Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

The initial phase in the development of a migraine is still poorly understood. Here, we describe a previously unknown signaling pathway between stressed neurons and trigeminal afferents during cortical spreading depression (CSD), the putative cause of migraine aura and headache. CSD caused neuronal Pannexin1 (Panx1) megachannel opening and caspase-1 activation followed by high-mobility group box 1 (HMGB1) release from neurons and nuclear factor κB activation in astrocytes. Suppression of this cascade abolished CSD-induced trigeminovascular activation, dural mast cell degranulation, and headache. CSD-induced neuronal megachannel opening may promote sustained activation of trigeminal afferents via parenchymal inflammatory cascades reaching glia limitans. This pathway may function to alarm an organism with headache when neurons are stressed.

High-mobility group box 1 (HMGB1), a nuclear factor released extracellularly as an inflammatory cytokine, is an endogenous ligand for Toll-like receptor 4 (TLR4). TLR4 activation mediates kidney ischemia-reperfusion injury (IRI), but whether HMGB1 contributes to IRI is unknown. Here, treating wild-type mice with neutralizing anti-HMGB1 antibody protected them against kidney IRI, evidenced by lower serum creatinine and less tubular damage than untreated mice. Mice treated with anti-HMGB1 had significantly less tubulointerstitial infiltration by neutrophils (day 1) and macrophages (day 5) and markedly reduced apoptosis of tubular epithelial cells. Furthermore, anti-HMGB1 antibody-treated IRI kidneys had significantly lower levels of IL-6, TNFα, and monocyte chemoattractant protein 1 (MCP1). mRNA, which are downstream of HMGB1. Conversely, administration of rHMGB1 after reperfusion exacerbated kidney IRI in wild-type mice. TLR4 deficient (TLR4(-/-)) mice were protected against kidney IRI; administration of neither anti-HMGB1 antibody nor rHMGB1 affected this renoprotection. In conclusion, endogenous HMGB1 promotes kidney damage after IRI, possibly through the TLR4 pathway. Administration of a neutralizing antibody to HMGB1 either before or soon after ischemia-reperfusion affords significant protection, suggesting therapeutic potential for acute kidney injury.

HMGB1 was originally identified as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells. Recent studies indicate that HMGB1 can be "passively released" into the extracellular milieu by necrotic and damaged somatic cells. Extracellular HMGB1 represents an optimal "necrotic marker" selected by the innate immune system to recognize tissue damage and initiate reparative responses. HMGB1 in the extracellular milieu promotes maturation of myeloid and plasmacytoid dendritic cells, and induces myocardial regeneration after infarction. However, extracellular HMGB1 also acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. A growing number of studies indicate that HMGB1 is a successful therapeutic target in experimental models of ischemia/reperfusion, acute respiratory distress syndrome, rheumatoid arthritis, sepsis, and cancer. From a clinical perspective, HMGB1 represents a current challenge that can be exploited orchestrate reparative responses while preventing its pathological potential. This article focus on the immuno-regulatory role of HMGB1 and its contribution to infectious and inflammatory disorders.

Established tumors are typified by an immunosuppresive microenvironment. Countering this naturally occurring phenomenon, emerging evidence suggests that radiation promotes a proimmunogenic milieu within the tumor capable of stimulating host cancer-specific immune responses. Three cryptic immunogenic components of cytotoxic-agent induced cell death-namely, calreticulin cell surface exposure, the release of high mobility group box 1 (HMGB1) protein, and the liberation of ATP-have been previously shown to be critical for dendritic cell (DC) activation and effector T-cell priming. Thus, these immune-mobilizing components commonly presage tumor rejection in response to treatment. We initially set out to address the hypothesis that radiation-induced immunogenic cell death (ICD) is dose-dependent. Next, we hypothesized that radiation would enhance chemotherapy-induced ICD when given concomitantly, as suggested by the favorable clinical outcomes observed in response to analogous concurrent chemoradiation regimens. Thus, we designed an in vitro assay to examine the 3 hallmark features of ICD at clinically relevant doses of radiation. We then tested the immunogenic-death inducing effects of radiation combined with carboplatin or paclitaxel, focusing on these combinations to mimic chemoradiation regimens actually used in clinical trials of early stage triple negative [NCT0128953/NYU-10-01969] and locally advanced [NYU-06209] breast cancer patients, respectively. Despite the obvious limitations of an in vitro model, radiotherapy produced both a dose-dependent induction and chemotherapeutic enhancement of ICD. These findings provide preliminary evidence that ICD stimulated by either high-dose radiotherapy alone, or concurrent chemoradiation regimens, may contribute to the establishment of a peritumoral proimmunogenic milieu.

