or TNF-α (20 ng/ml) treatment for an additional 20 h. Release of a IL-6, b CCL5, c CXCL10, d IL-1β, and e IFN-β into the supernatant of the poly (I:C)/LyoVec-treated coculture was determined. f Gating strategies for bronchial epithelial cells and eosinophils are shown. Bronchial epithelial cells and eosinophils in the cocultures were gated based on the FSC and SSC parameters. The cell surface expression of ICAM-1 on g HBE/EOS single-cultured cells and h cocultured cells was analyzed by flow cytometry. The levels of i IL-6, j CCL5, and k CXCL10 in the supernatant of the TNF-α-treated coculture were measured. The expression of ICAM-1 on l HBE/EOS single-cultured cells and m cocultured cells after stimulation with TNF-α was determined by flow cytometry. A negative control of 56 °C heat-inactivated human IL-38 (100 ng/ml) and a positive control of dexamethasone (1 μM) were included. Abbreviations: HBE, human primary bronchial epithelial cells; PLV, poly (I:C)/LyoVec; EOS, eosinophils; HBE-EOS, coculture of human bronchial epithelial cells and eosinophils; Co-HBE, human primary bronchial epithelial cells in coculture; and Co-EOS, eosinophils in coculture. The results are shown as the mean ± SEM of triplicate independent experiments with a total of three donors. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared between the denoted groups