HMGB proteins are members of the High Mobility Group (HMG) superfamily, possessing a unique DNA-binding domain, the HMG-box, which can bind non-B-type DNA structures (bent, kinked and unwound) with high affinity, and also distort DNA by bending/looping and unwinding. HMGBs (there are four HMGBs in mammals, HMGB1-4) are highly abundant and ubiquitously expressed non-histone proteins, acting as DNA chaperones influencing multiple processes in chromatin such as transcription, replication, recombination, DNA repair and genomic stability. Although HMGB1 is a nuclear protein, it can be secreted into the extracellular milieu as a signaling molecule when cells are under stress, in particular, when necrosis occurs. Mammalian HMGBs contain two HMG-boxes arranged in tandem, share more than 80% identity and differ in the length (HMGB1-3) or absence (HMGB4) of the acidic C-tails. The acidic tails consist of consecutive runs of only Glu/Asp residues of various length, and modulate the DNA-binding properties and functioning of HMGBs. HMGBs are subject to post-translational modifications which can fine-tune interactions of the proteins with DNA/chromatin and determine their relocation from the nucleus to the cytoplasm and secretion. Association of HMGBs with chromatin is highly dynamic, and the proteins affect the chromatin fiber as architectural factors by transient interactions with nucleosomes, displacement of histone H1, and facilitation of nucleosome remodeling and accessibility of the nucleosomal DNA to transcription factors or other sequence-specific proteins.

Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain.

Abstract High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi from interleukin (IL)-1 receptor type I-/-, Toll-like receptor 2 (TLR2-/-) and RAGE-/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mphi, with RAGE as the major activation-inducing receptor.

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, induces Beclin1-dependent autophagy and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA intercalators (oxaliplatin or adriamycin). On the contrary, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release, as well as its redox state, thus links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.

Sustained proinflammatory responses in rheumatoid arthritis, atherosclerosis, and diabetic retinopathy, as well as in cancer, are often associated with increased angiogenesis that contributes to tissue disruption and disease progression. High mobility group B1 (HMGB1) has been recognized as a proinflammatory cytokine and more recently, as a proangiogenic factor. HMGB1 can either be passively released from necrotic cells or actively secreted in response to angiogenic and inflammatory signals. HMGB1 itself may signal through the receptor for advanced glycation end products (RAGE), and via toll-like receptors, TLR2 and TLR4. Activation of these receptors results in the activation of NFkappaB, which induces the upregulation of leukocyte adhesion molecules and the production of proinflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation. Interestingly, HMGB1 seems to be involved in a positive feedback mechanism, that may help to sustain inflammation and angiogenesis in several pathological conditions, thereby contributing to disease progression. Endothelial cells express HMGB1, as well as the receptors RAGE, TLR2, and TLR4, and in diverse pathologies HMGB1 and its receptors are overexpressed. Furthermore, HMGB1-induced signaling can activate NFkappaB, which can subsequently induce the expression of HMGB1 receptors. Thus, HMGB1 can mediate amplification of inflammation and angiogenesis through increased secretion of HMGB1 and increased expression of the receptors it can interact with. In this review, we discuss signaling cascades that HMGB1 can induce via TLRs and RAGE, as well as its contribution to pathologies involving endothelial cells.

High mobility group box 1 (HMGB1) is an abundant and conserved nuclear protein that is released by necrotic cells and acts in the extracellular environment as a primary proinflammatory signal. In this study we show that human dendritic cells, which are specialized in Ag presentation to T cells, actively release their own HMGB1 into the extracellular milieu upon activation. This secreted HMGB1 is necessary for the up-regulation of CD80, CD83, and CD86 surface markers of human dendritic cells and for IL-12 production. The HMGB1 secreted by dendritic cells is also required for the clonal expansion, survival, and functional polarization of naive T cells. Using neutralizing Abs and receptor for advanced glycation end product-deficient (RAGE(-/-)) cells, we demonstrate that RAGE is required for the effect of HMGB1 on dendritic cells. HMGB1/RAGE interaction results in downstream activation of MAPKs and NF-kappaB. The use of an ancient signal of necrosis, HMGB1, by dendritic cells to sustain their own maturation and for activation of T lymphocytes represents a profitable evolutionary mechanism.