Physiological cell death, which occurs as a continuous byproduct of cellular turnover, is non-immunogenic or even tolerogenic, thereby avoiding autoimmunity. By contrast, cancer cell death elicited by radiotherapy and some chemotherapeutic agents such as anthracyclines is immunogenic. Recent data suggest that innate and cognate immune responses elicited by such anti-cancer agents are required for an optimal therapeutic outcome, underscoring the clinical relevance of immunogenic cell death. Here we discuss the concept that immunogenic death involves changes in the composition of the cell surface, as well as the release of soluble immunogenic signals that occur in a defined temporal sequence. This 'key' then operates on a series of receptors expressed by dendritic cells (DC, the 'lock') to allow for the presentation of tumor antigens to T cells and for the initiation of a productive immune response. Immunogenic cell death is characterized by the early cell surface exposure of chaperones including calreticulin and/or heat shock proteins, which determine the uptake of tumor antigens and/or affect DC maturation. Moreover, the late release of High mobility group box 1 (HMGB1), which acts on toll-like receptor 4 (TLR4), is required for optimal presentation of antigens from dying tumor cells. Nonetheless, numerous details on the molecular events that define immunogenicity remain to be defined, both at the level of the dying cancer cells and at the level of the responding innate effectors.

The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL-6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL-1beta, the TLR4 ligand LPS, the TLR9 ligand CpG-ODN, or the TLR1-TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A-box and B-box domains of HMGB1 had the ability to bind LPS and enhance IL-6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL-18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.

High mobility group box 1 (HMGB1) can be actively secreted by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli (such as bacterial endotoxin, TNF-alpha, IL-1, and IFN-gamma) or passively released by necrotic cells and mediates innate and adaptive inflammatory responses to infection and injury. Here, we demonstrated that a reactive oxygen species, hydrogen peroxide (H(2)O(2)), induces active and passive HMGB1 release from macrophage and monocyte cultures in a time- and dose-dependent manner. At nontoxic doses (e.g., 0.0125-0.125 mM), H(2)O(2) induced HMGB1 cytoplasmic translocation and active release within 3-24 h. At higher concentrations (e.g., 0.25 mM), however, H(2)O(2) exhibited cytotoxicity to macrophage and monocyte cell cultures and consequently, triggered active and passive HMGB1 release. In addition, H(2)O(2) stimulated potential interaction of HMGB1 with a nuclear export factor, chromosome region maintenance (CRM1), in macrophage/monocyte cultures. Inhibitors specific for the JNK (SP600125) and MEK (PD98059), but not p38 MAPK (SB203580), abrogated H(2)O(2)-induced, active HMGB1 release. Together, these data establish an important role for oxidative stress in inducing active HMGB1 release, potentially through a MAPK- and CRM1-dependent mechanism.

Osteosarcoma is the most commonly occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. In this study, we implicate the DNA-binding protein HMGB1, which also exerts immunoregulatory effects in its secreted form, in the development of drug resistance in osteosarcoma. Anticancer agents including doxorubicin, cisplatin, and methotrexate each induced HMGB1 upregulation in human osteosarcoma cells, and RNA interference-mediated knockdown of HMGB1 restored the chemosensitivity of osteosarcoma cells in vivo and in vitro. Mechanistic investigation revealed that HMGB1 increased drug resistance by inducing autophagy, an intracellular self-defense mechanism known to confer drug resistance. We found that HMGB1 bound to the autophagy regulator Beclin1 and regulated the formation of the Beclin1-PI3KC3 [PI3KC3, phosphatidylinositol 3-kinase class 3] complex that facilitates autophagic progression. In addition, we found that interaction between HMGB1 and Beclin1 relied upon the autophagic complex ULK1-mAtg13-FIP200. Therefore, through its role as a regulator of autophagy, HMGB1 is a critical factor in the development of chemoresistance, and it offers a novel target for improving osteosarcoma therapy.

