Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group–1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.

A neural circuit that involves a specialized population of memory T cells regulates the immune response.

A key question in immunology concerns how sterile injury activates innate immunity to mediate damaging inflammation in the absence of foreign invaders. The discovery that HMGB1, a ubiquitous nuclear protein, mediates the activation of innate immune responses led directly to the understanding that HMGB1 plays a critical role at the intersection of the host inflammatory response to sterile and infectious threat. HMGB1 is actively released by stimulation of the innate immune system with exogenous pathogen-derived molecules and is passively released by ischemia or cell injury in the absence of invasion. Established molecular mechanisms of HMGB1 binding and signaling through TLR4 reveal signaling pathways that mediate cytokine release and tissue damage. Experimental strategies that selectively target HMGB1 and TLR4 effectively reverse and prevent activation of innate immunity and significantly attenuate damage in diverse models of sterile and infection-induced threat.

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a “late” mediator of endotoxin lethality. Anti–HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1–induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1β] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1β. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 μg per mouse) or the DNA-binding A box (600 μg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%, P < 0.03; GST control protein = 28% vs. A box = 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.