In 1999, Wang and colleagues 12 identified HMGB1 as a cytokine-like mediator of lipopolysaccharide (LPS)-induced mortality in mice. Subsequently, these findings were extended by Yang and colleagues 13, who showed that HMGB1 is also a mediator of lethality in mice rendered septic by the induction of polymicrobial bacterial peritonitis. Additional studies documented that extracellular HMGB1 can promote tumor necrosis factor (TNF) release from mononuclear cells 14 and increase the permeability of Caco-2 monolayers 15.

One of the most interesting features of HMGB1 as a cytokine-like mediator of inflammation is that this protein is released much later in the inflammatory process than are the classical 'alarm-phase' cytokines, such as TNF and interleukin (IL)-1β. For example, in mice, injection of a bolus dose of LPS elicits a monophasic spike in circulating TNF which peaks within 60 to 90 minutes of the proinflammatory challenge and is over within 4 hours 16. The peak in IL-1β concentration occurs somewhat later (that is, 4 to 6 hours after the injection of LPS) 17. In contrast, after mice are injected with LPS, circulating levels of HMGB1 are not elevated until 16 hours after the proinflammatory stimulus but remain elevated for more than 30 hours 12. Furthermore, treatment with neutralizing anti-HMGB1 antibodies 1213 or various pharmacological agents that block HMGB1 secretion, such as nicotine 18 or ethyl pyruvate 19, is effective in preventing LPS- or sepsis-induced lethality, even when therapy is started 4 to 24 hours after the initiation of the disease process. Because of the delayed kinetics for release, HMGB1 is a very attractive drug target for acute, often lethal, syndromes such as severe sepsis and hemorrhagic shock because the 'treatment window' for anti-HMGB1 therapies should be longer than is the case for therapeutic agents directed at more proximal mediators of the inflammatory cascade (for example, TNF or IL-1β).

Data obtained by Scaffidi and colleagues 20 supported the view that HMGB1 is passively released by necrotic, but not apoptotic, cells. This process may depend, at least in part, on activation of the enzyme PARP (poly [ADP]-ribose polymerase), which is activated as a result of DNA damage and which upon activation promotes translocation of HMGB1 from the nucleus to the cytosol 21. In this fashion, the release of HMGB1 from necrotic tissue damaged by trauma or ischemia could serve as an endogenous 'danger signal' that alerts the immune system to the presence of injured cells 2223.

Recently, however, Jiang and colleagues 24 reported that macrophages and Jurkat T cells passively release HMGB1 during the process of apoptosis. Similarly, Bell and colleagues 25 reported that Jurkat cells, U937 human monocytic cells, Panc1 (human pancreatic cancer) cells, and HeLa cells all passively release HMGB1 when apoptosis is induced by agents, such as staurosporine, etoposide, or camptothecin. Furthermore, Qin and colleagues 26 showed that incubating RAW 264.7 murine macrophage-like cells with apoptotic or necrotic macrophages or apoptotic T lymphocytes triggers the active secretion of HMGB1 by the RAW 264.7 cells. Thus, it seems doubtful that passive release of HMGB1 occurs only when cells die a necrotic (rather than apoptotic) death. Also, it seems doubtful that only necrotic cells are capable of eliciting HMGB1 secretion by other (viable) macrophages.

HMGB1 is actively secreted by immunostimulated macrophages 12272829, natural killer cells 30, plasmacytoid dendritic cells 31, pituicytes 32, and enterocytes 33. As with members of the IL-1 family of cytokines, the primary amino acid sequence of HMGB1 lacks a signal peptide. Accordingly, secretion of HMGB1 by macrophages or monocytes presumably occurs via a nonclassical secretory pathway. Indeed, when monocytes are activated by exposure to LPS, HMGB1 relocalizes from the nucleus into cytoplasmic organelles that belong to the endolysosomal compartment 28. Gardella and colleagues 28 reported that 65% of HMGB1 is confined to the nucleus in resting monocytes but that only 26% of HMGB1 is nuclear and 74% is associated with cytoplasmic organelles in LPS-stimulated monocytes. In activated monocytes, the transfer of HMGB1 from the nucleus to the cytoplasm is mediated by hyperacetylation of critical lysine clusters that are components of nuclear localization signals 29. This acetylation prevents HMGB1 from interacting with the nuclear-importer protein complex, so re-entry to the nucleus is blocked. Acetylated, cytosolic HMGB1 subsequently migrates to cytoplasmic secretory vesicles. Currently, it is not known how cellular activation leads to acetylation of HMGB1.