The intranuclear architectural protein that is termed high mobility group box chromosomal protein 1 (HMGB1) was recently identified as a potent proinflammatory mediator when present extracellularly. HMGB1 has been demonstrated to be a long-searched-for nuclear danger signal passively released by necrotic, as opposed to apoptotic, cells that will induce inflammation. Furthermore, HMGB1 can also be actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of the molecule, which enables translocation from the nucleus to secretory lysosomes. Subsequent transport out of the cells depends on a secretion signal mediated by either extracellular lysophophatidyl-choline or ATP. HMGB1 passively released from necrotic cells and HMGB1 actively secreted by inflammatory cells are thus molecularly different. Extracellular HMGB1 acts as a cytokine by signaling via the receptor for advanced glycated end-products and via members of the Toll-like receptor family. The initiated inflammatory responses include the production of multiple cytokines, chemoattraction of certain stem cells, induction of vascular adhesion molecules and impaired function of intestinal epithelial cells. Therapeutic administration of HMGB1 antagonists rescues mice from lethal sepsis, even when initial treatment is delayed for 24 h after the onset of infection, establishing a clinically relevant therapeutic window that is significantly wider than for other known cytokines.

While studies in animal models have linked Toll-like receptor (TLR) 4 signaling to kidney injury induced by ischemia and reperfusion, the relevance of TLR4 activation to allograft injury in human kidney transplants is unknown. Here we show that TLR4 is constitutively expressed within all donor kidneys but is significantly higher in deceased-, compared with living-donor organs. Tubules from deceased- but not living-donor kidneys also stained positively for high-mobility group box-1 (HMGB1), a known endogenous TLR4 ligand. In vitro stimulation of human tubular cells with HMGB1, in a TLR4-dependent system, confirmed that HMGB1 can stimulate proinflammatory responses through TLR4. To assess the functional significance of TLR4 in human kidney transplantation, we determined whether TLR4 mutations that confer diminished affinity for HMGB1 influence intragraft gene-expression profiles and immediate graft function. Compared with kidneys expressing WT alleles, kidneys with a TLR4 loss-of-function allele contained less TNFalpha, MCP-1, and more heme oxygenase 1 (HO-1), and exhibited a higher rate of immediate graft function. These results represent previously undetected evidence that donor TLR4 contributes to graft inflammation and sterile injury following cold preservation and transplantation in humans. Targeting TLR4 signaling may have value in preventing or treating postischemic acute kidney injury after transplantation.

OBJECTIVE

High mobility group (HMG) B1 is a nonhistone nuclear protein that was recently identified as a late-acting mediator of lipopolysaccharide-induced lethality in mice. The proinflammatory actions of HMGB1 have been localized to a region of the molecule called the B box.

METHODS

To determine whether HMGB1 or B box are capable of causing derangements in intestinal barrier function, we incubated cultured Caco-2 human enterocytic monolayers with recombinant human HMGB1 or a 74-residue truncated form of the protein consisting of the B box domain.

RESULTS

Both HMGB1 and B box increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate-labeled dextran (FD4) in a time- and dose-dependent fashion. The increase in permeability was reversible following removal of the recombinant protein. Exposure of Caco-2 cells to B box resulted in increased expression of inducible nitric oxide synthase messenger RNA and increased production of NO. When we used various pharmacologic strategies to inhibit NO production or scavenge NO or peroxynitrite (ONOO(-)), we abrogated B box-induced hyperpermeability. Administration of B box to wild-type mice increased both ileal mucosal permeability to FD4 and bacterial translocation to mesenteric lymph nodes. These effects were not observed in inducible nitric oxide synthase knockout mice.

CONCLUSIONS

These data support the view that HMGB1 and B box are capable of causing alterations in gut barrier function via a mechanism that depends on the formation of NO and ONOO(-).

The high mobility group box-1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine-like activity in the extracellular space. Here we show that treatment with neutralizing anti-HMGB1 monoclonal antibody (mAb; 200 microg, twice) remarkably ameliorated brain infarction induced by 2-h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti-HMGB1 mAb inhibited the increased permeability of the blood-brain barrier, the activation of microglia, the expression of TNF-alpha and iNOS, and suppressed the activity of MMP-9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemical study revealed that HMGB1 immunoreactivity in the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an outstandingly suitable target for the treatment. Intravenous injection of neutralizing anti-HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.