OBJECTIVE

Individuals with type 2 diabetes have a myriad of metabolic aberrations including increased inflammation, increasing their cardiovascular risk. Toll-like receptors (TLRs) and their ligands play a key role in insulin resistance and atherosclerosis. However, there is a paucity of data examining the expression and activity of TLRs in type 2 diabetes. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression, their ligands, and signaling in monocytes of recently diagnosed type 2 diabetic patients.

METHODS

TLR mRNA, protein expression, TLR ligands, and TLR signaling were measured in freshly isolated monocytes from healthy human control subjects (n = 23) and type 2 diabetic subjects (n = 23) using real-time RT-PCR, Western blot, and flow cytometric assays.

RESULTS

Type 2 diabetic subjects had significantly increased TLR2, TLR4 mRNA, and protein in monocytes compared with control subjects (P < 0.05). Increased TLR2 and TLR4 expression correlated with BMI, homeostasis model assessment-insulin resistance (HOMA-IR), glucose, A1C, N(epsilon)-(carboxymethyl) lysine (CML), and free fatty acid (FFA). Ligands of TLR2 and TLR4, namely, HSP60, HSP70, HMGB1, endotoxin, and hyaluronan levels, were elevated in type 2 diabetic subjects and positively correlated with TLR2 and TLR4. Type 2 diabetic subjects showed increased MyD88, phosphorylated IRAK-1, Trif, TICAM-1, IRF-3, and NF-kappaB p65 expression in monocytes compared with control subjects. Furthermore, TLR-MyD88-NF-kappaB signaling resulted in elevated levels of cytokines (P < 0.05), but increased interleukin (IL)-1beta, interferon (IFN)-gamma, and endotoxin were not significant when adjusted for BMI.

CONCLUSIONS

In this comprehensive study, we make the novel observation that TLR2 and TLR4 expression and their ligands, signaling, and functional activation are increased in recently diagnosed type 2 diabetes and contribute to the proinflammatory state.

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

Complex genetic and physiological variations as well as environmental factors that drive emergence of chromosomal instability, development of unscheduled cell death, skewed differentiation, and altered metabolism are central to the pathogenesis of human diseases and disorders. Understanding the molecular bases for these processes is important for the development of new diagnostic biomarkers, and for identifying new therapeutic targets. In 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed high-mobility group (HMG) proteins. The HMG proteins include three superfamilies termed HMGB, HMGN, and HMGA. High-mobility group box 1 (HMGB1), the most abundant and well-studied HMG protein, senses and coordinates the cellular stress response and plays a critical role not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as the prototypic damage associated molecular pattern molecule (DAMP). This DAMP, in conjunction with other factors, thus has cytokine, chemokine, and growth factor activity, orchestrating the inflammatory and immune response. All of these characteristics make HMGB1 a critical molecular target in multiple human diseases including infectious diseases, ischemia, immune disorders, neurodegenerative diseases, metabolic disorders, and cancer. Indeed, a number of emergent strategies have been used to inhibit HMGB1 expression, release, and activity in vitro and in vivo. These include antibodies, peptide inhibitors, RNAi, anti-coagulants, endogenous hormones, various chemical compounds, HMGB1-receptor and signaling pathway inhibition, artificial DNAs, physical strategies including vagus nerve stimulation and other surgical approaches. Future work further investigating the details of HMGB1 localization, structure, post-translational modification, and identification of additional partners will undoubtedly uncover additional secrets regarding HMGB1's multiple functions.

The receptor for advanced glycation endproducts (RAGE) mediates responses to cell danger and stress. When bound by its many ligands (which include advanced glycation endproducts, certain members of the S100/calgranulin family, extracellular high-mobility group box 1, the integrin Mac-1, amyloid beta-peptide and fibrils), RAGE activates programs responsible for acute and chronic inflammation. RAGE is therefore also involved in cancer progression, diabetes, atherosclerosis, and Alzheimer's disease. RAGE has several isoforms deriving from alternative splicing, including a soluble form called endogenous secretory RAGE (esRAGE). We show here that most soluble RAGE, either produced by cell lines or present in human blood, is not recognized by an anti-esRAGE antibody. Cells transfected with the cDNA for full-length RAGE, and thus not expressing esRAGE, produce a form of soluble RAGE, cleaved RAGE (cRAGE) that derives from proteolytic cleavage of the membrane-bound molecules and acts as a decoy receptor. By screening chemical inhibitors and genetically modified mouse embryonic fibroblasts (MEFs), we identify the sheddase ADAM10 as a membrane protease responsible for RAGE cleavage. Binding of its ligand HMGB1 promotes RAGE shedding. Our data do not disprove the interpretation that high levels of soluble forms of RAGE protect against chronic inflammation, but rather suggest that they correlate with high levels of ongoing inflammation.

