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Keywords:

  • HMGB1;
  • Nuclear protein;
  • Inflammation;
  • Necrosis

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

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

Abbreviations:
CLP:

Cecal ligation and puncture

HMGB1:

High mobility group box chromosomal protein 1

RA:

Rheumatoid arthritis

RAGE:

Receptor for advanced glycated end-products

TLR:

Toll-like receptor

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

The innate immune system is rapidly activated and initiates an inflammatory cascade in response to infection, tissue injury and trauma. During infections, the activation signals, i.e.danger signals, are microbial components that are well conserved among microorganisms 14. LPS, proteoglycans and bacterial DNA are well-studied examples of suchsignals and are known to bind to and stimulate a class of activating cell surface receptors denoted Toll-like receptors (TLR) 5. Binding of a danger molecule to a TLR induces production of inflammatory mediators, including cytokines, and increases phagocytosis and expression of costimulatory molecules 6.

An inflammatory reaction can also be triggered in a non-infectious environment, indicating that damaged cells can themselves produce and release molecules functioning as danger signals 711. Cytokines 12, 13, oxidized mitochondrial DNA 14, heat-shock proteins 15 and uric acid 16 are examples of a diverse set of endogenous molecules with known danger-signaling capacities. Release of such molecules can either be the result of an active process in living cells or be due to leakage from dying cells.

A common denominator for the above-mentioned molecules is their extranuclear location within a normal cell. Intranuclear components should also be able to act as danger signals. Only one single nuclear protein, high mobility group box chromosomal protein 1 (HMGB1), has so far been identified to have the capacity to induce cytokines and activate inflammatory cells when it is applied extracellularly 17, 18. The recent discovery of extracellular HMGB1 as a proinflammatory mediator has already generated a number of scientific reports. This review provides a brief background of the HMGB1 protein itself and its reported intracellular functions, and explores the role of HMGB1 as a cytokine and its potential as a target molecule for anti-inflammatory therapy.

2 HMGB1 basics

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

High mobility group proteins were discovered as nuclear proteins more than 30 years ago and are named for their high mobility in electrophoretic polyacrylamide gels 19. There are three families of HMG proteins (HMGA, HMGB and HMGN), all having the capacity to bind and distort DNA but not being transcriptions factors per se. The HMGB family consists of three members — HMGB1, HMGB2 and HMGB3 — with an 80% amino acid identity among the three proteins. All HMGB proteins bind to DNA via their HMG boxes, made up by amino acid sequences formed in α-helical structures with a nonspecific binding affinity for minor grooves of DNA (reviewed in 20, 21).

HMGB1 is highly conserved through evolution, and has 99% identity among all mammals. Out of its 215 amino acids, only two residues are substituted in the rodent and human versions. HMGB1 has two DNA-binding domains — the A-box (aa 1–79) and the B-box (aa 89–163) — and a highly acidic, repetitive C-terminal tail (aa 186–215) (Fig. 1) 2225. The cytokine-inducing part of the HMGB1 molecule when it is released extracellularly has been defined as comprising the first 20 amino acids of the B-box domain 26. There is a so-far-unexplored possibility that HMGB2 or HMGB3 might also mediate extracellular functions in an analogous way to HMGB1.

HMGB1 occurs in high copy numbers in all eukaryotic cells and is vital for ex utero growth. HMGB1 gene deficient mice are born with several defects and die shortly after birth because of hypoglycemia caused by deficient glucocorticoidreceptor function 27. The main intranuclear function of HMGB1 is to distort the double helix sharply to allow proper physical interactions between many different transcription factors and the chromatin. HMGB1 modulates the transcriptional activity of steroid hormone receptors 28, 29, NF-κB, p53 30, RAG1 recombinase 31 and homeobox-containing proteins. Furthermore, HMGB1 interacts with histones in a manner that affects the chromatin structure, with HMGB1 opening up the chromatin coils. HMGB1 is also known to shield cisplatin-damaged DNA from repair mechanisms 32. Because of its low-affinity nonspecific binding to DNA, HMGB1 is a highly motile protein within the nucleus and can actually shuttle between the nucleus and cytosol via nuclear pores. The fact that HMGB1 detaches from condensed chromosomes and diffuses to the cytoplasm during metaphase of cell division further emphasizes the unstable association between HMGB1 and the chromatin 33.