Epithelial cells, including enterocytes, also secrete HMGB1 following immune stimulation. Kuniyasu and colleagues 34 recently reported that WiDr human colon cancer cells constitutively release HMGB1 into culture supernatants. In contrast, Liu and colleagues 33 observed only very low levels of HMGB1 in the media of unstimulated Caco-2 human transformed enterocyte-like cells. However, following stimulation of the cells with a mixture of TNF, IL-1β, and interferon-gamma [IFN-γ]), there was a large increase in the amount of HMGB1 released into the culture media. Liu and colleagues 33 also showed that incubating Caco-2 cells with the synthetic Toll-like receptor (TLR) 2 ligand, FSL-1, or the TLR5 ligand, flagellin, caused a large increase in the amount of HMGB1 released into the media. Interestingly, the TLR4 agonist, LPS, failed to stimulate HGMB1 secretion by Caco-2 cells.

Data obtained by Gardella and colleagues 28 support the notion that the secretion of HMGB1 by stimulated monocytes occurs when secretory lysosomes undergo exocytosis. In contrast, secretion of HMGB1 from Caco-2 cells apparently depends on the release of exosomes into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface 33. Exosomes are 30- to 90-nm membrane-bound vesicles that are secreted by numerous cell types, including reticulocytes 35, platelets 36, B lymphocytes 37, dendritic cells 38, and epithelial cells 39. Exosomes are formed when multivesical bodies in the cytoplasm fuse with the plasma membrane, releasing the vesicles into the extracellular compartment 40.

HMGB1 is expressed in virtually all nucleated cells. In general, the HMGB1 gene appears to be tightly regulated, being expressed at a basal level in most cells and tissues. In proliferating tissues and actively dividing cells, there is a slight increase in expression level (approximately twofold) 4142. Expression of HMGB1 increases by about the same extent when estrogen-responsive breast cancer cells are treated with estrogen 43 or synchronized Chinese hamster ovary cells progress from the G₁ to the S phase 44.

Transcription of the human HMGB1 gene starts at a major site located 57 nucleotides upstream from the first exon–intron boundary 45. The core promoter of the human HMGB1 gene lacks a TATA box and is located within the -219 to +154 region. Immediately upstream of the core promoter, there is a silencer element that contains a putative growth factor independence-1 (GFI-1)-binding site. Since GFI-1 is a known repressor 46, it is possible that GFI-1 binds to this site and represses the expression of HMGB1. Constitutive activity of this repressor may be important for maintaining HMGB1 expression at basal levels in most cells. Intron 1 is highly conserved between the human and the mouse HMGB1 genes. The region of intron 1 between +155 to +2061 contains enhancer activity, and the most potent enhancer elements are located between +1043 and +1429. Within this region of intron 1, there are several binding sites of putative Sp1, activator protein (AP) 1, AP4, and upstream stimulatory factor. Sp1, in particular, is known to enhance the expression of genes with TATA-less core promoters 4748 and is known to be crucial for the transcriptional regulation of IL-10 secretion by LPS-stimulated macrophages 49. Furthermore, signaling via members of the AP1 family of transcription factors is known to be important in the transcriptional regulation of a number of genes, such as heme oxygenase-1 5051 and IL-18 52, in LPS- and/or IFN-γ stimulated macrophages.

