Human recombinant HMGB1 increases alveolar epithelial wound closure via an IL-1β-dependent signaling pathway.

<p>(<b>A</b>) Human recombinant HMGB1 increases the rate of wound closure of primary rat ATII cell monolayers. HMGB1 (10 ng/ml) or its vehicle was added to the monolayers after scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 16 h (right panels t = 16 h). Scale bar: 100 µm. In some experiments, glycyrrhizin or its vehicle was added to the monolayers after the scratch. (<b>B</b>) IL-1 receptor antagonist (IL-1RA) prevents HMGB1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. HMGB1 (10 ng/ml) and/or IL-1 receptor antagonist (IL-1RA, 20 µg/ml) or their respective vehicles were added to the monolayers after the scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 16 h (right panels t = 16 h). Scale bar: 100 µm. (<b>C</b>) HMGB1 increases the secretion of IL-1β by primary rat ATII cell monolayers. HMGB1 (10, 50 and 250 ng/ml, 6 h) or fully oxidized HMGB1 (ox HMGB1, 250 ng/ml) or their respective vehicles was added to the cell monolayers, and IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. In some experiments, zVAD or its vehicle was added to the cell medium 30 min prior to HMGB1. (<b>D</b>) Glycyrrhizin prevents HMGB1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. (<b>E</b>) High dose of Human recombinant HMGB1 (1 µg/ml) is required to increase the rate of wound closure of 3T3 fibroblasts. HMGB1 (50, 250, 1000 ng/ml) or its vehicle was added to the monolayers after scratch. (<b>F</b>) HMGB1 does not increase the secretion of IL-1β by 3T3 fibroblasts. HMGB1 (50, 250 and 1000 ng/ml, 6 h) or its vehicle was added to the cell monolayers, and IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. IL-1β (10 ng/ml) and glycyrrhizin (20 µg/ml) or their respective vehicles were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to HMGB1 or IL-1β vehicles. **<i>p</i><0,005 from monolayers exposed to HMGB1.