High-mobility group box 1 (HMGB1) is a 30-kDa DNA-binding protein that displays proinflammatory cytokine-like properties. HMGB1-dependent inflammatory processes have been demonstrated in models of sterile injury, including ischemia-reperfusion injury and hemorrhagic shock. Here, we tested the hypothesis that the systemic inflammatory response and associated remote organ injury that occur after peripheral tissue injury are highly dependent on HMGB1. Toll-like receptor 4 (TLR4) wild-type (WT) mice subjected to bilateral femur fracture after treatment with neutralizing antibodies to HMGB1 had lower serum IL-6 and IL-10 levels compared with mice treated with nonimmune control IgG. Similarly, compared with injured mice treated with control IgG, anti-HMGB1 antibody-treated mice had lower serum alanine aminotransferase levels and decreased hepatic and gut mucosal NF-kappaB DNA binding. TLR4 mutant (C3H/HeJ) mice subjected to bilateral femur fracture had less systemic inflammation and liver injury than WT controls. Residual trauma-induced systemic inflammation and hepatocellular injury were not ameliorated by treatment with a polyclonal anti-HMGB1 antibody, even though HMGB1 levels were transiently elevated just 1 h after injury in both WT and C3H/HeJ mice. Collectively, these data demonstrate a critical role for a TLR4-HMGB1 pathway in the initiation of systemic inflammation and end-organ injury following isolated peripheral tissue injury.

HMGB1 (high mobility group box chromosomal protein 1), historically known as an abundant, nonhistone architectural chromosomal protein, is extremely conserved across species. As a nuclear protein, HMGB1 stabilizes nucleosomes and allows bending of DNA that facilitates gene transcription. Unexpectedly, recent studies identified extracellular HMGB1 as a potent macrophage-activating factor, signaling via the receptor for advanced glycation end-products to induce inflammatory responses. It is released as a late mediator during inflammation and participates in the pathogenesis of systemic inflammation after the early mediator response has resolved. HMGB1 occupies a critical role as a proinflammatory mediator passively released by necrotic but not apoptotic cells. Necrotic Hmgb1(-/-) cells mediate minimal inflammatory responses. Stimulated macrophages actively secrete HMGB1 to promote inflammation and in turn, stimulate production of multiple, proinflammatory cytokines. HMGB1 mediates endotoxin lethality, acute lung injury, arthritis induction, activation of macrophages, smooth muscle cell chemotaxis, and epithelial cell barrier dysfunction. HMGB1 is structurally composed of three different domains: two homologous DNA-binding sequences entitled box A and box B and a highly, negatively charged C terminus. The B box domain contains the proinflammatory cytokine functionality of the molecule, whereas the A box region has an antagonistic, anti-inflammatory effect with therapeutic potential. Administration of highly purified, recombinant A box protein or neutralizing antibodies against HMGB1 rescued mice from lethal sepsis, even when initial treatment was delayed for 24 h after the onset of infection, establishing a clinically relevant therapeutic window that is significantly wider than for other known cytokines.

OBJECTIVE

To investigate the association between markers of acute endothelial glycocalyx degradation, inflammation, coagulopathy, and mortality after trauma.

BACKGROUND

Hyperinflammation and acute coagulopathy of trauma predict increased mortality. High catecholamine levels can directly damage the endothelium and may be associated with enhanced endothelial glycocalyx degradation, evidenced by high circulating syndecan-1.

METHODS

Prospective cohort study of trauma patients admitted to a Level 1 Trauma Centre in 2003 to 2005. Seventy-five patients were selected blindly post hoc from 3 predefined injury severity score (ISS) groups (<16, 16-27, >27). In all patients, we measured 17 markers of glycocalyx degradation, inflammation, tissue and endothelial damage, natural anticoagulation, and fibrinolysis (syndecan-1, IL-6, IL-10, histone-complexed DNA fragments, high-mobility group box 1 (HMGB1), thrombomodulin, von Willebrand factor, intercellular adhesion molecule-1, E-selectin, protein C, tissue factor pathway inhibitor (TFPI), antithrombin, D-dimer, tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), soluble uPA receptor, and plasminogen activator inhibitor-1), hematology, coagulation, catecholamines, and assessed 30-day mortality. Variables were compared in patients stratified according to syndecan-1 median.

