High-mobility group box (HMGB)-1 (also referred to as amphoterin) was originally identified as a nuclear DNA-binding protein that functions as a structural cofactor critical for proper transcriptional regulation and gene expression (Fig. 1). Recent studies indicate that immune cells can liberate HMGB-1 into the extracellular milieu where it functions as a proinflammatory cytokine (1-4). Indeed, HMGB-1 is a member of a novel family of inflammatory cytokines composed of intracellular proteins that, when present in the extracellular milieu, are recognized by the innate immune system as “necrotic markers” to signal tissue damage (3-4). HMGB-1 is liberated to the extracellular milieu through two alternative mechanisms: it can be “passively released” from damaged or necrotic cells in injured tissues (5-7) or “actively secreted” by immune cells including macrophages and neutrophils (8-12). Although the biological consequences of the mechanism of liberation remain unknown, extracellular HMGB-1 can trigger an inflammatory response and contribute to the pathologic progression of infectious and inflammatory disorders (2-4).
HMGB-1 induces a concentration-dependent activity that varies from beneficial to pathologic. Similar to other proinflammatory cytokines, moderate amounts of HMGB-1 induce a beneficial immune response to confine infection or tissue damage and promote wound healing and tissue regeneration (12-16). However, excessive levels of HMGB-1 result in an uncontrolled inflammatory response that can be more dangerous than the original insult, producing tissue injury and organ failure (2-5). HMGB-1 is a signal of chemotaxis and activation for inflammation-mediating immune cells such as neutrophils, monocytes, and macrophages (17-19). During infection or trauma, extracellular HMGB-1 activates endothelial cells to express cellular adhesion molecules and tissue-type plasminogen activator (20, 21). These molecules promote the adhesion of immune cells to the endothelial vessel wall and permit their extravasation into tissues. Although this process is critical for the proper resolution of infection or injury, overwhelming levels of HMGB-1 result in the disruption of endothelial barrier functions, leading to vascular leakage and tissue hypoperfusion similar to that observed in sepsis (20, 21). Likewise, HMGB-1 can also alter the permeability of cultured enterocytes, impair the intestinal barrier function in mice, and increase bacterial translocation and systemic dissemination of the infection (22). Hence, uncontrolled extracellular HMGB-1 causes multiple organ failure and can be more dangerous to the host than the original pathologic scenario.
Unlike other proinflammatory cytokines, HMGB-1 is a “late” appearing inflammatory mediator, and therefore, it provides a wider time frame for clinical intervention against progressive inflammatory disorders. Other cytokines such as tumor necrosis factor (TNF) are produced within minutes after stimulation, and their circulating levels revert to near-baseline levels within the first few hours during the progression of the disease (2, 23, 24). Thus, specific inhibition of these cytokines provides a narrow time frame for clinical intervention. In many progressive scenarios similar to sepsis, systemic levels of these cytokines are resolved even before the disorder is diagnosed. In contrast to this “early” response, HMGB-1 is secreted from macrophages approximately 20 h poststimulation (10-12). In endotoxemia and other experimental models of systemic inflammation, elevated serum HMGB-1 levels are detected in a “late” plateau beginning only 20-72 h after the onset of the disease (8, 10, 19). In fact, several HMGB-1-directed therapies have proven effective to “rescue” experimental animals from established sepsis, even when the treatment is started after the onset of the disease (10, 12). These studies suggest that strategies targeting HMGB-1 can expand the time frame for therapeutic intervention in clinical settings. Despite its clinical relevance, little is known about the pathologic mechanisms induced by HMGB-1 in different tissues and clinical scenarios. This article analyzes the pathophysiologic potential of HMGB-1 and its interest for clinical trials against infectious and inflammatory disorders.