According to Kalinina and colleagues 53, steady-state levels of HMGB1 mRNA are increased in THP-1 human promonocyte-like cells stimulated with IFN-γ or TNF. These authors reported that TNF- or IFN-γ-induced upregulation of HMGB1 mRNA expression is not affected in THP-1 cells by pharmacological inhibition of extracellular signal-regulated kinase (ERK) 1/ERK2 mitogen-activated protein kinase (MAPK)- or PKC (protein kinase C)-dependent signaling but is inhibited by treating the cells with wortmannin, an inhibitor of PI3K (phosphatidyl inositol-3-kinase). Liu and colleagues 54 reported that incubating RAW 264.7 murine macrophage-like cells with LPS leads to increased HMGB1 mRNA expression. LPS-induced upregulation of HMGB1 mRNA expression was blocked by several pharmacological inhibitors of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway. Increased HMGB1 mRNA expression also has been observed in animal models of acute or chronic inflammation, including collagen-induced arthritis in rats 55, murine cardiac allograft rejection 56, and LPS injection in rats pretreated with ethanol 57.

The TLR 4 agonist, LPS, and the cytokines TNF, IFN-γ, and TWEAK (

T

WEAK

Other data argue against an important role for NF-κB-dependent signaling. In a study of TNF- or IFN-γ-stimulated THP-1 cells, Kalinina and colleagues 53 reported that HMGB1 secretion is not inhibited by the NF-κB inhibitor, iso-helanin. Similarly, Killeen and colleagues 59 showed that treating RAW 264.7 cells with PDTC (pyrollidine diothiocarbamate), SN50 (amino acid sequence AAVALLPAVLLA-LLAPVQRKRQKLMP), or 5-(thien-3-yl)-3-aminothiophene-2-carboxamide (SC-514) blocks LPS-induced NF-κB DNA binding but fails to inhibit LPS-induced HMGB1 secretion.

To date, four transmembrane proteins have been identified as potential cellular receptors for HMGB1. These proteins are the

R

A

G

E

RAGE, a member of the immunoglobulin superfamily of proteins, is activated by a wide variety of ligands, including products of the non-enzymatic oxidation of glucose (

A

G

E

The recognition that HMGB1 is capable of activating RAGE-dependent signaling was prompted by a series of publications by Rauvala and Pihlaskari 68. In 1987, they identified a 27- to 30-kDa heparin-binding protein that promotes neurite outgrowth in rat brain neurons. Subsequently, this research group cloned this protein from a cDNA library constructed from rat brain mRNA 69. The protein, which was called amphoterin because of its positively charged N-terminal region and negatively charged C-terminal domain, was shown to have the same primary amino acid sequence as HMGB1 69. Amphoterin/HMGB1 was shown to be localized in the cytoplasm and filopodia of neurons 69.

During the course of tissue surveys to assess RAGE distribution in vivo, it became evident that expression of the receptor occurs in early development, especially in the central nervous system where AGEs, the presumed primary ligands for RAGE, are unlikely to be present. Accordingly, these investigators entertained the hypothesis that AGEs might be accidental ligands for a receptor that has other functions. Toward this end, they sought to define putative natural ligands for RAGE. Starting with homogenates prepared from bovine lung tissue, protein fractions obtained using a heparin-Sepharose column were evaluated for RAGE-binding activity. Ultimately, two polypeptides (molecular masses of 12 and 23 kDa) were identified. The 23-kDa polypeptide was identified as amphoterin/HMGB1 63. Moreover, authentic amphoterin/HMGB1 was shown to bind to RAGE with high affinity 63. Subsequently, it was shown that amphoterin induces neurite outgrowth in neuroblastoma cells transfected with a plasmid encoding RAGE but not in cells transfected with a plasmid encoding a mutant RAGE missing the intracytoplasmic portion of the receptor 70.

More recently, the pathogen-associated molecular pattern (PAMP) receptors, TLR2 717273 and TLR4 717273747576, also have been identified as HMGB1 receptors. Nevertheless, a number of studies have shown that treatment of various cell types with anti-RAGE antibodies inhibits HMGB1-mediated effects by 50% to 100% 15317778.