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Figure 1.  Schematic drawing of the HMGB1 molecule. The DNA-binding domains — A-box and B-box — are denoted with yellow fill color. The acidic C-terminal tail is denoted with pink fill color. Acidic amino acids are denoted with red letters, basic amino acids are denoted with blue letters. The shortest peptide with demonstrated cytokine-inducing capacity is denoted with bold letters.

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3 Post-translational modifications

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

After translation, newly synthesized HMGB1 is extensively modified; it may be subjected to acetylation, phosphorylation, methylation, ADP-ribosylation and to glycosylation to a lower extent. Acetylation within the HMG-boxes is functionally very important and will influence the DNA-binding avidity, histone interaction and shuttling between the nucleus and cytoplasm 34. Acetylation of certain lysine residues of HMGB1 will promote the relocation from the nucleus to the cytoplasm and prevent a nuclear reentry, which is a prerequisite for extracellular secretion — to be discussed later. Acetylated HMGB1 will also facilitate intranuclear histone assembly during cell replication. Phosphorylation of HMGB1 has been reported to influence the regulatory effect on transcription factor activity as well as influencing the stability, conformation and DNA-binding affinity of the HMGB1 protein itself 35.

4 Extranuclear localization of HMGB1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

Apart from being present in the nucleus of cells, HMGB1 has also been detected extranuclearly with a tissue-specific localization. Lymphoid tissues and testis have high levels in both nuclei and cytoplasm, whereas brain and liver have low levels, mainly in the cytosol 36. The levels also correlate with the differentiation stage of cells, with a higher expression in undifferentiated cells 37. The fact that HMGB1 is detected in most tumor types and at a level that is higher than in normal tissue is in line with this notion 36. Furthermore, a cell surface expressed form of HMGB1 (called amphoterin) has been described in the developing brain and in growing neurites, where it induces cell proliferation and outgrowth by interacting and signaling through the multi-ligand receptor for advanced glycated end-products (RAGE) 38, 39.

Resting human platelets express HMGB1 cytoplasmically 40. When platelets are activated, HMGB1 is transported to the cell surface. The function of this transport is not fully understood, but it is well established that HMGB1 associates with plasminogen and tissue plasminogen activator on cell surfaces and enhances plasmin generation and proteolysis 41. Membrane-bound HMGB1 has also been reported on the cell surface of murine erythroleukemia (MEL) cells with an implicated role in cell differentiation. Upon stimulation, MEL cells can also release HMGB1 extracellularly 42.

5 Extracellular release of HMGB1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

5.1 HMGB1 is released in two different ways

To act as a danger signal and inflammatory mediator, HMGB1 must be transported extracellularly. This occurs in two principally different manners: active secretion from living inflammatory cells or passive release from necrotic cells.

5.2 Active secretion of HMGB1

Active secretion of HMGB1 from TNF-stimulated macrophages was discovered during studies of endotoxin-induced systemic inflammation 17. Since then, it has been demonstrated that several proinflammatory stimuli induce an active release of HMGB1 from macrophages and monocytes. In comparison with the production and secretion of TNF and IL-1 in activated macrophages/ monocytes, the release of HMGB1 is delayed, with a lag phase of 12–18 h.

HMGB1 lacks a leader peptide and is thus not secreted via the Golgi/ER pathway. HMGB1 secretion from monocytes/macrophages depends on a relocalization from the nucleus to special cytoplasmic organelles, the secretory lysosomes 43. The secretion of IL-1β is also dependent on secretory lysosomes. The initial phase of HMGB1 secretion requires an inflammatory signal such as LPS, IL-1 or TNF to the monocyte (Fig. 2) 17. This signal will lead to acetylation of specified lysine residues, which causes an accumulation of HMG1 in the cytoplasm and blocks a reentry to the nuclear compartment 34. Cytoplasmic HMGB1 will then be taken up by secretory endolysosomes in hematopoietic cells by a mechanism that is presently not known. The HMGB1-containing secretory endolysosomes can then be fused with the cell membrane and secreted after cellular activation by extracellular lysphosphatidylcholine or ATP 43. The majority of the HMGB1 released during the first 16 h of secretion originates from the preformed pool in the nucleus. Only after this period of time does an increased cellular synthesis lead to incorporation of radiolabeled amino acids into the secreted protein 17.