THE PATHOPHYSIOLOGIC EFFECTS OF HMGB-1
Even though the pathogenesis of tissue injury and organ failure during the development and progression of systemic inflammation is a complex process that is unlikely attributable to a single agent, several studies indicate that HMGB-1 is a sufficient and necessary mediator of the lethal multiple organ failure associated with severe sepsis (2, 15, 24): (a) patients with severe sepsis, as well as experimental animals of sepsis-induced organ dysfunction, show elevated systemic HMGB-1 levels (8); (b) administration of recombinant HMGB-1 to mice is sufficient to recapitulate the symptoms of severe sepsis including vascular leakage, derangement of the intestinal barrier function, acute lung injury, and lethal multiple organ failure (8, 22, 25); and (c) inhibition of HMGB-1 prevents both endotoxin- and bacteremia-induced multiple organ failure, “rescues” experimental animals from established sepsis, and improves survival in polymicrobial severe sepsis (8, 10, 11). These results indicate that HMGB-1 contributes to the pathogenesis of severe sepsis, and there is a great deal of interest in the characterization of its pathologic effects on specific organs. This section reviews the pathologic effects of HMGB-1 in specific organs in several clinical scenarios (Fig. 2).
Hemorrhagic shock, ischemia/reperfusion, endotoxemia, and sepsis often result in hepatic injury as a critical factor contributing to lethality. Initial studies on this topic focused on the analysis of HMGB-1 mRNA levels. Hepatic injury caused by either concanavalin A (a carbohydrate-binding lectin molecule that induces hepatocyte death in an interferon-γ-dependent manner ), or endotoxin, induced an early but unsustained up-regulation of HMGB-1 mRNA levels in hepatic tissue as determined by real time-PCR (27). In contrast to this early septic response, hepatic HMGB-1 mRNA levels were elevated only after 24 h postinduction in experimental severe burn (28). In this scenario, the increased hepatic HMGB-1 mRNA levels correlated tightly with the indicators of hepatic injury (serum alanine aminotransferase and aspartate aminotransferase) and with markers for hepatic neutrophil infiltration (myeloperoxidase activity) (28).
Specific neutralizing antibodies have proven HMGB-1 as a therapeutic target against hepatic tissue damage (7, 29). In a mouse experimental model of hepatic injury-induced by ischemia/reperfusion, hepatic HMGB-1 protein expression increased as early as 1 h after reperfusion of ischemic liver, suggesting that it may act as an early initiator of inflammation and organ damage (29). In contrast to septic models, serum HMGB-1 protein accumulation was also observed at a relatively early phase (3 h post-ischemia) of the hepatic ischemia/reperfusion injury (7). The levels of serum HMGB-1 also showed a “late” further increase 12 h post-ischemia/reperfusion injury (7). This late phase might be more significant for causing pathophysiologic effects because delaying the administration of neutralizing antibody against HMGB-1 still confers protection against hepatic injury (29). The pathologic contribution of HMGB-1 to hepatic ischemia was established in lethal experimental models of hepatic ischemia/reperfusion injury (7). Administration of neutralizing anti-HMGB-1 antibodies improved the survival time of animals suffering from hepatic ischemia/reperfusion injury (29). Recombinant HMGB-1 administered to mice immediately after reperfusion significantly increased serum hepatic enzyme levels and exacerbated ischemia/reperfusion-induced hepatic injury (29). HMGB-1 blockade using neutralizing polyclonal antibodies protected mice against hepatic ischemia/reperfusion injury, even when administered after the ischemic insult (7, 29). However, anti-HMGB-1 antibody failed to provide protection in toll-like receptor (TLR)-4-defective mice (29), implicating this receptor in the pathologic potential of HMGB-1. These results are in agreement with previous studies indicating that TLR-2 and TLR-4 were both involved in HMGB-1-induced activation of NF-κB in macrophages. In contrast to the TLR receptors, receptor for advanced glycation end products (RAGE) appears to play a minor role in macrophage activation by HMGB-1 (18).
Hypoxic exposure of primary rat hepatocytes, which mimics in vivo ischemic injury, increased the steady-state levels of HMGB-1 in the growth medium of these cells (29). These results suggest that hepatocytes themselves could be responsible for the elevated HMGB-1 levels in ischemic liver and support earlier predictions that hypoxic hepatocytes can be a major source for HMGB-1 released in injured hepatic tissue produced by toxic levels of acetaminophen (5). The role for necrosis in inducing HMGB-1 extracellular release remains controversial because there was no appreciable loss in cell viability detected in these hepatocytes (29). Future studies are needed to determine the mechanism of HMGB-1 release from hypoxic hepatocytes, as these cells do not possess secretory lysosomes, which are thought to be the process that immune cells use to actively secrete HMGB-1.