BACKGROUND

Gout is a prevalent inflammatory arthritis affecting 1-2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.

RESULTS

Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.

CONCLUSIONS

These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1beta and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1beta-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1beta/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1beta production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1beta secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.

Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD(P)H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD(P)H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD(P)H oxidase activation, in the form of phosphorylation of p47phox subunit of NAD(P)H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD(P)H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD(P)H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.

Acetaminophen overdose is a common reason for hospital admission and the most frequent cause of hepatotoxicity in the Western world. Early identification would facilitate patient-individualized treatment strategies. We investigated the potential of a panel of novel biomarkers (with enhanced liver expression or linked to the mechanisms of toxicity) to identify patients with acetaminophen-induced acute liver injury (ALI) at first presentation to the hospital when currently used markers are within the normal range. In the first hospital presentation plasma sample from patients (n = 129), we measured microRNA-122 (miR-122; high liver specificity), high mobility group box-1 (HMGB1; marker of necrosis), full-length and caspase-cleaved keratin-18 (K18; markers of necrosis and apoptosis), and glutamate dehydrogenase (GLDH; marker of mitochondrial dysfunction). Receiver operator characteristic curve analysis and positive/negative predictive values were used to compare sensitivity to report liver injury versus alanine transaminase (ALT) and International Normalized Ratio (INR). In all patients, biomarkers at first presentation significantly correlated with peak ALT or INR. In patients presenting with normal ALT or INR, miR-122, HMGB1, and necrosis K18 identified the development of liver injury (n = 15) or not (n = 84) with a high degree of accuracy and significantly outperformed ALT, INR, and plasma acetaminophen concentration for the prediction of subsequent ALI (n = 11) compared with no ALI (n = 52) in patients presenting within 8 hours of overdose.

CONCLUSIONS

Elevations in plasma miR-122, HMGB1, and necrosis K18 identified subsequent ALI development in patients on admission to the hospital, soon after acetaminophen overdose, and in patients with ALTs in the normal range. The application of such a biomarker panel could improve the speed of clinical decision-making, both in the treatment of ALI and the design/execution of patient-individualized treatment strategies.

High mobility group box protein 1 (HMGB1) has been considered as a ubiquitous nuclear protein with an architectural function, but even early reports have described its presence outside of the nucleus. Today, we have only started to understand the extranuclear and extracellular functions of HMGB1: we know that it participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may even be involved in the metastatic invasion programme of cancer cells. Given such diverse roles, it is important to know which cells express HMGB1, where, and how much. The present review deals with the expression pattern of HMGB1 and provides evidence that, far from being housekeeping, the HMGB1 gene is tightly regulated. This can have implications for therapeutic intervention on inflammatory diseases as well as cancer.

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein present in the nuclei and cytoplasm of nearly all cell types, is a necessary and sufficient mediator of inflammation during sterile and infection-associated responses. Elevated levels of HMGB1 in serum and tissues occur during sterile tissue injury and during infection, and targeting HMGB1 with antibodies or specific antagonists is protective in established preclinical inflammatory disease models including lethal endotoxemia or sepsis, collagen-induced arthritis, and ischemia-reperfusion induced tissue injury. Future advances in this field will stem from understanding the biological basis for the success of targeting HMGB1 to therapeutic improvement in the treatment of inflammation, infection and ischemia-reperfusion induced injury.

Autophagy, a tightly regulated lysosome-dependent catabolic pathway, is important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. However, the role of autophagy in leukemia still remains largely unknown. Here we show that high-mobility group box 1 (HMGB1), the best characterized damage-associated molecular pattern, was released from leukemia cell lines after chemotherapy-induced cytotoxicity and activated autophagy to protect against injury. Treatment with HMGB1-neutralizing antibodies increased the sensitivity of leukemia cells to chemotherapy; whereas, exogenous HMGB1 rendered these cells more resistant to drug-induced cytotoxicity. Moreover, exogenous HMGB1 increased autophagy as evaluated by increased expression of the autophagic marker microtubule-associated protein light chain 3-II, degradation of sequestosome 1 (p62) and autophagosome formation. Furthermore, knockdown or pharmacological inhibition of either phosphoinositide 3-kinase-III or extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase inhibited HMGB1-induced autophagy. Taken together, these results suggest that HMGB1 release after chemotherapy is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia.

