Proinflammatory Effects of High Mobility Group B1 (HMGB1) Versus LPS and the Mechanism of IL-8 Promoter Stimulation by HMGB1

Background: High mobility group box 1 (HMGB1) is a novel, late mediator of inflammation.This study compared the pro-inflammatory effects of LPS and HMGB1.The transcriptional factors that play an important role in mediating the HMGB1-induced stimulation of IL-8 were also evaluated.Methods: RAW264.7 cells were stimulated with either LPS (100 ng/ml) or HMGB1 (500 ng/ml).The TNF-α, MIP-2 and IL-1β levels in the supernatant were evaluated by ELISA at 0, 2, 4, 8, 12 and 24h after stimulation.An acute lung injury was induced by an injection of LPS (5 mg/kg) or HMGB1 (2.5 mg/kg) into the peritoneum of the Balb/c mice.The lung cytokines and MPO activity were measured at 4h (for LPS) or 24h (for HMGB1) after the injection.The transcriptional factor binding sites for NF-IL6, NF-κB and AP-1 in the IL-8 promoter region were artificially mutated.Each mutant was ligated with pIL-6luc and transfected into the RAW264.7 cells.One hour after stimulation with HMGB1 (500 ng/ml), the cell lysate was analyzed for the luciferase activity.Results: The expression of MIP-2, which peaked at 8h with LPS stimulation, increased sequentially until 24h after HMGB1 stimulation.An intraperitoneal injection of HMGB1, which induced a minimal increased in IL-1β expression, provoked the accumulation of neutrophils the lung.A mutation of AP-1 as well as NF-κB in the IL-8 promoter region resulted in a lower luciferase activity after HMGB1 stimulation.Conclusion: The proinflammatory effects of HMGB1, particularly on IL-8, are mediated by both NF-κB and AP-1.(