Secretory endolysosomes have been described in myeloid cells, NK cells and cytotoxic T cells. However, HMGB1 secretion via secretory endolysosomes has so far only been described for macrophages and monocytes. Apart from macrophages and monocytes, active release of HMGB1 from pituicytes 44, endothelial HUVEC cells (G. Mullins et al., Activation of human umbilical vein endothelial cells leads to relocation and release of HMGB1; whose pro-inflammatory activity is dexamethasone sensitive, submitted manuscript) and MEL cells 45 has also been reported by as-yet-uncharacterized mechanisms.

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Figure 2.  Cultured, resting macrophages stained by immunofluorescence for intracellular HMGB1. The strongest green FITC signals generated by HMGB1 are located in the nucleus of each cell, with a weak contribution from cytoplasmic HMGB1 (A). The same cells counterstained in the nuclei by blue DAPI-generated fluorescence are demonstrated in (B). A substantial proportion of the green-labeled nuclear HMGB1 has been translocated to the cytoplasm of each cell after a co-culture period of 24 h with endotoxin, which activates the macrophages (C). The nuclei of the corresponding cells are demonstrated by blue DAPI staining (D).

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HMGB1 mRNA after LPS and TNF stimulation in vitro in macrophages was first analyzed by Wang et al. 17 who could not detect any up-regulation of HMGB1 mRNA levels at any of the investigated timepoints (0, 8, 12 and 16 h after stimulation). In contrast, Sass et al. reported that HMGB1 mRNA levels were increased in liver tissue already 1 h after LPS or Con A injections 46. Thermal injury, which is known to increase mRNA levels of proinflammatory cytokines in various tissues, also induced up-regulated HMGB1 mRNA levels 24 h after the burn 47.

5.3 Passive release of HMGB1

Passive release of HMGB1 from necrotic cells was demonstrated by Scaffidi et al. in 2002 10. It was reported that cells rendered necrotic by repeated freeze-thawing releasedtheir nuclear HMGB1 into the supernatant. This was in contrast to apoptotic cells, which retained their HMGB1 bound to DNA. The authors could further demonstrate that the addition of apoptotic wild-type fibroblasts to macrophage cultures did not induce any TNF production whereas the addition of necrotic wild-type fibroblasts induced a strong TNF production. In contrast, necrotic hmgb1gene deficient fibroblasts only induced a very low production of TNF in macrophage cultures, thus indicating that HMGB1 is a major factor in necrosis-induced inflammation and fulfills the criteriaof an endogenous danger signal.

The mechanism through which HMGB1 was retained in the nuclei of apoptotic cells was demonstrated to involve the underacetylation of histones occurring during the apoptotic process. The affinity of HMGB1 for DNA is dependent on the acetylation degree of the histones, with a lower degree of acetylation causing a stronger binding of HMGB1 to DNA. If apoptotic cells were prevented from modifying their chromatin by treatment with the deacetylase inhibitor trichstatin A, they no longer retained HMGB1 and were then as inflammatory as necrotic cells 10.

The fact that there are two distinct mechanisms of release of this endogenous danger signal implies the importance of HMGB1 as an endogenous mediator of inflammation and demonstrates that HMGB1 can act both as an early initiator (passive release from necrotic cells) and a late promotor (active, late release from macrophages as well as passive release) of inflammation. It is also important to remember that HMGB1 that is passively released from necrotic cells and HMGB1 that is actively secreted by macrophages are molecularly different, since the actively secreted molecule is acetylated 34. The functional consequences of this are presently unknown.

6 Signaling receptors for HMGB1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

Membrane HMGB1 signals the outgrowth of neurites by binding to RAGE 38, 39. RAGE belongs to the immunoglobulin superfamily of transmembrane proteins and is expressed on a wide variety of cells including endothelial cells, vascular smooth muscle cells, neurons and monocytes/macrophages (reviewed in 48). Originally, RAGE was defined asthe receptor that bound advanced glycated end-products (AGE), which among other things results in a nuclear translocation of NF-κB. Ligand-binding studies revealed that HMGB1 binds RAGE with a 7-fold higher affinity than do AGE 38 and when applied to a rat glioma cell line, results in activation of p21ras, MEK and MAPK kinases as well as NF-κB nuclear translocation 49. Similar intracellular signaling pathways were stimulated in rat smooth muscle cells stimulated with HMGB1 50. The same pathways are also activated in primary rat macrophages and dendritic cells after HMGB1 exposure (R. Kokkola et al., RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages, submitted manuscript).

