Protective effects of an anti-4-HNE monoclonal antibody against liver injury and lethality of endotoxemia in mice.

4-hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product that is known to be elevated during oxidative stress. During systemic inflammation and endotoxemia, plasma levels of 4-HNE are elevated in response to lipopolysaccharide (LPS) stimulation. 4-HNE is a highly reactive molecule due to its generation of both Schiff bases and Michael adducts with proteins, which may result in modulation of inflammatory signaling pathways. In this study, we report the production of a 4-HNE adduct-specific monoclonal antibody (mAb) and the effectiveness of the intravenous injection of this mAb (1 mg/kg) in ameliorating LPS (10 mg/kg, i.v.)-induced endotoxemia and liver injury in mice. Endotoxic lethality in control mAb-treated group was suppressed by the administration of anti-4-HNE mAb (75 vs. 27%). After LPS injection, we observed a significant increase in the plasma levels of AST, ALT, IL-6, TNF-α and MCP-1, and elevated expressions of IL-6, IL-10 and TNF-α in the liver. All these elevations were inhibited by anti-4-HNE mAb treatment. As to the underlining mechanism, anti-4-HNE mAb inhibited the elevation of plasma high mobility group box-1 (HMGB1) levels, the translocation and release of HMGB1 in the liver and the formation of 4-HNE adducts themselves, suggesting a functional role of extracellular 4-HNE adducts in hypercytokinemia and liver injury associated with HMGB1 mobilization. In summary, this study reveals a novel therapeutic application of anti-4-HNE mAb for endotoxemia.

Treatment of Marmoset Intracerebral Hemorrhage with Humanized Anti-HMGB1 mAb.

Intracerebral hemorrhage (ICH) is recognized as a severe clinical problem lacking effective treatment. High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity once released into the extracellular space from the nuclei. We previously demonstrated that intravenous injection of rat anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain injury in a rat ICH model. Therefore, we developed a humanized anti-HMGB1 mAb (OKY001) for clinical use. The present study examined whether and how the humanized anti-HMGB1 mAb ameliorates ICH injury in common marmosets. The results show that administration of humanized anti-HMGB1 mAb inhibited HMGB1 release from the brain into plasma, in association with a decrease of 4-hydroxynonenal (4-HNE) accumulation and a decrease in cerebral iron deposition. In addition, humanized anti-HMGB1 mAb treatment resulted in a reduction in brain injury volume at 12 d after ICH induction. Our in vitro experiment showed that recombinant HMGB1 inhibited hemoglobin uptake by macrophages through CD163 in the presence of haptoglobin, suggesting that the release of excess HMGB1 from the brain may induce a delay in hemoglobin scavenging, thereby allowing the toxic effects of hemoglobin, heme, and Fe2+ to persist. Finally, humanized anti-HMGB1 mAb reduced body weight loss and improved behavioral performance after ICH. Taken together, these results suggest that intravenous injection of humanized anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

Histamine induced high mobility group box-1 release from vascular endothelial cells through H1 receptor.

Background: Systemic allergic reaction is characterized by vasodilation and vascular leakage, which causes a rapid, precipitous and sustained decrease in arterial blood pressure with a concomitant decrease of cardiac output. Histamine is a major mediator released by mast cells in allergic inflammation and response. It causes a cascade of inflammation and strongly increases vascular permeability within minutes through its four G-protein-coupled receptors (GPCRs) on endothelial cells. High mobility group box-1 (HMGB1), a nonhistone chromatin-binding nuclear protein, can be actively secreted into the extracellular space by endothelial cells. HMGB1 has been reported to exert pro-inflammatory effects on endothelial cells and to increase vascular endothelial permeability. However, the relationship between histamine and HMGB1-mediated signaling in vascular endothelial cells and the role of HMGB1 in anaphylactic-induced hypotension have never been studied. Methods and results: EA.hy 926 cells were treated with different concentrations of histamine for the indicated periods. The results showed that histamine induced HMGB1 translocation and release from the endothelial cells in a concentration- and time-dependent manner. These effects of histamine were concentration-dependently inhibited by d-chlorpheniramine, a specific H1 receptor antagonist, but not by H2 or H3/4 receptor antagonists. Moreover, an H1-specific agonist, 2-pyridylethylamine, mimicked the effects of histamine, whereas an H2-receptor agonist, 4-methylhistamine, did not. Adrenaline and noradrenaline, which are commonly used in the clinical treatment of anaphylactic shock, also inhibited the histamine-induced HMGB1 translocation in endothelial cells. We therefore established a rat model of allergic shock by i.v. injection of compound 48/80, a potent histamine-releasing agent. The plasma HMGB1 levels in compound 48/80-injected rats were higher than those in controls. Moreover, the treatment with anti-HMGB1 antibody successfully facilitated the recovery from compound 48/80-induced hypotension. Conclusion: Histamine induces HMGB1 release from vascular endothelial cells solely through H1 receptor stimulation. Anti-HMGB1 therapy may provide a novel treatment for life-threatening systemic anaphylaxis.