The intestinal epithelium functions as a selective gastrointestinal barrier to permit the absorption of nutrients while restricting the diffusion of microbes. A common consequence of sepsis is the impairment of intestinal barrier function, leading to bacterial translocation into the bloodstream and systemic dissemination of the infection. Administration of HMGB-1 causes gastrointestinal barrier dysfunction with increased mucosal permeability and augmented bacterial translocation in both naive and endotoxin-instilled mice (22). The contribution of HMGB-1 to gastrointestinal dysfunction was confirmed in vivo by using anti-HMGB-1 antibodies in experimental models of sepsis (13). In addition to antibodies, ethyl pyruvate, an inhibitor of HMGB-1 secretion from macrophages (11, 12), also provides protection against endotoxin-induced gastrointestinal barrier dysfunction in a mouse model of endotoxemia (30). In addition to sepsis, recent studies also suggest that HMGB1 contributes to tumor growth and it is closely associated with invasion and metastasis of colarectal cancer (62, 63). These results suggest that HMGB-1 contributes to gastrointestinal derangements in different clinical scenarios.
The pathologic effect of HMGB-1 on intestinal epithelial cells was studied in cultured Caco-2 human enterocytic monolayers using either full-length recombinant HMGB-1 or the truncated “B-box” inflammatory domain (corresponding to amino acid residues 89-108 of full-length HMGB-1). HMGB-1 impairs gastrointestinal barrier function by increasing the permeability of enterocytic monolayers. Both HMGB-1 and the truncated “B-box” increased the permeability of Caco-2 monolayers in a time- and dose-dependent manner, and the effect was reversible following the removal of the recombinant HMGB-1 protein (22). HMGB-1-induced hyperpermeability of enterocyte monolayers was not caused by cell death, as shown by trypan blue staining at 24 or 48 h after treatment. In vivo, “B-box” administration increased both ileal mucosal permeability and bacterial translocation to mesenteric lymph nodes (22). “B-box” induces intestinal dysfunction through a mechanism dependent on the NF-κB pathway and inducible nitric oxide synthase (iNOS). Accordingly, mice defective in inducible nitric oxide synthase are protected from “B-box”-induced gastrointestinal barrier dysfunction. Pharmacological strategies inhibiting either the NF-κB pathway (pyrrolidine diothiocarbamate or ethyl pyruvate) or nitric oxide production (L-NIL or C-PTIO) abrogated the “B-box”-induced hyper-permeability in these cells (22, 30). These results indicate that HMGB-1 induces gastrointestinal derangements and can contribute to the systemic dissemination of an infection.
Acute lung injury
Acute lung injury and its more severe form, the acute respiratory distress syndrome, represent one of the major causes of mortality and morbidity in intensive care units and affect more than 150,000 people annually in the United States (25). Despite intense research efforts, no effective treatment of acute lung injury is currently available. A major advance in the understanding of the pathogenesis of acute lung injury was the establishment of a link between tissue injury and inflammation, indicating that proinflammatory cytokines are potential therapeutic targets against lung injury. Several studies indicate that HMGB-1 may represent this link between a pathologic inflammatory response and lung injury. From a clinical perspective, HMGB-1 appears at elevated levels in both plasma and in lung epithelial lining fluids in patients with acute lung injury (31). The direct contribution of HMGB-1 to acute lung injury was first demonstrated by the intratracheal administration of recombinant HMGB-1 and later confirmed in diverse experimental models of lung inflammation (25, 31-33). HMGB-1 induces a dose-dependent interstitial and intra-alveolar neutrophil accumulation and lung edema at 8 and 24 h post-administration of recombinant purified protein (25). HMGB-1-induced lung injury was correlated with increased concentrations of proinflammatory cytokines including MIP-2, TNF, and IL-1β (25, 33). These studies suggest that HMGB-1 causes acute lung injury by inducing neutrophil infiltration and activation. Acute lung injury is consistently associated with a marked increase in the numbers of neutrophils in the lung, and they can constitute up to 80% of the total cells obtained by bronchoalveolar lavage as compared with the 2-3% found in normal subjects (25, 34). The neutrophil influx into the lung is driven, at least in part, by α-chemokines (33), which function as potent chemoattractants and cellular activators (9). A specific hexapeptide that blocks both type 1 and type 2 α-chemokine receptors inhibits neutrophil infiltration and can reduce HMGB-1-induced lung injury (9, 33). These studies suggest that HMGB-1 causes acute lung injury in part through α-chemokine-induced neutrophil infiltration. In agreement with this hypothesis, treatment of endotoxin-instilled mice with antibodies against HMGB-1 significantly decreased neutrophil accumulation in the lungs, and attenuated the severity of lung edema and lung permeability (31-33).