RESULTS

Patients with high circulating syndecan-1 had higher catecholamines, IL-6, IL-10, histone-complexed DNA fragments, HMGB1, thrombomodulin, D-dimer, tPA, uPA (all P < 0.05), and 3-fold increased mortality (42% vs. 14%, P = 0.006) despite comparable ISS (P = 0.351). Only in patients with high glycocalyx degradation was higher ISS correlated with higher adrenaline, IL-6, histone-complexed DNA fragments, HMGB1, thrombomodulin, and APTT, lower protein C (all P < 0.05), unchanged TFPI and blunted D-dimer response (P < 0.001) because D-dimer was profoundly increased even at low ISS. After adjusting for age and ISS, syndecan-1 was an independent predictor of mortality (OR: 1.01 [95%CI, 1.00-1.02]; P = 0.043).

CONCLUSIONS

In trauma patients, high circulating syndecan-1, a marker of endothelial glycocalyx degradation, is associated with inflammation, coagulopathy and increased mortality.

The success of some chemo- and radiotherapeutic regimens relies on the induction of immunogenic tumor cell death and on the induction of an anticancer immune response. Cells succumbing to immunogenic cell death undergo specific changes in their surface characteristics and release pro-immunogenic factors according to a defined spatiotemporal pattern. This stimulates antigen presenting cells such as dendritic cells to efficiently take up tumor antigens, process them, and cross-prime cytotoxic T lymphocytes, thus eliciting a tumor-specific cognate immune response. Such a response can also target therapy-resistant tumor (stem) cells, thereby leading, at least in some instances, to tumor eradication. In this review, we shed some light on the molecular identity of the factors that are required for cell death to be perceived as immunogenic. We discuss the intriguing observations that the most abundant endoplasmic reticulum protein, calreticulin, the most abundant intracellular metabolite, ATP, and the most abundant non-histone chromatin-binding protein, HMGB1, can determine whether cell death is immunogenic as they appear on the surface or in the microenvironment of dying cells.

Muscle regeneration following injury is characterized by myonecrosis accompanied by local inflammation, activation of satellite cells, and repair of injured fibers. The resolution of the inflammatory response is necessary to proceed toward muscle repair, since persistence of inflammation often renders the damaged muscle incapable of sustaining efficient muscle regeneration. Here, we show that local expression of a muscle-restricted insulin-like growth factor (IGF)-1 (mIGF-1) transgene accelerates the regenerative process of injured skeletal muscle, modulating the inflammatory response, and limiting fibrosis. At the molecular level, mIGF-1 expression significantly down-regulated proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, and modulated the expression of CC chemokines involved in the recruitment of monocytes/macrophages. Analysis of the underlying molecular mechanisms revealed that mIGF-1 expression modulated key players of inflammatory response, such as macrophage migration inhibitory factor (MIF), high mobility group protein-1 (HMGB1), and transcription NF-kappaB. The rapid restoration of injured mIGF-1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF-1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair.

