High mobility group 1 (HMGB1) protein is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (the receptor for advanced glycation end products) and is a potent mediator of inflammation. HMGB1 is secreted by activated monocytes and macrophages, and is passively released by necrotic or damaged cells. Here we report that Hmgb1(-/-) necrotic cells have a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell to its neighbours. Apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not cleared promptly by phagocytic cells. In apoptotic cells, HMGB1 is bound firmly to chromatin because of generalized underacetylation of histone and is released in the extracellular medium (promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal that is broadcast by cells that have been damaged or killed by trauma.

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

High-mobility group box 1 protein (HMGB1), which previously was thought to function only as a nuclear factor that enhances transcription, was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation. These observations have led to the emergence of a new field in immunology that is focused on understanding the mechanisms of HMGB1 release, its biological activities and its pathological effects in sepsis, arthritis, cancer and other diseases. Here, we discuss these features of HMGB1 and summarize recent advances that have led to the preclinical development of therapeutics that modulate HMGB1 release and activity.

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP(3)R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy.

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-kappaB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-kappaB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKbeta, HMGB1 exposure resulted in activation of both IKKalpha and IKKbeta. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-kappaB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-kappaB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS.

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

The immune system has evolved to respond not only to pathogens, but also to signals released from dying cells. Cell death through necrosis induces inflammation, whereas apoptotic cell death provides an important signal for tolerance induction. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 can associate with other molecules, including TLR ligands and cytokines, and activates cells through the differential engagement of multiple surface receptors including TLR2, TLR4, and RAGE. RAGE is a multiligand receptor that binds structurally diverse molecules, including not only HMGB1, but also S100 family members and amyloid-beta. RAGE activation has been implicated in sterile inflammation as well as in cancer, diabetes, and Alzheimer's disease. While HMGB1 through interactions with TLRs may also be important, this review focuses on the role of the HMGB1-RAGE axis in inflammation and cancer.

A key question in immunology concerns how sterile injury activates innate immunity to mediate damaging inflammation in the absence of foreign invaders. The discovery that HMGB1, a ubiquitous nuclear protein, mediates the activation of innate immune responses led directly to the understanding that HMGB1 plays a critical role at the intersection of the host inflammatory response to sterile and infectious threat. HMGB1 is actively released by stimulation of the innate immune system with exogenous pathogen-derived molecules and is passively released by ischemia or cell injury in the absence of invasion. Established molecular mechanisms of HMGB1 binding and signaling through TLR4 reveal signaling pathways that mediate cytokine release and tissue damage. Experimental strategies that selectively target HMGB1 and TLR4 effectively reverse and prevent activation of innate immunity and significantly attenuate damage in diverse models of sterile and infection-induced threat.

High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic cells, triggers inflammation. HMGB1 can also be secreted by activated monocytes and macrophages, and functions as a late mediator of inflammation. Secretion of a nuclear protein requires a tightly controlled relocation program. We show here that in all cells HMGB1 shuttles actively between the nucleus and cytoplasm. Monocytes and macrophages acetylate HMGB1 extensively upon activation with lipopolysaccharide; moreover, forced hyperacetylation of HMGB1 in resting macrophages causes its relocalization to the cytosol. Cytosolic HMGB1 is then concentrated by default into secretory lysosomes, and secreted when monocytic cells receive an appropriate second signal.

High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH(2)-terminal kinase and higher nuclear factor-kappaB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process.

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1beta] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1beta. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 microg per mouse) or the DNA-binding A box (600 microg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%, P < 0.03; GST control protein = 28% vs. A box = 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)-induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)-dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid-recognizing antibodies and type I IFN production in this disease.

Although it was thought that apoptotic cells, when rapidly phagocytosed, underwent a silent death that did not trigger an immune response, in recent years a new concept of immunogenic cell death (ICD) has emerged. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface-exposed calreticulin (CRT), secreted ATP and released high mobility group protein B1 (HMGB1). Most DAMPs can be recognized by pattern recognition receptors (PRRs). In this Review, we discuss the role of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in regulating the immunogenicity of dying cancer cells and the effect of therapy-resistant cancer microevolution on ICD.

