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...

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.

Although our data suggest that 1:1 FFP:RBC reduced coagulopathy, this did not translate into a survival benefit. Our findings indicate that the relationship between coagulopathy and mortality is more complex, and further clinical investigation is necessary before recommending routine 1:1 in the exsanguinating trauma patient.

High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations after trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early significant elevations of plasma HMGB1. Trauma patients at risk for multiple organ failure (ISS ≥15) were identified for inclusion (n = 23), and postinjury plasma samples were assayed for HMGB1 by enzyme-linked immunosorbent assay. Comparison of postinjury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused per first 24 h, and base deficit) was performed. To investigate whether postinjury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leuko-reduced and non–leuko-reduced packed RBCs. Plasma HMGB1 was elevated more than 30-fold above healthy controls within 1 h of injury (median, 57.76 vs. 1.77 ng/mL; P < 0.003), peaked from 2 to 6 h postinjury (median, 526.18 ng/mL; P < 0.01 vs. control), and remained elevated above control through 136 h. No clear relationship was evident between postinjury HMGB1 levels and markers for patient outcome. High-mobility group box 1 levels increase with duration of RBC storage, although concentrations did not account for postinjury plasma levels. Leuko-reduced attenuated HMGB1 levels in packed RBCs by approximately 55% (P < 0.01). Plasma HMGB1 is significantly increased within 1 h of trauma in humans with marked elevations occurring from 2 to 6 h postinjury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event after traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.