[High mobility group box-1 stimulates proinflammatory cytokine production in endothelial cells via MAP kinases]

Abstract

Objective: To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.

Methods: HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.

Results: The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng<ml) or HMGB1 (15 ng/ml) alone resulted in significantly increased levels of IL-8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.

Conclusion: HMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.