It is also possible that, in analogy to the LPS-binding protein, HMGB1 also functions as a carrier for other substances. With its high degree of charged amino acids, it is likely to be "sticky" and might bind other substances such as DNA. It is reported that HMGB1 significantly increases the uptake of exogenous DNA by transporting the DNA through the cell membrane of mammalian cells 51.

Blocking studies performed with RAGE-blocking antibodies never completely prevent cellular activation. It is thus likely that there are alternative HMGB1-binding receptors than RAGE (52 and R. Kokkola et al., submitted manuscript, as above). This is substantiated by the recent finding that HMGB1-induced differentiation of erythroleukemia cells is a RAGE-independent event 42 and that extracellular HMGB1 induces mesangioblast migration and proliferation even when the RAGE receptor has been disabled 53.

In line with this thinking, it has very recently been reported that both TLR2 and TLR4 are involved in HMGB1-induced NF-κB activation in macrophages and in neutrophils. Transfection of macrophages with dominant negative constructs for TLR2 and TLR4 indicated that RAGE signaling only plays a minor role in NFκB activation in this cell type 54. In contrast, stimulation of RAGE-gene-deficient macrophages with recombinant HMGB1 resulted in a strongly suppressed TNF and IL-6 production (R. Kokkola et al., submitted manuscript as above), indicating a discrepancy between the observations. The relative functional significance of different HMGB1 receptors will be a central issue to resolve in future studies. It is a principally very important observation that TLR, meant for microbial danger signals, may interact with an endogenous mammalian molecule. Taking all the available data together, it thus appears that although RAGE is a potent receptor for HMGB1, other means of HMGB1 stimulation of cells exist, and that these mechanisms may differ between different cell types or between the same cell type from different strains of animals. Thus, despite its highly conserved structure, the regulatory control of the actions mediated by HMGB1 may vary.

7 Extracellular, proinflammatory functions of HMGB1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

7.1 HMGB1 as a proinflammatory mediator

The discovery of HMGB1 as a cytokine was made during studies of endotoxemia and sepsis 17. Numerous attempts have been made to treat sepsis by targeting well-known proinflammatory cytokines, including TNF. Although these approaches have led to the development of successful therapies for rheumatoid arthritis (RA) and Crohn's disease, they have not proven effective regarding sepsis. A major obstacle in targeting TNF or other proinflammatory cytokines in the treatment of sepsis is that most of these cytokines are released early during the septic response, usually within hours after the exposure to the sepsis-inducing stimulus. This fast kinetic pattern does not allow sufficient time for therapeutic intervention in clinical settings, despite the successfulresults in experimental systems.

The fact that death in sepsis may occur long after the systemic levels of proinflammatory cytokines have returned to normal levels prompted K. J. Tracey and coworkers to systematically search for as-yet-undiscovered mediators of sepsis lethality 17. They thus started to define proteins released from activated macrophages after stimulating cells with TNF, IL-1 or LPS and subsequently analyzed secreted proteins, using the techniques of denaturing protein gel electrophoresis and sequencing. HMGB1 could be demonstrated in the cell supernatant at time points later than 8 h after LPS-stimulation, with peak concentrations occurring at 18 h. Similarly, in mice injected with LPS or TNF, HMGB1 could be detected in serum samples after 8–32 h. Recombinant HMGB1 produced in Escherichia coli was lethal to both LPS-sensitive and LPS-resistant mice when injected intraperitoneally, indicating that the effect was not caused by LPS contamination. The injection of toxic HMGB1 doses leads to fever, weight loss, piloerection, shivering and microthrombi formation in liver and lungs. It also causes an increase in serum TNF levels in vivo. Thus extracellular HMGB1 appears to have a strong cytokine-like effect, inducing a proinflammatory response in macrophages and monocytes.