Autophagy is rapidly developing into a new immunological paradigm. The latest links now include overlaps between autophagy and innate immune signaling via TBK-1 and IKKα/β, and the role of autophagy in inflammation directed by the inflammasome. Autophagy's innate immunity connections include responses to pathogen and damage-associated molecular patterns including alarmins such as HMGB1 and IL-1β, Toll-like receptors, Nod-like receptors including NLRC4, NLRP3 and NLRP4, and RIG-I-like receptors. Autophagic adaptors referred to as SLRs (sequestosome 1/p62-like receptors) are themselves a category of pattern recognition receptors. SLRs empower autophagy to eliminate intracellular microbes by direct capture and by facilitating generation and delivery of antimicrobial peptides, and also serve as inflammatory signaling platforms. SLRs contribute to autophagic control of intracellular microbes, including Mycobacterium tuberculosis, Salmonella, Listeria, Shigella, HIV-1 and Sindbis virus, but act as double-edged sword and contribute to inflammation and cell death. Autophagy roles in innate immunity continue to expand vertically and laterally, and now include antimicrobial function downstream of vitamin D3 action in tuberculosis and AIDS. Recent data expand the connections between immunity-related GTPases and autophagy to include not only IRGM but also several members of the Gbp (guanlyate-binding proteins) family. The efficacy with which autophagy handles microbes, microbial products and sterile endogenous irritants governs whether the outcome will be with suppression of or with excess inflammation, the latter reflected in human diseases that have strong inflammatory components including tuberculosis and Crohn's disease.

Human malignant mesothelioma is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor because of late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival. Malignant mesothelioma cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in malignant mesothelioma patient sera were higher than that found in healthy individuals. The motility, survival, and anchorage-independent growth of HMGB1-secreting malignant mesothelioma cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products, a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of malignant mesothelioma xenografts in severe-combined immunodeficient mice and extended host survival. Taken together, our findings indicate that malignant mesothelioma cells rely on HMGB1, and they offer a preclinical proof-of-principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for malignant mesothelioma treatment.

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged or dying cells and activate the innate immune system by interacting with pattern recognition receptors (PRRs). Although DAMPs contribute to the host's defense, they promote pathological inflammatory responses. Recent studies have suggested that various DAMPs, such as high-mobility group box 1 (HMGB1), S100 proteins, and heat shock proteins (HSPs), are increased and considered to have a pathogenic role in inflammatory diseases. Here, we review current research on the role of DAMPs in inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, atherosclerosis, Alzheimer's disease, Parkinson's disease, and cancer. We also discuss the possibility of DAMPs as biomarkers and therapeutic targets for these diseases.

The balance between apoptosis ("programmed cell death") and autophagy ("programmed cell survival") is important in tumor development and response to therapy. Here, we show that high mobility group box 1 (HMGB1) and p53 form a complex that regulates the balance between tumor cell death and survival. We show that knockout of p53 in HCT116 cells increases expression of cytosolic HMGB1 and induces autophagy. Conversely, knockout of HMGB1 in mouse embryonic fibroblasts increases p53 cytosolic localization and decreases autophagy. p53 is thus a negative regulator of the HMGB1/Beclin 1 complex, and HMGB1 promotes autophagy in the setting of diminished p53. HMGB1-mediated autophagy promotes tumor cell survival in the setting of p53-dependent processes. The HMGB1/p53 complex affects the cytoplasmic localization of the reciprocal binding partner, thereby regulating subsequent levels of autophagy and apoptosis. These insights provide a novel link between HMGB1 and p53 in the cross-regulation of apoptosis and autophagy in the setting of cell stress, providing insights into their reciprocal roles in carcinogenesis.

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

OBJECTIVE

Electrical vagus nerve stimulation inhibits proinflammatory cytokine production and prevents shock during lethal systemic inflammation through an alpha7 nicotinic acetylcholine receptor (alpha7nAChR)-dependent pathway to the spleen, termed the cholinergic anti-inflammatory pathway. Pharmacologic alpha7nAChR agonists inhibit production of the critical proinflammatory mediator high mobility group box 1 (HMGB1) and rescue mice from lethal polymicrobial sepsis. Here we developed a method of transcutaneous mechanical vagus nerve stimulation and then investigated whether this therapy can protect mice against sepsis lethality.

METHODS

Prospective, randomized study.

METHODS

Institute-based research laboratory.

METHODS

Male BALB/c mice.