HMGB1 is a ubiquitous nuclear protein present in almost all cell types. In addition to its intracellular functions, HMGB1 can be extracellularly released, where it mediates activation of innate immune responses, including chemotaxis and cytokine release. HMGB1 contains three conserved redox-sensitive cysteines (C23, C45, and C106); modification of these cysteines determines the bioactivity of extracellular HMGB1. Firstly, the cytokine-stimulating activity of HMGB1 requires C23 and C45 to be in a disulfide linkage, at the same time that C106 must remain in its reduced form as a thiol. This distinctive molecular conformation enables HMGB1 to bind and signal via the TLR4/MD-2 complex to induce cytokine release in macrophages. Secondly, for HMGB1 to act as a chemotactic mediator, all three cysteines must be in the reduced form. This all-thiol HMGB1 exerts its chemotactic activity to initiate inflammation by forming a heterocomplex with CXCL12; that complex binds exclusively to CXCR4 to initiate chemotaxis. Thirdly, binding of the HMGB1 to CXCR4 or to TLR4 is completely prevented by all-cysteine oxidation. Also, the initial post-translational redox modifications of HMGB1 are reversible processes, enabling HMGB1 to shift from acting as a chemotactic factor to acting as a cytokine and vice versa. Lastly, post-translational acetylation of key lysine residues within NLSs of HMGB1 affects HMGB1 to promote inflammation; hyperacetylation of HMGB1 shifts its equilibrium from a predominant nuclear location toward a cytosolic and subsequent extracellular presence. Hence, post-translational modifications of HMGB1 determine its role in inflammation and immunity.

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK-treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Here, we show that extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesoangioblasts), and thus may play a role in muscle tissue regeneration. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are unresponsive to control beads. Interestingly, alpha-sarcoglycan null dystrophic muscle contains elevated levels of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled. This implies that the HMGB1-RAGE interaction is sufficient, but not necessary, for mesoangioblast homing; a different pathway might coexist. Although the role of endogenous HMGB1 in the reconstruction of dystrophic muscle remains to be clarified, injected HMGB1 may be used to promote tissue regeneration.

The function of the C5a receptors, C5ar (encoded by C5ar) and C5l2 (encoded by Gpr77), especially of C5l2, which was originally termed a 'default receptor', remains a controversial topic. Here we investigated the role of each receptor in the setting of cecal ligation and puncture-induced sepsis by using antibody-induced blockade of C5a receptors and knockout mice. In 'mid-grade' sepsis (30-40% survival), blockade or absence of either C5ar or C5l2 greatly improved survival and attenuated the buildup of proinflammatory mediators in plasma. In vivo appearance or in vitro release of high mobility group box 1 protein (HMGB1) required C5l2 but not C5ar. In 'high-grade' sepsis (100% lethality), the only protective condition was the combined blockade of C5l2 and C5ar. These data suggest that C5ar and C5l2 contribute synergistically to the harmful consequences in sepsis and that C5l2 is required for the release of HMGB1. Thus, contrary to earlier speculation, C5l2 is a functional receptor rather than merely a default receptor.

BACKGROUND

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.

RESULTS

Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4(+) and CD8(+) T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.

CONCLUSIONS

Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.

HMGB1, outside of a cell, is a trigger of inflammation and a stimulus for tissue reconstruction; the balance may depend on the complexes it forms with other molecules. HMGB1 is the prime example of a danger signal that originates from the damaged self rather than from invading pathogens. HMGB1 is released by cells that die traumatically and is secreted by cells destined to die and by activated cells of the innate immunity system. As a danger signal, HMGB1 is expected to trigger inflammation, but recent reports indicate that pure recombinant HMGB1 has no proinflammatory activity and only acts as a chemoattractant and a mitogen. However, HMGB1 forms highly inflammatory complexes with ssDNA, LPS, IL-1beta, and nucleosomes, which interact with TLR9, TLR4, IL-1R, and TLR2 receptors, respectively. Thus, HMGB1 has dual activities, solo or in company; I speculate that this may serve our body's necessity to sacrifice or reconstruct tissues as required by the presence or absence of pathogens.

