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.

This document reflects a process whereby a group of experts and opinion leaders revisited the 1992 sepsis guidelines and found that apart from expanding the list of signs and symptoms of sepsis to reflect clinical bedside experience, no evidence exists to support a change to the definitions. This lack of evidence serves to underscore the challenge still present in diagnosing sepsis in 2003 for clinicians and researchers and also provides the basis for introducing PIRO as a hypothesis-generating model for future research.

1. Current concepts of sepsis, severe sepsis and septic shock remain useful to clinicians and researchers. 2. These definitions do not allow precise staging or prognostication of the host response to infection. 3. While SIRS remains a useful concept, the diagnostic criteria for SIRS published in 1992 are overly sensitive and non-specific. 4. An expanded list of signs and symptoms of sepsis may better reflect the clinical response to infection. 6. PIRO, a hypothetical model for staging sepsis is presented, which, in the future, may better characterize the syndrome on the basis of predisposing factors and premorbid conditions, the nature of the underlying infection, the characteristics of the host response, and the extent of the resultant organ dysfunction.

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-B and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Tolllike receptor ( HMGB11 (formerly HMG1) was originally described as a non-histone, chromatin-associated nuclear protein (1-4).HMGB1 has a highly conserved sequence among species, with murine HMGB1 differing from the human form by only two amino acids. HMGB1-deficient mice die within a few hours of birth, demonstrating the crucial role of this protein in cellular function. HMGB1 consists of two tandem L-shaped domains, HMGB boxes A and B, each ϳ75 amino acids in length, and a highly acidic carboxyl terminus of 30 amino acids in length.HMGB1 appears to have two distinct functions in cellular systems. First, it has been shown to be an intracellular regulator of transcription, and, second, HMGB1 can occupy an extracellular role in which it promotes tumor metastasis and inflammation (2-9). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (10, 11). Monocytes and macrophages stimulated by lipopolysaccharide (LPS), tumor necrosis factor (TNF)-␣, or interleukin-1 (IL-1) secrete HMGB1 (5, 11). Culture of monocytes with HMGB1 results in the release of TNF-␣, IL-1␣, IL-1␤, IL-1Ra, IL-6, IL-8, macrophage inflammatory protein-1␣, macrophage inflammatory protein-1␤, but not IL-10 or IL-12 (5, 11). Production of proinflammatory cytokines after exposure to HMGB1 occurs with delayed kinetics as compared with LPS-induced stimulation. For example, culture of macrophages with LPS results in increases in TNF-␣ that are apparent within less than 1 h, whereas TNF-␣ synthesis following HMGB1 exposure only begins to occur after 2 h and then persists for as long as 8 h (8, 11). The signaling mechanisms responsible for the delayed expression of proinflammatory cytokines by HMGB1-stimulated cells remain incompletely explained but appear to involve the p38, ERK, JNK, and Akt kinases and to lead to enhanced nuclear translocation of .In recent studies (13), we found that the magnitude and kinetics of cytokine expression and nuclear translocation of NF-B after culture of neutrophils with HMGB1 or LPS were similar, suggesting that overlapping mechanisms of cellular activation might be involved. Comparison of gene expression arrays also demonstrated substantial but not complete homology in response to HMGB1 and LPS. Signaling via the TLR 4 receptor is responsible for LPS-induced activation of the IKK kinase complex, including IKK...

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High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-Bdependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS. fluorescence resonance energy transfer; receptor of advanced glycation end products HIGH MOBILITY GROUP BOX 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly by monocytes and macrophages stimulated by LPS, TNF-␣, or IL-1 (2, 44). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (1,44,46), and also appears to be involved in pathophysiological processes associated with cellular necrosis, such as acetaminophen-induced liver injury (34).Although HMGB1 and LPS appear to initiate similar intracellular events, including activation of kinases such as p38, ERK1/2, and Akt and transcriptional factors including NF-B, that lead to production of proinflammatory cytokines, gene arrays demonstrated differences in expression profiles with each of these stimuli (12, 30). Unlike LPS, which primarily increased the activity of IKK-␤, HMGB1 exposure resulted in activation of both IKK-␣ and IKK-␤ (31). In addition, culture of neutrophils lacking Toll-like receptor (TLR)4 with HMGB1, but not with LPS, still resulted in enhanced nuclear translocation of NF-B (31). Such results suggest that the receptors interacting with HMGB1 and leading to cellular activation and gene transcription are likely to be distinct from TLR4, which is responsible for LPS-induced responses (40). Recent data indicate that HMGB1 interacts not only with TLR4 but also with TLR2 and the receptor for advanced glycation end products (RAGE) (31, 46). In particular, a decrease in NF-B-dependent reporter gene expression after transfection with dominantnegative constructs to TLR2, TLR4, or both, demonstr...

Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group-1 (HMG-1) protein, in addition to its role as a transcriptional regulatory factor, has recently been identified as a late mediator of endotoxin lethality. In the present studies, HMG-1 given intratracheally produced acute inflammatory injury to the lungs, with neutrophil accumulation, the development of lung edema, and increased pulmonary production of IL-1β, TNF-α, and macrophage-inflammatory protein-2. In endotoxin-induced acute lung inflammation, administration of anti-HMG-1 Abs either before or after endotoxin exposure decreased the migration of neutrophils to the lungs as well as lung edema. These protective effects of anti-HMG-1 were specific, because pulmonary levels of IL-1β, TNF-α, or macrophage-inflammatory protein-2 were not decreased after therapy with anti-HMG-1. Together, these findings indicate that HMG-1 is a distal mediator of acute inflammatory lung injury.

Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.