METHODS

Mice received lipopolysaccharide to induce lethal endotoxemia or underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive electrical, transcutaneous, or sham vagus nerve stimulation and were followed for survival or euthanized at predetermined time points for cytokine analysis.

RESULTS

Transcutaneous vagus nerve stimulation dose-dependently reduced systemic tumor necrosis factor levels during lethal endotoxemia. Treatment with transcutaneous vagus nerve stimulation inhibited HMGB1 levels and improved survival in mice with polymicrobial sepsis, even when administered 24 hrs after the onset of disease.

CONCLUSIONS

Transcutaneous vagus nerve stimulation is an efficacious treatment for mice with lethal endotoxemia or polymicrobial sepsis.

Increasing evidence supports the involvement of immune and inflammatory processes in the etiopathogenesis of seizures. In particular, activation of innate immune mechanisms and the subsequent inflammatory responses, that are induced in the brain by infection, febrile seizures, neurotrauma, stroke are well documented conditions associated with acute symptomatic seizures and with a high risk of developing epilepsy. A decade ago, pharmacological experiments showed that elevated brain levels of the anti-inflammatory molecule IL-1 receptor antagonist reduced seizures in epilepsy models. This observation, together with the evidence of in situ induction of inflammatory mediators and their receptors in experimental and human epileptogenic brain tissue, established the proof-of-concept evidence that the activation of innate immunity and inflammation in the brain are intrinsic features of the pathologic hyperexcitable tissue. Recent breakthroughs in understanding the molecular organization of the innate immune system first in macrophages, then in the different cell types of the CNS, together with pharmacological and genetic studies in epilepsy models, showed that the activation of IL-1 receptor/Toll-like receptor (IL-1R/TLR) signaling significantly contributes to seizures. IL-1R/TLR mediated pro-excitatory actions are elicited in the brain either by mimicking bacterial or viral infections and inflammatory responses, or via the action of endogenous ligands. These ligands include proinflammatory cytokines, such as IL-1beta, or danger signals, such as HMGB1, released from activated or injured cells. The IL-1R/TLR signaling mediates rapid post-translational changes in voltage- and ligand-gated ion channels that increase excitability, and transcriptional changes in genes involved in neurotransmission and synaptic plasticity that contribute to lower seizure thresholds chronically. The anticonvulsant effects of inhibitors of the IL-1R/TLR signaling in various seizures models suggest that this system could be targeted to inhibit seizures in presently pharmaco-resistant epilepsies.

LPS-binding protein (LBP) is a central mediator that transfers LPS to CD14 to initiate TLR4-mediated proinflammatory response. However, a possibility of another LPS transfer molecule has been suggested because LBP-deficient mice showed almost normal inflammatory response after LPS injection. In this study, we describe the novel finding that high mobility group box 1 protein (HMGB1) recently identified as a mediator of sepsis has a function of LPS transfer for a proinflammatory response. We used ELISA and surface plasmon resonance to show that HMGB1 binds LPS in a concentration-dependent manner and that the binding is stronger to lipid A moiety than to the polysaccharide moiety of LPS. This binding was inhibited by LBP and polymyxin B. Using native PAGE and fluorescence-based LPS transfer analyses, we show that HMGB1 can catalytically disaggregate and transfer LPS to both soluble CD14 protein and to human PBMCs in a dose-dependent manner. However, this effect was dramatically reduced to the baseline level when HMGB1 was heat inactivated. Furthermore, a mixture of HMGB1 and LPS treatment results in a higher increase in TNF-alpha production in human PBMCs and peripheral blood monocytes than LPS or HMGB1 treatment alone or their summation. Thus, we propose that HMGB1 plays an important role in Gram-negative sepsis by catalyzing movement of LPS monomers from LPS aggregates to CD14 to initiate a TLR4-mediated proinflammatory response.