OBJECTIVE

To study the systemic release and kinetics of high mobility group box-1 protein (HMGB1) in relation to clinical features in a population of patients with severe sepsis or septic shock and to compare these with the kinetics of the cytokines interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-alpha.

METHODS

Prospective study of two cohorts of patients.

METHODS

Intensive care unit and infectious disease clinic at Karolinska University Hospital Huddinge.

METHODS

Twenty-six patients with severe sepsis, 33 patients with septic shock, and a reference group of five patients with sepsis.

METHODS

None.

RESULTS

Sixty-four patients were included, ten of whom died within 28 days. Cytokine levels were measured at five time points during the first week after admission and were correlated to Acute Physiology and Chronic Health Evaluation II and Sepsis-related Organ Failure Assessment scores. Two HMGB1 assays were used. Both demonstrated delayed kinetics for HMGB1 with high levels on inclusion that remained high throughout the study period. Serum concentration at 144 hrs, the last sampling point, was 300 times higher, 34,000 +/- 76,000 pg/mL (mean +/- sd), than any of the other cytokines. This study, however, found no predictable correlation between serum levels of HMGB1 and severity of infection. We did quite unexpectedly find significantly lower levels of HMGB1 in nonsurvivors compared with survivors as measured by our main assay, but the other showed no difference between the two groups. Levels of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-alpha correlated significantly with severity of disease, and all were significantly higher in patients with septic shock compared with those with severe sepsis. Neither of these comparisons showed significant correlations for HMGB1.

CONCLUSIONS

This is the first prospective study assessing the release over time of HMGB1 in a population of patients with sepsis, severe sepsis, or septic shock. Levels remained high in the majority of patients up to 1 wk after admittance, indicating that the cytokine indeed is a downstream and late mediator of inflammation. Further studies are required to fully define the relationship of HMGB1 to severity of disease.

Forty years ago, high mobility group box 1 (HMGB1) was discovered in calf thymus and named according to its electrophoretic mobility in polyacrylamide gels. Now, we know that HMGB1 performs dual functions. Inside the cell, HMGB1 is a highly conserved chromosomal protein acting as a DNA chaperone. Outside of the cell, HMGB1 is a prototypical damage-associated molecular pattern, acting with cytokines, chemokines, and growth factors. During tumor development and in cancer therapy, HMGB1 has been reported to play paradoxical roles in promoting both cell survival and death by regulating multiple signaling pathways, including inflammation, immunity, genome stability, proliferation, metastasis, metabolism, apoptosis, and autophagy. Here, we review the current knowledge of both HMGB1's oncogenic and tumor-suppressive roles and the potential strategies that target HMGB1 for the prevention and treatment of cancer.

Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunologic process with a wide array of roles in immunity. The immunologic functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunologic effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a stepwise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors, Nod-like receptors, RIG-I-like receptors, damage-associated molecular patterns such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors that act as autophagy adapters, immunity-related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T- and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.

High mobility group box-1 (HMGB1) protein is a nonhistone, DNA-binding protein that plays a critical role in regulating gene transcription. Recently, HMGB1 has also been shown to act as a late mediator of endotoxic shock and to exert a variety of proinflammatory, extracellular activities. Here, we report that HMGB1 simultaneously acts as a chemoattractant and activator of dendritic cells (DCs). HMGB1 induced the migration of monocyte-derived, immature DCs (Mo-iDCs) but not mature DCs. The chemotactic effect of HMGB1 on iDCs was pertussis toxin-inhibitable and also inhibited by antibody against the receptor of advanced glycation end products (RAGE), suggesting that HMGB1 chemoattraction of iDCs is mediated by RAGE in a Gi protein-dependent manner. In addition, HMGB1 treatment of Mo-iDCs up-regulated DC surface markers (CD80, CD83, CD86, and HLA-A,B,C), enhanced DC production of cytokines (IL-6, CXCL8, IL-12p70, and TNF-alpha), switched DC chemokine responsiveness from CCL5-sensitive to CCL21-sensitive, and acquired the capacity to stimulate allogeneic T cell proliferation. Based on its dual DC-attracting and -activating activities as well as its reported capacity to promote an antigen-specific immune response, we consider HMGB1 to have the properties of an immune alarmin.