Physiological anti-inflammatory mechanisms can potentially be exploited for the treatment of inflammatory disorders. Here we report that the neurotransmitter acetylcholine inhibits HMGB1 release from human macrophages by signaling through a nicotinic acetylcholine receptor. Nicotine, a selective cholinergic agonist, is more efficient than acetylcholine and inhibits HMGB1 release induced by either endotoxin or tumor necrosis factor-alpha (TNF-alpha). Nicotinic stimulation prevents activation of the NF-kappaB pathway and inhibits HMGB1 secretion through a specific 'nicotinic anti-inflammatory pathway' that requires the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In vivo, treatment with nicotine attenuates serum HMGB1 levels and improves survival in experimental models of sepsis, even when treatment is started after the onset of the disease. These results reveal acetylcholine as the first known physiological inhibitor of HMGB1 release from human macrophages and suggest that selective nicotinic agonists for the alpha7nAChR might have therapeutic potential for the treatment of sepsis.

Advanced glycation end products (AGEs), S100/calgranulins, HMGB1-proteins, amyloid-beta peptides, and the family of beta-sheet fibrils have been shown to contribute to a number of chronic diseases such as diabetes, amyloidoses, inflammatory conditions, and tumors by promoting cellular dysfunction via binding to cellular surface receptors. The receptor for AGEs (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules acting as counter-receptor for these diverse molecules. Engagement of RAGE converts a brief pulse of cellular activation to sustained cellular dysfunction and tissue destruction. The involvement of RAGE in pathophysiologic processes has been demonstrated in murine models of chronic disease using either a receptor decoy such as soluble RAGE (sRAGE), RAGE neutralizing antibodies, or a dominant-negative form of the receptor. Studies with RAGE-/- mice confirmed that RAGE contributes, at least in part, to the development of late diabetic complications, such as neuropathy and nephropathy, macrovascular disease, and chronic inflammation. Furthermore, deletion of RAGE provided protection from the lethal effects of septic shock caused by cecal ligation and puncture (CLP). In contrast, deletion of RAGE had no effect on the host response in delayed-type hypersensitivity (DTH). Despite the lack of effect seen in adaptive immunity by the deletion of RAGE, administration of the receptor decoy, sRAGE, still afforded a protective effect in RAGE-/- mice. Thus, sRAGE is likely to sequester ligands, thereby preventing their interaction with other receptors in addition to RAGE. These data suggest that, just as RAGE is a multiligand receptor, its ligands are also likely to recognize several receptors in mediating their biologic effects.

BACKGROUND

Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo.

RESULTS

Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands.

CONCLUSIONS

Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection.

In eukaryotes, the core promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or downstream promoter element (DPE) found in most core promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived positive cofactors (PC1/PARP-1, PC2, PC3/DNA topoisomerase I, and PC4) and negative cofactor 1 (NC1/HMGB1), normally function independently or in combination to fine-tune the promoter activity in a gene-specific or cell-type-specific manner. In addition, other cofactors, such as TAF1, BTAF1, and negative cofactor 2 (NC2), can also modulate TBP or TFIID binding to the core promoter. In general, these cofactors are capable of repressing basal transcription when activators are absent and stimulating transcription in the presence of activators. Here we review the roles of these cofactors and GTFs, as well as TBP-related factors (TRFs), TAF-containing complexes (TFTC, SAGA, SLIK/SALSA, STAGA, and PRC1) and TAF variants, in pol II-mediated transcription, with emphasis on the events occurring after the chromatin has been remodeled but prior to the formation of the first phosphodiester bond.

High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-kappaB-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS.

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.

Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

Recent studies indicate that IL-1alpha functions intracellularly in pathways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-associated protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines, is an endothelium-derived, chromatin-associated nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-associated nuclear factor in HEV endothelial cells in vivo. Association of IL-33 with heterochromatin was also observed in human and mouse cells under living conditions. In addition, colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-association, and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, associated with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1alpha and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.

During infection, vertebrates develop "sickness syndrome," characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.