Chemokines regulate the migration and the maturation of dendritic cells (DC) licensed by microbial constituents. We have recently found that the function of DC, including their ability to activate naïve, allogeneic CD4+ T cells, requires the autocrine/paracrine release of the nuclear protein high mobility group box 1 (HMGB1). We show here that human myeloid DC, which rapidly secrete upon maturation induction their own HMGB1, remodel their actin-based cytoskeleton, up-regulate the CCR7 and the CXCR4 chemokine receptors, and acquire the ability to migrate in response to chemokine receptor ligands. The events are apparently causally related: DC challenged with LPS in the presence of HMGB1-specific antibodies fail to up-regulate the expression of the CCR7 and CXCR4 receptors and to rearrange actin-rich structures. Moreover, DC matured in the presence of anti-HMGB1 antibodies fail to migrate in response to the CCR7 ligand CCL19 and to the CXCR4 ligand CXCL12. The blockade of receptor for advanced glycation end products (RAGE), the best-characterized membrane receptor for HMGB1, impinges as well on the up-regulation of chemokine receptors and on responsiveness to CCL19 and CXCL12. Our data suggest that the autocrine/paracrine release of HMGB1 and the integrity of the HMGB1/RAGE pathway are required for the migratory function of DC.

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcriptional regulation, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair damage recognition proteins to triplex-directed psoralen DNA interstrand cross-links (ICLs). Thus, we hypothesized that HMGB1 modulates the repair of DNA damage in mammalian cells. We demonstrate here that mammalian cells lacking HMGB1 are hypersensitive to DNA damage induced by psoralen plus UVA irradiation (PUVA) or UVC radiation, showing less survival and increased mutagenesis. In addition, nucleotide excision repair efficiency is significantly decreased in the absence of HMGB1 as assessed by the repair and removal of UVC lesions from genomic DNA. We also explored the role of HMGB1 in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no histone acetylation upon DNA damage. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. These results reveal a role for HMGB1 in the error-free repair of DNA lesions. Its absence leads to increased mutagenesis, decreased cell survival, and altered chromatin reorganization after DNA damage. Because strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases.

HMGB1 (High-Mobility Group Box-1) is a nuclear protein that acts as an architectural chromatin-binding factor involved in the maintenance of nucleosome structure and regulation of gene transcription. It can be released into the extracellular milieu from immune and non-immune cells in response to various stimuli. Extracellular HMGB1 contributes to the pathogenesis of numerous chronic inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, atherosclerosis, chronic kidney disease, systemic lupus erythematosus (SLE), as well as cancer pathogenesis. Interaction of released HMGB1 with the cell-surface receptor for advanced glycation end products (RAGE) is one of the main signaling pathways triggering these diseases. It has been also demonstrated that the inhibition of the HMGB1-RAGE interaction represents a promising approach for the modulation of the inflammatory and tumor-facilitating activity of HMGB1. In this review we describe various approaches recently proposed in the literature to inhibit HMGB1 and the related inflammatory processes, especially focusing on the block of RAGE-HMGB1 signaling. Several strategies are based on molecules which mainly interact with RAGE as competitive antagonists of HMGB1. As an alternative, encouraging results have been obtained with HMGB1-targeting, leading to the identification of compounds that directly bind to HMGB1, ranging from small natural or synthetic molecules, such as glycyrrhizin and gabexate mesilate, to HMGB1-specific antibodies, peptides, proteins as well as bent DNA-based duplexes. Future perspectives are discussed in the light of the overall body of knowledge acquired by a large number of research groups operating in different but related fields.