Since key receptors for HMGB1, such as TLR2 and TLR4, are localized to the apical surface of enteroyctes 7179, the observation that HMGB1 is secreted apically by intestinal epithelial cells supports the idea that release of this protein might serve an autocrine role to amplify the activation of enterocytes by other factors. This notion is supported by our previously reported observation that HMGB1 promotes activation of NK-κB in Caco-2 cells and also increases the permeability of Caco-2 monolayers 15. To specifically test this hypothesis, Liu and colleagues 33 stimulated Caco-2 monolayers in the absence or presence of a polyclonal neutralizing anti-HMGB1 antibody added to the apical compartment of Transwell chambers. Treatment with anti-HMGB1 antibody significantly blunted the development of hyperpermeability 33. Thus, secretion of HMGB1 may be an important positive feedback phenomenon that promotes the development of intestinal epithelial barrier dysfunction due to inflammation.

Recently, it has become apparent that highly purified HMGB1 has only minimal cytokine-like activity in vitro, whereas Escherichia coli-derived recombinant HMGB1, presumably contaminated with trace amounts of various microbial products, is more effective at triggering TNF secretion by cultured macrophages 8081. Since HMGB1 tends to avidly bind bacterial products and DNA, it is possible that the proinflammatory effects of HMGB1 are mediated not by the pure protein per se, but rather by complexes formed when the protein interacts with other proinflammatory substances 82. This notion is supported by findings reported by Tian and colleagues 82, who showed that although HMGB1 binds to a RAGE-like man-made fusion protein (RAGE-Fc), binding is much better when HMGB1 is complexed with CpG-rich oligodeoxynucleotides.

TLR9 is a PAMP receptor that is localized within cells in the endoplasmic reticulum and endosomal compartments 8384. TLR9 recognizes methylated (bacterial) or unmethylated (eukaryotic) CpG oligodeoxynucleotides 85. Tian and colleagues 82 have presented data indicating that complexes of RAGE, CpG-rich oligodeoxynucleotides, and HMGB1 are transported into cells. These complexes are localized within an endosomal compartment and are physically associated with TLR9. Thus, TLR9 may be another 'HMGB1 receptor,' at least when HMGB1 is complexed with CpG-rich oligodeoxynucleotides and RAGE.

Circulating concentrations of HMGB1 are increased in rodent models of sepsis 121319868788 or hemorrhagic shock 7589. Furthermore, treatment with anti-HMGB1 neutralizing antibodies has been shown to ameliorate organ dysfunction and/or improve survival in rodent models of sepsis 121387, hemorrhagic shock 8990, acute pancreatitis 91, and hepatic ischemia/reperfusion injury 74. Similarly, drugs that block HMGB1 secretion have been shown to improve survival and/or ameliorate organ dysfunction in mice subjected to cecal ligation and perforation to induce sepsis 181992. Finally, administration of authentic HMGB1 (or the B box fragment of the protein) has been shown to induce lethality and/or induce organ damage in experimental animals 121593. Thus, HMGB1 appears to fulfill a modern version of Koch's postulates for being a mediator of various forms of acute illness.

Wang and colleagues 12 reported that circulating levels of HMGB1 are increased in patients with severe sepsis, particularly among patients with a lethal form of the syndrome. Similar findings were reported by Hatada and colleagues 94, who measured plasma immunoreactive HMGB1 levels in patients with proven or suspected disseminated intravascular coagulation (DIC) by means of an enzyme-linked immunosorbent assay (ELISA) system. In that study, circulating concentrations of HMGB1 were below the detection limit in normal subjects but were moderately elevated in patients with infectious diseases, cancer, and trauma. DIC was associated with even greater plasma HMGB1 levels, and the highest HMGB1 levels were detected in patients with organ failure and nonsurvivors.

Other investigators, studying patients with infections and/or sepsis, have obtained qualitatively different findings. For example, Gaïni and colleagues 95 reported that circulating HMGB1 levels are increased (relative to healthy controls) in intensive care unit (ICU) patients with infections, sepsis, or severe sepsis (that is, sepsis with organ dysfunction). In that study, HMGB1 levels were measured by means of a commercially available ELISA kit. Importantly, these authors found that HMGB1 levels failed to discriminate between ICU patients with infections and those without infections. Thus, in that study at least, a high circulating level of HMGB1 appeared to be more of an indicator of 'sickness' rather than a marker of infection.