It was later demonstrated that HMGB1 is not only released in response to proinflammatory stimuli, but itself induces the production of inflammatory mediators by macrophages and neutrophils 18, 55. HMGB1 stimulation of macrophages induced de novo synthesis of TNF, as mRNA levels were also increased. Addition of HMGB1 to monocyte cultures activated the formation of TNF, IL-1α, IL-1β, IL-1ra, IL-6, IL-8, MIP-1α and MIP-1β but not IL-10 or IL-12. Interestingly, HMGB1-induced TNF production was significantly delayed compared with LPS-induced TNF production. HMGB1-induced TNF release was biphasic, with the first peak at 3 h and a second peak after 8–10 h. LPS-induced TNF production peaked after 2-h stimulation and was virtually undetectable by 8 18.

HMGB1-stimulation of neutrophils has been reported to induce an activated phenotype as determined by gene expression profiling, including increased production of TNF, IL-1β and IL-8 55. Endothelial HUVEC cells stimulated with full-length protein or the B-box of HMGB1 dose-dependently upregulated adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and released IL-8 and G-CSF 52. Human microvascular endothelial cells also responded with up-regulation of adhesion molecules and production of proinflammatory cytokines upon HMGB1-stimulation56.

HMGB1 is thus able to induce production of multiple proinflammatory mediators by a variety of cells, and importantly it is possible that it might induce its own release from monocytes and macrophages. This implicates HMGB1 as a central player in proinflammatory reactions.

7.2 HMGB1 has chemoattractant properties

HMGB1 stimulates migration of vascular smooth muscle cells and induces cytoskeleton reorganization 50. Furthermore, HMGB1 induces migration and proliferation of vessel-associated stem cells (mesoangioblasts), indicating an important role in muscle tissue regeneration 53. HMGB1 recruits neutrophils, monocytes and macrophages to sites of inflammation as demonstrated in models of acute lung injury and synovitis 57, 58. It needs to be clarified whether these activities depend on direct or indirect mechanisms induced by HMGB1.

7.3 Bactericidal activity of HMGB1

Recent studies of antibacterial factors purified by tissue extraction from human adenoid glands without signs of inflammation revealed that HMGB1 may mediate potent, direct antibacterial effects. Reversed-phase HPLC purification of adenoid-derived HMGB1 or rHMGB1 demonstrated that HMGB1 functionally belongs to the growing family of antibiotic peptides. The mechanism for this activity is presently unknown, but must be different from the cytokine-inducing activity, as HPLC fractionation of HMGB1 destroys the cytokine-inducing effects whereas the antibacterial effects remain intact 59.

It is thus clear that a single molecule, HMGB1, is capable of a plethora of activities associated with inflammation, not only including recruitment and activation of immune competent cells, but also acting as an immediate toxic first-line defense against microbial invaders. That such a small molecule can have such an array of properties implicates an intriguing structure.

8 The cytokine-inducing domain of HMGB1

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

HMGB1 protein is composed of three different domains: the DNA-binding A- and B-boxes, and the C-terminal tail 20, 60, 61. The structural basis for the cytokine-inducing capacity of HMGB1 was defined by stimulating macrophage cell-line cultures with full-length and truncated forms of recombinant HMGB1 as well as with synthetic peptides.B-box-containing variants of HMGB1 induced production of TNF, IL-1 and IL-6. The B-box consists of 74 amino acids and the cytokine-inducing part was subsequently mapped to amino acids 1–20 (corresponding to HMGB1 aa 89–108) 26. Interestingly, it has been demonstrated that purified recombinant A-box works as an antagonist to B-box-induced cytokine-production and as such the A-box has been successfully applied in experimental therapeutic settings (as discussed below). It is presently unknown whether isolated A-box or B-box peptides are produced in vivo during inflammatory processes.

9 HMGB1 in disease

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

A growing number of scientific reports describe the presence of extracellular/cytoplasmic HMGB1 during various inflammatory conditions. With respect to acute inflammation, HMGB1 has been demonstrated to be of pathogenic relevance in sepsis, pneumonia and endotoxemia. Serum levels of HMGB1 significantly increased 16–32 h following systemic LPS challenge of mice and, as systemic injection of rHMGB1 to mice is lethal, this further supports the pathogenic role of HMGB1 in endotoxemia 17. Induction of experimental sepsis in mice by caecal ligation and puncture (CLP) also resulted in increased serum levels of HMGB1 62. A study performed on sera collected from septic patients demonstrated increased levels of HMGB1 compared with sera from healthycontrols, with the highest increase in HMGB1 levels in patients who succumbed to disease 17. Intra-tracheal administration of HMGB1 to mice induced acute lung inflammation with accumulation of neutrophils, edema and production of proinflammatory cytokines 57. An increase in serum HMGB1 levels has also been reported in patients with hemorrhagic shock 63.