Mammalian preimplantation embryonic development (PED) is thought to be governed by highly conserved processes. While it had been suggested that some plasticity of conserved signaling networks exists among different mammalian species, it was not known to what extent modulation of the genomes and the regulatory proteins could "rewire" the gene regulatory networks (GRN) that control PED. We therefore generated global transcriptional profiles from three mammalian species (human, mouse, and bovine) at representative stages of PED, including: zygote, two-cell, four-cell, eight-cell, 16-cell, morula and blastocyst. Coexpression network analysis suggested that 40.2% orthologous gene triplets exhibited different expression patterns among these species. Combining the expression data with genomic sequences and the ChIP-seq data of 16 transcription regulators, we observed two classes of genomic changes that contributed to interspecies expression difference, including single nucleotide mutations leading to turnover of transcription factor binding sites, and insertion of cis-regulatory modules (CRMs) by transposons. About 10% of transposons are estimated to carry CRMs, which may drive species-specific gene expression. The two classes of genomic changes act in concert to drive mouse-specific expression of MTF2, which links POU5F1/NANOG to NOTCH signaling. We reconstructed the transition of the GRN structures as a function of time during PED. A comparison of the GRN transition processes among the three species suggested that in the bovine system, POU5F1's interacting partner SOX2 may be replaced by HMGB1 (a TF sharing the same DNA binding domain with SOX2), resulting in rewiring of GRN by a trans change.

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2-deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2-HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4-MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness.

HMGB1 (amphoterin) is a 30-kDa heparin-binding protein that mediates transendothelial migration of monocytes and has proinflammatory cytokine-like activities. In this study, we have investigated proinflammatory activities of both highly purified eukaryotic HMGB1 and bacterially produced recombinant HMGB1 proteins. Mass analyses revealed that recombinant eukaryotic HMGB1 has an intrachain disulphide bond. In mass analysis of tissue-derived HMGB1, two forms were detected: the carboxyl terminal glutamic acid residue lacking form and a full-length form. Cell culture studies indicated that both eukaryotic and bacterial HMGB1 proteins induce TNF-alpha secretion and nitric oxide release from mononuclear cells. Affinity chromatography analysis revealed that HMGB1 binds tightly to proinflammatory bacterial substances. A soluble proinflammatory substance was separated from the bacterial recombinant HMGB1 by chloroform-methanol treatment. HMGB1 interacted with phosphatidylserine in both solid-phase binding and cell culture assays, suggesting that HMGB1 may regulate phosphatidylserine-dependent immune reactions. In conclusion, HMGB1 polypeptide has a weak proinflammatory activity by itself, and it binds to bacterial substances, including lipids, that may strengthen its effects.

OBJECTIVE

Immunotherapy with ipilimumab improves the survival of patients with metastatic melanoma. Because only around 20% of patients experience long-term benefit, reliable markers are needed to predict a clinical response. Therefore, we sought to determine if some myeloid cells and related inflammatory mediators could serve as predictive factors for the patients' response to ipilimumab.

METHODS

We performed an analysis of myeloid cells in the peripheral blood of 59 stage IV melanoma patients before the treatment and at different time points upon the therapy using a clinical laboratory analysis and multicolor flow cytometry. In addition, the production of related inflammatory factors was evaluated by ELISA or Bio-Plex assays.

RESULTS

An early increase in eosinophil count during the treatment with ipilimumab was associated with an improved clinical response. In contrast, elevated amounts of monocytic myeloid-derived suppressor cells (moMDSC), neutrophils, and monocytes were found in nonresponders (n = 36) as compared with basal levels and with responding patients (n = 23). Moreover, in nonresponders, moMDSCs produced significantly more nitric oxide, and granulocytic MDSCs expressed higher levels of PD-L1 than these parameters at baseline and in responders, suggesting their enhanced immunosuppressive capacity. Upon the first ipilimumab infusion, nonresponders displayed elevated serum concentrations of S100A8/A9 and HMGB1 that attract and activate MDSCs.

CONCLUSIONS

These findings highlight additional mechanisms of ipilimumab effects and suggest levels of eosinophils, MDSCs, as well as related inflammatory factors S100A8/A9 and HMGB1 as novel complex predictive markers for patients who may benefit from the ipilimumab therapy. Clin Cancer Res; 21(24); 5453-9. ©2015 AACR.