Several lines of investigation indicate that HMGB-1 contributes to the development of acute lung injury in different pathologic scenarios including endotoxemia and sepsis. Instillation of endotoxin into mice causes migration of inflammatory-mediating cells into the lung, destruction of pulmonary parenchyma cells, pulmonary hemorrhage, and acute lung injury (31). Increased levels of HMGB-1 were observed in both plasma and bronchoalveolar lavage fluid in endotoxin-instilled mice (30). HMGB-1 was redistributed from the nucleus into the cytoplasm in most alveolar macrophages of the experimental mice (31). Because cytoplasmic translocation of HMGB-1 from the nucleus is an early step leading to its secretion, these results suggest that alveolar macrophages are a major source of extracellular HMGB-1 in this experimental model of endotoxin-induced acute lung injury. Similar findings have been reported in experimental models of severe sepsis using cecal ligation and puncture (33), suggesting that HMGB-1 contributes to acute lung injury not only during endotoxemia but also in experimental models of polymicrobial peritonitis.
Elevated serum levels of HMGB-1 and acute lung injury are not exclusively related to infection, as they both appear in patients suffering from other inflammatory disorders. Increased serum concentrations of HMGB-1 were found in a patient suffering from life-threatening intra-abdominal hemorrhagic shock. The elevated HMGB-1 levels decreased as the clinical condition improved (35). Increased systemic HMGB-1 levels also contribute to acute lung injury in experimental models of hemorrhagic shock (32). HMGB-1 expression in the lung increased within 4 h after hemorrhage and remained elevated for more than 72 h after the blood loss. Hemorrhage-induced acute lung injury was correlated with increased pulmonary levels of proinflammatory cytokines and neutrophil accumulation. The development of pulmonary edema was effectively diminished by administration of neutrolizing anti-HMGB-1 antibodies, even when given after the hemorrhagic insult (32). These studies established a critical role for HMGB-1 in the development of acute lung injury that is not caused by infectious agents, and support HMGB-1 as a therapeutic target against acute lung injury in diverse clinical scenarios (Table 1).
Taken together, these studies mount convincing evidence of the critical role of HMGB-1 in causing injurious effects on multiple organs including lung, liver, and intestine (2, 3). The pathologic effects of uncontrolled levels of extracellular HMGB-1 might be a unifying mechanism for infection- or injury-induced deregulation of proinflammatory responses leading to a failure in parenchyma cell function, which often culminates in the lethal dysfunction of multiple organs.
THERAPEUTIC STRATEGIES AGAINST HMGB-1
HMGB-1 is a necessary mediator of severe sepsis because independent strategies that inhibit either HMGB-1 cytokine activity (neutralizing antibodies or specific HMGB-1 antagonists) or secretion (ethyl pyruvate or agonists for α7-nicotinic acetylcholine receptors [α7-nAChR]) can prevent multiple organ failure and rescue mice in experimental models of severe sepsis (8, 10-13, 36). In contrast to previous published reviews, this article emphasizes the clinical relevance of HMGB-1 including the therapeutic strategies inhibiting its pathologic potential. These strategies are of special interest because they are currently in clinical trials and support HMGB-1 as a therapeutic target for infectious and inflammatory disorders. These studies support the preclinical relevance of HMGB-1 as a therapeutic target for the treatment of infectious and inflammatory disorders related to severe sepsis, and each strategy is discussed independently (Fig. 3).