Interaction of natural killer (NK) cells with autologous immature dendritic cells (DCs) results in reciprocal activation; however, the underlying mechanisms are so far elusive. We show here that NK cells trigger immature DCs to polarize and secrete interleukin 18 (IL-18), a cytokine lacking a secretory leader sequence. This occurs through a Ca2+-dependent and tubulin-mediated recruitment of IL-18-containing secretory lysosomes toward the adhering NK cell. Lysosome exocytosis and IL-18 secretion are restricted at the synaptic cleft, thus allowing activation of the interacting NK cells without spreading of the cytokine. In turn, DC-activated NK cells secrete the proinflammatory cytokine high mobility group B1 (HMGB1), which induces DC maturation and protects DCs from lysis. Also HMGB1 is a leaderless cytokine that undergoes regulated secretion. Differently from IL-18, soluble HMGB1 is consistently detected in NK/DC supernatants. These data point to secretion of leaderless cytokines as a key event for the reciprocal activation of NK cells and DCs. DCs initiate NK cell activation by targeted delivery of IL-18, thus instructing NK cells in the absence of adaptive-type cytokines; in turn, activated NK cells release HMGB1, which promotes inflammation and induces DC maturation, thus favoring the onset of the adaptive immune response.

The receptor for advanced glycation end-product (RAGE) is the signal transduction receptor which senses a variety of signalling molecules including advanced glycation end products (AGEs), HMGB1, S100/calgranulins, β-amyloid, phosphatidylserine, C3a and advanced oxidation protein products (AOPPs). It is usually abnormally up-regulated and plays crucial roles during the development of many human diseases such as diabetes, cardiovascular diseases, osteoarthritis and cancer. RAGE regulates a number of cell processes of pivotal importance like inflammation, apoptosis, proliferation and autophagy. Therapeutic strategies to block RAGE may represent great therapeutic potentials and therefore it has been under extensive investigation during the last decade. Accordingly, there is a growing interest of unraveling the intracellular signalling pathways by which RAGE controls these disease-related processes. Early studies are mainly focused on inflammatory pathways involving the NFκB and the MAPK pathways. Nevertheless, many novel signalling pathways implicated in other cell processes, such as autophagy, have also recently been found to be activated upon RAGE stimulation and contribute to the detrimental effects of RAGE. In this review, we aim to provide a comprehensive summary of previous and recent studies relating to the complex molecular network of RAGE signalling, with a particular emphasis on RAGE transgenic mouse models.

BACKGROUND

High mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear binding protein that has recently been shown to be an early trigger of sterile inflammation in animal models of trauma-hemorrhage via the activation of the Toll-like-receptor 4 (TLR4) and the receptor for the advanced glycation endproducts (RAGE). However, whether HMGB1 is released early after trauma hemorrhage in humans and is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of the present study.

METHODS

One hundred sixty eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level 1 Trauma center. Blood was drawn within 10 minutes of arrival to the emergency room before the administration of any fluid resuscitation. HMGB1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, von Willebrand Factor (vWF), angiopoietin-2 (Ang-2), Prothrombin time (PT), prothrombin fragments 1+2 (PF1+2), soluble thrombomodulin (sTM), protein C (PC), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and D-Dimers were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared to outcome measures obtained from the electronic medical record and trauma registry.

RESULTS

Plasma levels of HMGB1 were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, tissue hypoperfusion, early posttraumatic coagulopathy and hyperfibrinolysis as well with a systemic inflammatory response and activation of complement. Non-survivors had significantly higher plasma levels of HMGB1 than survivors. Finally, patients who later developed organ injury, (acute lung injury and acute renal failure) had also significantly higher plasma levels of HMGB1 early after trauma.

CONCLUSIONS

The results of this study demonstrate for the first time that HMGB1 is released into the bloodstream early after severe trauma in humans. The release of HMGB1 requires severe injury and tissue hypoperfusion, and is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.