Somewhat similar findings were reported by Sunden-Cullberg and colleagues 96, who detected persistently high serum levels of HMGB1 in patients with sepsis or septic shock but found no predictable correlation between HMGB1 concentration and severity of infection. Similarly, in a prospective study of patients with community-acquired pneumonia, Angus and colleagues 97 found that plasma HMGB1 concentrations remained elevated throughout the hospital course and did not differ between those with and without severe sepsis. In that study, HMGB1 concentrations were slightly (and statistically significantly) higher in nonsurvivors than survivors 97. Remarkably, half of the patients who were alive at 90-day follow-up had HMGB1 concentrations greater than three times the upper 95th percentile of the range for normal controls.

Thus, in our current state of knowledge, we must conclude that even though HMGB1 is an important mediator of lethal sepsis in mice and circulating levels of HMGB1 are elevated in septic humans, there is at best a weak relationship between the magnitude of 'HMGB1-emia' and clinical prognosis. The story – at least as it stands right now – is indeed puzzling.

High circulating levels of HMGB1 also have been detected in patients with hemorrhagic shock and/or trauma. Ombrellino and colleagues 98 described a patient with high circulating levels of HMGB1 following an episode of hemorrhagic shock. This finding was confirmed by Yang and colleagues 90, who showed that circulating HMGB1 levels are significantly greater in victims of trauma with hemorrhagic shock than those measured in normal volunteers. High circulating levels of HMGB1 also have been detected during the first few days after a major surgical procedure (esophagectomy) 99. Plasma or serum HMGB1 levels are increased in patients with acute coronary syndrome or cerebral vascular ischemia (transient ischemic attack or cerebral vascular accident) 100, human immunodeficiency virus infection 101, multiple organ failure associated with critical illness 94, acute lung injury 102, and severe acute pancreatitis 103.

All of the available clinical data regarding HMGB1 levels in plasma or serum in patients with various forms of acute or chronic illness have been obtained by measuring immuno-reactive levels of the protein. Unfortunately, detecting HMGB1 by ELISA or Western blot assay fails to provide information about the functional activity of the protein. It is possible that the circulating form of HMGB1 changes over time. For example, in the first 48 hours or so after the onset of an acute infection, HMGB1 might be present as a pro-inflammatory mediator, whereas later on the protein might be biologically inactive (or even, potentially, anti-inflammatory). Clearly, additional clinical studies that seek to correlate immunoreactive protein levels with HMGB1-mediated biological responses are needed.

As yet, of course, no anti-HMGB1 therapeutic is available for clinical administration to humans. Nevertheless, a number of agents have been shown to be capable of blocking HMGB1 secretion by immunostimulated cells, including various nicotinic cholinergic agonists 18104; stearoyl lysophos-phatidylcholine 105; ethyl pyruvate 19; the serine protease inhibitor, nafamostat mesilate 86; several steroid-like pigments (tanshinone I, tanshinone IIA, and cryptotanshinone) derived from a Chinese medicinal herb, danshen (Salvia miltiorrhiza) 92; and the diuretic, ethacrynic acid, as well as other drugs that are known to be 'phase 2 enzyme' inducers 59. Some of these pharmacological agents, as well as various polyclonal neutralizing anti-HMGB1 antibodies, have been shown to ameliorate organ dysfunction and/or improve survival in various animal models of critical illness (see above). Because the HMGB1-as-cytokine story is still less than a decade old, it probably will be several more years before any of these approaches for targeting HMGB1 will be tested in a proof-of-principle trial in human patients. However, because HMGB1 is such an attractive drug target, it seems likely that such trials eventually will be performed. Additionally, it is possible that approaches such as using hemoperfusion through a column packed with the LPS-binding agent, polymyxin B 106107, can indirectly decrease circulating levels of HMGB1 by removing the upstream stimulus for secretion of the protein.