Neutralizing anti-HMGB-1 antibodies inhibit HMGB-1-induced TNF and IL-6 production but do not affect the ability of TNF to induce IL-6 release in macrophages (8). These results indicate that anti-HMGB-1 antibodies specifically inhibit extracellular HMGB-1 activity rather than blocking the ability of cells to respond to an immune stimulus. Treatment with neutralizing anti-HMGB-1 antibodies also protects mice against polymicrobial peritonitis-induced sepsis in a concentration-dependent manner (8). The survival advantage conveyed by anti-HMGB-1 antibodies is in contrast to previous studies using specific antibodies against other cytokines. For instance, anti-TNF antibodies are ineffective to rescue animals from experimental models of peritonitis-induced sepsis (39, 40). However, antibodies are difficult to administer and produce antigen-antibody complexes that can affect the inflammatory response by further activating innate immune pathways promoting further tissue damage (41-44). Accordingly, previous antibody-based blockade strategies have shown limited efficacy in clinical trials against sepsis (41-43). Therefore, more recent efforts have focused on alternative strategies that do not involve antibodies to neutralize the cytokine activity of HMGB-1.
Structure-function analyses conducted indicate that partial peptides of HMGB-1 may define specific functional domains of clinical interest. The C-terminal DNA-binding “B-box” (amino acids 88-162) represents an inflammatory motif sufficient to stimulate TNF production in macrophages (19). Further mapping of the cytokine-inducing activity of the “B-box” indicates that the first 20 amino acids of this motif constitute the minimal proinflammatory cytokine-eliciting domain of HMGB-1. In contrast to the C-terminal “B-box,” the N-terminal “A-box” (amino acids 1-85) has little or no stimulatory effect on macrophages (19). Instead of activating macrophages, the “A-box” acts as a competitive antagonist to the full-length HMGB-1 protein. This effect is also observed in vivo, as dose-dependent administration of purified “A-box” protects experimental animals from lethal endotoxemia (19). An interesting consideration is that the “A-box” may represent a physiological anti-HMGB-1 activity that protects against further tissue damage at sites of inflammation. “A-box” may be liberated locally at sites of inflammation by proteolytic cleavage of the full-length HMGB-1 protein and protect against further tissue damage from HMGB-1 toxicity. It is noteworthy that HMGB-1 has been found to activate tissue-type plasminogen activator, which stimulates plasmin generation, and that HMGB-1 itself is a plasmin-sensitive protein (45). The region between the “A-box” and the “B-box” of HMGB-1 contains preferred plasmin digestion sites that would potentially liberate the “A-box” peptide (46). However, it remains unknown whether the “A-box” is expressed as a discrete peptide in vivo, either as a separate translation product or as a cleavage product of HMGB-1 (19). Although the extracellular processing of HMGB-1 can generate peptides that induce erythroleukemia cell differentiation, it remains controversial whether distinctive HMGB-1-derived proteolytic peptides actually have physiologic implications (47). The observation that purified A-box can neutralize the toxicity of full-length HMGB-1 in experimental animals supports HMGB-1 as a therapeutic target for the treatment of severe sepsis and other inflammatory disorders like rheumatoid arthritis (56-60). Nonetheless, future studies are required to define its therapeutic potential and the specificity and mechanism of action of “A-box.” Further efforts have focused not on neutralizing extracellular HMGB-1 but rather on developing pharmacologic strategies to inhibit HMGB-1 secretion.
