Journal List > Tuberc Respir Dis > v.62(4) > 1001078

Jeon, Kwak, Song, Lee, Chung, Choi, Shin, Park, Choi, and Kim: Proinflammatory Effects of High Mobility Group B1 (HMGB1) Versus LPS and the Mechanism of IL-8 Promoter Stimulation by HMGB1

Abstract

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.

Figures and Tables

Figure 1
The structure of human HMGB1 protein (Li J et al, J Immunol Methods, 2004).
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Figure 2
Flow sheet of luciferase assay.
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Figure 3
The chronological expression of TNF-α in the supernatant of RAW264.7 cell after stimulation with LPS (100 ng/ml)(A) or HMGB1 (500 ng/ml)(B).
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Figure 4
The chronological expression of MIP-2 in the supernatant of RAW264.7 cell after stimulation with LPS (100 ng/ml)(A) or HMGB1 (500 ng/ml)(B).
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Figure 5
The expression of lung TNF-α (A), MIP-2 (B) and IL-1β (C) at 24 hour after HMGB1 (2.5 mg/kg) or 4 hour after LPS (5 mg/kg) injection into peritoneum.
*p<.05 to control, p<.05 to HMGB1
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Figure 6
The MPO activity of lung at 24 hour after HMGB1 (2.5 mg/kg) or 4 hour after LPS (5 mg/kg) injection into peritoneum.
*p<.05 to control, p<.05 to HMGB1
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Figure 7
Luciferase activity of control (pIL-6luc), wild type (-133luc), mutant of NF-IL6 (-133 lucmNF-IL6), mutant of NF-κB (-133lucmNF-κB) and mutant of AP-1 (-133lucmAP-1) at one hour after HMGB1 (500 ng/ml) stimulation.
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