Furthermore, in a model of thermal injury (burn), HMGB1 mRNA levels were reported to be increased in liver and lung, similarly to other proinflammatory cytokine mRNA 47. Additionally, in two experimental models of hepatitis (Con A- and GalN/LPS-induced hepatitis), liver HMGB1 mRNA levels increased after exposure 46.

Administration of HMGB1 in close proximity to the sciatic nerve induced lowered pain thresholds (allodynia), as did administration of TNF and administration of killed bacteria. HMGB1 has recently been reported to have proinflammatory activity within the central nervous system, including acting as an endogenous pyrogen 64.

HMGB1 has also been reported as having a possible pathogenic role in chronic inflammatory conditions. The presence of cytoplasmic and extracellular HMGB1 has been reported in experimental arthritis models as well as in human RA. HMGB1 is strictly localized to the nucleus of each cell in normal synovial tissue (as analyzed by immunohistochemistry), whereas in arthritic synovial tissue a cytoplasmic localization of HMGB1 as well as extracellular depositions are abundant. HMGB1 could be detected in a majority of investigated synovial fluid samples from RA patients 65, 66. In muscle biopsies from chronic myositis patients HMGB1 could be detected cytoplasmically in muscle fibers, in inflammatory infiltrates and in small vessel endothelial cells.Biopsies taken before and after systemic corticosteroid treatment revealed a diminished extranuclear HMBG1 expression 67.

It therefore appears that the potential central role of HMGB1 in inflammatory conditions, as indicated from studies of in vitro cellular systems, does indeed seem to be substantiated in an in vivo setting in both mouse and man. This is a significant conclusion, as it thus implicates HMGB1 as a potential target molecule for therapeutic intervention.

10 HMGB1-targeted therapies during inflammatory conditions

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

With the discovery of HMGB1 as a potent mediator of inflammation and the presence of extranuclear HMGB1 in several inflammatory conditions, investigations of possible beneficial effects of HMGB1-targeted therapies were initiated. The appearance of HMGB1 much later than TNF and IL-1 in the inflammatory cascade suggested that HMGB1-targeted therapies might be initiated even at late stages of acute inflammatory conditions.

In the first reported study, polyclonal rabbit antibodies directed against HMGB1 were evaluated in LPS-induced endotoxemia 17. As hypothesized, the antibodies prevented endotoxemia-induced lethality, even when a delayed treatment regimen was followed. Treatment with HMGB1-specific polyclonal antibodies could also, in another study, greatly improve disease outcome of endotoxin-induced lung inflammation 57. It is of great clinical interest that in experimental sepsis induced by CLP, HMGB1-targeted therapy had a much broader therapeutic window than other cytokine-targeted therapies previously investigated. Polyclonal anti-HMGB1 antibodies could be administered up to 24 h following CLP with maintained efficacy 62, whereas previous attempts with similar interventions with anti-TNF or anti-MIF (macrophage migration inhibitory factor) antibodies had a much shorter therapeutic window 68, 69. As sepsis patients are only admitted to hospital when sepsis is fully developed, these novel experimental results are encouraging for future human clinical trials.

The promising results with polyclonal anti-HMGB1 antibodies also initiated development of other HMGB1 antagonists. To date two different HMGB1 antagonists — HMGB1 A-box protein and ethyl pyruvate — have been successfully tested in experimental systems. Monoclonal anti-HMGB1 antibodies with neutralizing capacity have proven difficult to produce but are now available and are currently being tested in experimental models of sepsis and arthritis.

The antagonizing role of A-box protein regarding HMGB1-induced cytokine production was discovered while defining which part of the HMGB1 molecule had the cytokine-inducing capacity 26. Various truncated forms of rHMGB1 were tested in in vitro cultures and protein mutants containing the isolated A-box were demonstrated to be able to replace iodine-labeled full-length HMGB1 in binding studies. Neither did the A-box have any intrinsic cytokine-inducing capacity, despite its 40% homology with the B-box 62. Thus, purified A-box protein fulfilled the requirements of an HMGB1 antagonist.