Pharmacological strategies: ethyl pyruvate
Ethyl pyruvate, a stable aliphatic ester derived from pyruvic acid, was the first described pharmacologic inhibitor of HMGB-1 secretion and provided an effective therapeutic alternative to methods targeting blockade of extracellular HMGB-1. Ethyl pyruvate treatment inhibited HMGB-1 secretion from endotoxin-challenged cultured macrophages, and this in vitro effect correlated with a block in HMGB-1 serum accumulation in ethyl pyruvate-treated mice that were challenged with a lethal dose of endotoxin (11, 48). This single dose of ethyl pyruvate administered 30 min before endotoxin infusion protected the mice from lethal endotoxemia (survival rates: control 3/20; ethyl pyruvate 20/20 [P < 0.05]) (11). The survival advantage conveyed by ethyl pyruvate was significant because further experiments showed that it did not have to be administered prophylactically, as it rescued mice from lethal endotoxemia (survival rate: control 20%; ethyl pyruvate 80%; P < 0.05) or polymicrobial sepsis (survival rate: control 30%; ethyl pyruvate 88%, P < 0.05) even when the treatment began 1 day after the onset of disease, a time at which 10% of the mice had already died (20). Similar benefits of ethyl pyruvate treatment have been reported in various other models of acute critical illness related to HMGB-1, including hemorrhagic shock, ischemia/reperfusion injury, and acute lung injury (reviewed in ). Recently, NF-κB inhibition and the prevention of inflammatory mediator release (for example NO, TNF, and HMGB-1) emerged as a key mechanism underlying ethyl pyruvate antiinflammatory action (42-44, 49). The precise link between NF-κB activity and the secretion of inflammatory mediators such as HMGB-1, whose production and secretion are not regulated at the transcriptional level (9, 39), remains to be elucidated. Regardless of the precise mechanism, ethyl pyruvate has proven safe in phase I clinical trials and is currently in phase II trials for the prevention of systemic inflammation during cardiopulmonary bypass surgery (50). These studies collectively indicate that small molecules such as ethyl pyruvate that inhibit HMGB-1 secretion may have therapeutic value for treating “established” sepsis and other progressive inflammatory disorders in a clinically relevant time frame.
Selective α7-nicotinic acetylcholine receptor agonists
Recent studies indicate that the nervous system regulates the innate immune response and controls the production of inflammatory cytokines from macrophages. Activation of the vagus nerve can inhibit the release of HMGB-1 from tissue macrophages (51). This physiologic antiinflammatory mechanism can be exploited to control HMGB-1 secretion and represents a pharmacologic target for the treatment of inflammatory disorders (1, 10, 52). Acetylcholine, the principal neurotransmitter of the vagus nerve, was the first physiological inhibitor described for HMGB-1 secretion from human macrophages (1, 10). This anti-inflammatory mechanism, the “nicotinic anti-inflammatory pathway,” is dependent on the α7-nicotinic acetylcholine receptor, and can be modulated therapeutically by exogenous nicotinic receptor agonists such as nicotine (50). Treatment of cultured macrophages with nicotine, a more selective α7-nAChR agonist, dose-dependently prevents the activation of the NF-κB pathway and inhibits HMGB-1 secretion (1, 10). Likewise, treatment of mice with nicotine attenuates serum HMGB-1 levels in response to both experimental endotoxemia and polymicrobial sepsis and improves sepsis survival (survival rate after 5 days: control 50%; nicotine 85%, P < 0.05) even when the treatment is delayed 24 h after the onset of disease (10). However, the general application of nicotine as an antiinflammatory therapy is limited because of its nonspecific and potentially toxic effects when administered at clinically relevant doses. Efforts are currently under way to develop more selective agonists for the α7-nAChR that might provide a less toxic pharmacologic approach to inhibit HMGB-1 secretion.
Stearoyl (18:0) lysophosphatidylcholine
Stearoyl (18:0) lysophosphatidylcholine is a potential therapeutic agent to control systemic inflammation that inhibits HMGB-1 secretion from activated macrophages, and protects mice from lethal sepsis (36, 53). This inhibitory property is specific to stearoyl lysophosphatidylcholine, as the caproyl (6:0) and lauroyl (12:0) lysophosphatidylcholine derivatives do not inhibit HMGB-1 secretion (36). In fact, caproyl lysophosphatidylcholine stimulates HMGB-1 secretion from macrophages in a dose-dependent manner through a mechanism that is synergistic with endotoxin stimulation (36). Although the mechanism by which lysophosphatidylcholine inhibits HMGB-1 secretion remains elusive, lysophosphatidylcholine binds to the G protein-coupled G2A receptor and causes increases in intracellular calcium ions (3, 37). Treatment of activated macrophages with lysophosphatidylcholine abrogates the cytoplasmic accumulation and subsequent extracellular secretion of HMGB-1 (10). It is noteworthy that treatment with nicotine also evokes increases in intracellular calcium levels and inhibits HMGB-1 secretion in a manner that correlates with inhibition of NF-κB activity (10).
