The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion

Abstract

High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH(2)-terminal kinase and higher nuclear factor-kappaB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process.

Figure 1.

Pretreatment with neutralizing antibody to HMGB1 protects against liver I/R injury. (a) Sham mice and mice that underwent ischemia and 6 h of reperfusion were treated with anti-HMGB1 antibody (60 μg or 600 μg) or control antibody i.p. 1 h before ischemia. Serum ALT levels were analyzed as a measure of hepatocellular injury. Data represent means ± SE, n = six mice per group. *P < 0.05 versus mice subjected to I/R given control antibody. (b) Sham mice and mice that underwent ischemia and 24 h of reperfusion were treated with anti-HMGB1 antibody (600 μg) or control antibody i.p. 1 h before ischemia. Data represent means ± SE, n = six mice per group. *P < 0.05 versus mice that were subjected to I/R and given control antibody. (c) Hematoxylin-eosin–stained liver sections from control antibody and anti-HMGB1 antibody–treated animals 24 h after reperfusion (original magnification ×200). Images are representative liver sections from six mice per group.

Figure 2.

HMGB1 expression is up-regulated in hepatocytes by hypoxia and in the liver after I/R. (a) Mice underwent 60 min of ischemia and various lengths of reperfusion. Western blot analysis for cellular HMGB1 was performed for hepatic protein lysates of the ischemic lobes at the time points shown, with each lane representing a separate animal. Blot shown is representative of three experiments with similar results. (b) Immunofluorescent stain of HMGB1 from sections of normal liver and (c) liver subjected to 60 min of ischemia and 6 h of reperfusion (original magnification ×400). Images are representative liver sections from six mice per group. Red, HMGB1; blue, nuclei; green, F-actin. (d) Cultured rat hepatocytes were exposed to hypoxia (1% O₂) from 0 to 24 h. Whole cell lystate and media were subjected to Western blot analysis of HMGB1. Blot shown is representative of three experiments with similar results.

Figure 3.

Delayed administration of neutralizing antibody to HMGB1 protects against liver I/R injury. Sham mice and mice that underwent ischemia and 6 hours of reperfusion were treated with anti-HMGB1 antibody (600 μg) or control antibody immediately after reperfusion. Serum ALT levels were analyzed as a measure of hepatocellular injury. Data represent means ± SE, n = six mice per group. *P < 0.05 versus mice that were subjected to I/R and given control antibody.

Figure 4.

Neutralizing antibody to HMGB1 decreases production of inflammatory mediators. Hepatic TNF-α (a), IL-6 (b), and iNOS (c) mRNA expression were measured after ischemia and 1 h of reperfusion in mice that were treated with anti-HMGB1 antibody or control antibody. Results were obtained using real time RT-PCR and expressed as relative increase of mRNA expression compared with sham animals. Data represents means ± SE, n = eight mice per group. *P < 0.05 versus mice that were subjected to I/R and given control antibody.

Figure 5.

Neutralizing antibody to HMGB1 modulates inflammatory signaling pathways. (a) Mitogen-activated protein kinase activation was determined in sham mice and mice that underwent ischemia and 1 h of reperfusion. Animals were treated with anti-HMGB1 antibody or control antibody. Western blot analysis for phosphorylated and total ERK, p38, and JNK was performed. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. Blot shown is representative of three experiments with similar results. (b) NF-κB activation during hepatic I/R injury was assessed. Mice that underwent ischemia and 1 h of reperfusion were treated with anti-HMGB1 antibody or control antibody. Nuclear extracts were prepared from the ischemic livers and subjected to electrophoretic mobility shift assay. Assay shown is representative of three experiments with similar results.

Figure 6.

Administration of recombinant HMGB1 worsens liver I/R injury. Serum ALT levels were measured in sham mice and mice that underwent ischemia and 6 h of reperfusion. Animals were treated with a nonlethal dose of recombinant HMGB1 (20 μg) or vehicle PBS immediately after reperfusion. Data represent means ± SE, n = four mice per group. *P < 0.05 versus mice that were subjected to I/R and given vehicle PBS.

Figure 7.

Hepatic I/R injury involves TLR4 system and is independent of LPS-TLR4 interaction. (a) TLR4-defective mice (C3H/Hej) and their wild-type (WT) mice counterparts (C3H/HeOuj) were subjected to liver ischemia and 6 h of reperfusion. Animals were treated with anti-HMGB1 antibody or control antibody 1 h before ischemia and sALT levels were measured. Data represent means ± SE, n = four to six mice per group. *P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given control antibody. (b) CD14^+/+ and CD14^−/− mice were subjected to liver ischemia and 6 h of reperfusion. Animals were treated with anti-HMGB1 antibody or control antibody at reperfusion. Data represent means ± SE, n = 5 mice per group. *, P < 0.05 versus CD14^+/+ mice subjected to I/R given control antibody. **, P < 0.05 versus CD14^−/− mice subjected to I/R given control antibody.

Figure 8.

HMGB1-mediated I/R injury involves TLR4 system. (a) Hepatic TNF-α and IL-6 mRNA expression were obtained in TLR4-defective mice and wild-type mice after ischemia and 6 h of reperfusion. Mice were treated with anti-HMGB1 antibody or control antibody. Results are expressed as relative increase of mRNA expression compared with sham animals. Data represent means ± SE, n = four to six mice per group. *P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given control antibody. (b) Serum ALT levels were determined in TLR4-defective mice and wild-type mice after ischemia and 6 h of reperfusion. Animals were treated with recombinant HMGB1 or vehicle PBS immediately after reperfusion. Data represent means ± SE, n = four to six mice per group. *P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given vehicle PBS.