A-box treatment has been evaluated in both the CLP model and in collagen-induced arthritis. Daily therapeutic A-box treatment lead to equally beneficial effects in both models as did treatmentwith polyclonal anti-HMGB1 antibodies 62, 70. Thus, purified A-box protein has a clear potential for further clinical testing. A problem, though, with all treatment strategies based on small proteins or peptides is the short half-life of such small molecules in the circulation, necessitating frequently repeated administration. Modification of the A-box protein rendering it a prolonged accessibility would therefore be desirable.

Ethyl pyruvate, a stable lipophilic pyruvate derivative, has been demonstrated in vitro to inhibit the release of TNF and HMGB1 from endotoxin-stimulated macrophage cell lines. Ethyl pyruvate addition inhibited NF-κB translocation and p38 MAPK signaling, but the exact mechanism by which it inhibits HMGB1 release is not understood. Treatment with ethyl pyruvate was tested in the CLP sepsis model and was demonstrated to decrease lethality, even when administrated as late as 24 h after initiation of peritonitis. Ethyl pyruvate treatment also reduced the systemic levels of HMGB1 in these animals 71, 72. Ethyl pyruvate might not be as HMGB1-specific as anti-HMGB1 antibodies or purified A-box protein, but is clearly of clinical interest as a therapeutic compound since it is classified by the American Food and Drug Administration as a "generally regarded as safe" substance. Indeed, ethyl pyruvate is commonly used as a food additive.

11 Concluding remarks

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 HMGB1 basics
  5. 3 Post-translational modifications
  6. 4 Extranuclear localization of HMGB1
  7. 5 Extracellular release of HMGB1
  8. 6 Signaling receptors for HMGB1
  9. 7 Extracellular, proinflammatory functions of HMGB1
  10. 8 The cytokine-inducing domain of HMGB1
  11. 9 HMGB1 in disease
  12. 10 HMGB1-targeted therapies during inflammatory conditions
  13. 11 Concluding remarks

HMGB1, a nuclear protein that for a long time was studied for its intranuclear functions only, has during the last few years been discovered to have multiple extracellular functions. Dependingon responding cell type, HMGB1 may be mitogenic, induce cell differentiation, act as a chemoattractant, enhance expression of vascular adhesion molecules, impair the barrier function of intestinal epithelial cells 73 or act as a proinflammatory molecule. In the innate immune system, the release of HMGB1 from necrotic cells has been adapted as a danger signal instructing other cells that unphysiological cell death has occurred. Macrophages seem to have acquired the capacity to utilize this danger signaling in an active fashion, avoiding its release being associated with macrophage cell death. Thus, HMGB1 is a central molecule in initiating and sustaining the inflammatory cascade.

The discovery of HMGB1 as a general proinflammatory mediator has opened up a new venue for anti-inflammatory intervention. In comparison with previously investigated TNF- and IL-1-blocking therapy in acute inflammatory conditions such as sepsis, HMGB1-targeted therapies have a wider temporal treatment window, which is a critical necessity. It is theoretically possible that HMGB1-blockingtherapy may provide additional clinical benefit to TNF- and IL-1-blocking therapy in chronic arthritis and inflammatory bowel disease, since HMGB1 activates a broader set of genes than TNF and IL-1do. One-third of patients with chronic arthritis do not respond to TNF-blocking treatment, arguing for a search for further treatment modalities.

The recent finding of TLR2 and TLR4 as signaling receptors for HMGB1 that are distinct from RAGE highlights the diversified function of this protein as well as its intricate extracellular regulation. Our understanding of HMGB1-induced cellular activation and its regulation is still superficial and many questions remain. For example, what are the stimuli that induce HMGB1 release? How is the HMGB1 response down-regulated? How come that surface expressed HMGB1 (amphoterin) does not induce inflammation but only cell growth and motility? Will HMGB1-targeted anti-inflammatory therapy have detrimental effects on neurite outgrowth, platelet functions and fundamental repair mechanisms? Research in the near future will hopefully provide the answers to these questions as well as evaluate the consequences of HMGB1-targeted therapy in clinical trials.

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