Different pharmacological strategies that prevent HMGB-1 secretion (ethyl pyruvate, nicotine, and lysophosphatidylcholine) converge in the inhibition of the NF-κB pathway, and the studies discussed above strongly suggest a link between these two processes. The connection between NF-κB activity and HMGB-1 secretion represents a challenging conundrum, because NF-κB is a transcription factor, and HMGB-1 secretion is not regulated at the level of transcription. Another important consideration is that NF-κB contributes directly to pathologic inflammatory responses, but it also protects parenchyma cells from cytotoxic reagents and hepatocytes from cell death (38, 54). In vivo, inhibition of NF-κB after partial hepatectomy results in massive hepatocyte apoptosis associated with impaired liver function and decreased survival (55). Moreover, treatment with anti-HMGB-1 antibodies to prevent hepatic injury in response to ischemic insult was associated with enhanced activation of the NF-κB pathway (29). However, anti-HMGB-1 antibodies abrogate the activation of NF-κB in HMGB-1-challenged enterocytes (22). This double-edged sword issue makes it challenging to predict the clinical outcome of nonspecific inhibition of NF-κB in human inflammatory diseases and injuries. Unless the therapy is specifically targeted to the control of HMGB-1 secretion from macrophages and monocytes, inhibition of NF-κB activity may not generate an overall beneficial effect, especially in tissue injuries such as acute lung injury or hepatic injury induced by ischemia/reperfusion (29, 61). Future studies will need to determine the molecular links between NF-κB activity and the secretion of HMGB-1 from activated macrophages.
HMGB-1 is a nuclear protein critical for survival and an extracellular lethal mediator of systemic inflammation (2, 3, 65). From an immunologic perspective, HMGB-1 represents a “necrotic marker” characterized by intracellular proteins that function as inflammatory cytokines when released into the extracellular milieu (65, 66). HMGB-1 is “passively released” from necrotic cells, and this mechanism represents an optimal process adopted by the innate immune system to recognize imminent tissue damage and initiates a reparative response (5-7, 29, 64). HMGB-1 can also be “actively secreted” by stimulated immune cells, suggesting that during evolution, the immune system has copied this signal to activate the innate response (8-12). Extracellular HMGB-1 is a prototype marker of severe sepsis and is associated with the characteristic organ failure of this disorder and other related clinical conditions (2). As an inflammatory cytokine, HMGB-1 has been identified as a late mediator of severe sepsis that can provide a wider therapeutic time window for clinical intervention. Accordingly, effective treatment with ethyl pyruvate or nicotine, which prevent HMGB-1 secretion and protect mice from lethal polymicrobial sepsis, can be delayed to a clinically relevant time frame (10-12). To date, no other pathogenic mediators of lethal sepsis have been successfully targeted this late after the onset of disease to promote survival. In fact, anti-TNF antibodies increase mortality in experimental polymicrobial sepsis (40), and antibodies against macrophage migration inhibitory factor (MIF) are ineffective if administered more than 8 h after the induction of disease (67). Although these results in experimental models of severe sepsis are encouraging, caution is warranted because, as opposed to the experimental models using young and previously healthy animals, the clinical sepsis scenario typically involves older patients with previous disorders. Therefore, specific studies will be needed to determine the pathologic effect of HMGB-1 on physiologic variables including cardiovascular homeostasis and its contribution to the cardiovascular collapse observed in sepsis. These studies will be critical to determine the translational potential of experimental strategies directed against HMGB-1 as therapeutic approaches for the treatment of infectious and inflammatory diseases.
L.U. is supported by grants from the Faculty Award Program of the North Shore Health System and the North Shore-LIJ GCRC.
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