[High mobility group box-1 protein activates endothelial cells to produce cytokines and has synergistic effect with lipopolysaccharide in inducing interleukin-6 release]

Abstract

Objective: To observe the effects of high mobility group box 1 (HMGB1) protein on the release of cytokines of human umbilic vein endothelial cells (HUVECs) and the expression of interleukin-6 (IL-6) induced by lipopolysaccharide (LPS).

Methods: HUVECs were cultured and then divided into the following groups. (1) Recombinant HMGB1 protein of the terminal concentration of 15 ng/ml was added into the culture fluid of HUVECs, 24 hours later the supernatant was collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and interleukin-10, IL-12, IL-1beta, IL-2, IL-4, IL-6, and IL-8, by using LiquiChip system. (2) HMGB1 protein of the terminal concentrations of 3, 15, or 75 ng/ml was added into the culture fluid of the HUVECs, 24 hours later the supernatants were collected to detect the level of IL-6. (3) HMGB1 protein of the terminal concentration of 15 ng/ml was added into the culture fluid of HUVECs, 1, 3, 6, 12, and 24 hours after the stimulation the supernatant was collected to detect the level of IL-6. (4) The culture fluid of HUVECs was added with HMGB1 protein of the terminal concentration of 15 ng/ml and/or LPS of the concentration of 10 ng/ml, 24 hours later the supernatant was collected to detect the level of IL-6. Culture fluid not added with HMGB1 protein was used as control in any kind of test.

Results: (1) The levels of GM-CSF, IFN-gamma, IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) secreted by the HMGB1 stimulated HUVECs were 5, 7, 4.2, 27.8, 12.8, and 5.4 times that of those control group (all P < 0.01) (2) The levels of IL-6 secreted by the HUVECs stimulated with HMGB1 protein of the concentrations of 3, 15 and 75 ng/ml respectively were 155 pg/ml +/- 33 pg/ml, 901 pg/ml +/- 184 pg/ml, and 1508 pg/ml +/- 379 pg/ml respectively, all significantly higher than that of the control group (32 pg/ml +/- 21 pg/ml, all P < 0.01). (3) Since 3 h after the stimulation of HMGB1 protein the level of IL-6 began to increase gradually. 6, 12, and 24 hours after the stimulation the levels of IL-6 secreted by the HUVEC were 75 pg/ml +/- 22 pg/ml, 453 pg/ml +/- 78 pg/ml, and 901 pg/ml +/- 184 pg/ml respectively, all significantly higher than that of the control group (32 pg/ml +/- 21 pg/ml, all P < 0.01). (4) When the HUVECs were stimulated individually by LPS (10 ng/ml) or HMGB1 (15 ng/ml), the levels of IL-6 increased to 289 pg/ml +/- 42 pg/ml and 901 pg/ml +/- 183.6 pg/ml respectively (both P < 0.01); when the HUVECs were costimulated by LPS and HMGB1, the level of IL-6 increased to 2360 pg/ml +/- 299 pg/ml, which showing that there was a synergistic effect between HMGB and LPS (F = 69.405, P < 0.001).

Conclusion: HMGB1 activates HUVECs to produce multiple inflammatory cytokines and induces HUVEC to secret IL-6 in a dose- and time-dependent manner. HMGB1 can also act synergistically with LPS in inducing IL-6 release, which may play an important role in the development of sepsis.