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


CONCLUSIONS
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.
Liu
Jing-Hua
JH
Key Laboratory of Transcriptomics and Proteomics of Human Diseases of the Ministry of Education of China and Key Laboratory of Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China.
Li
Zhi-Jie
ZJ
Tang
Jing
J
Liu
Ya-Wei
YW
Zhao
Lei
L
Deng
Peng
P
Jiang
Yong
Y
D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D024243:HMGB1 Protein / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D016207:Cytokines; D024243:HMGB1 Protein; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor