Published online before print October 15, 2007
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Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
1Correspondence: Department of Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street, Presbyterian Hospital F1200, Pittsburgh, PA 15213, USA. E-mail: tsunga{at}upmc.edu
ABSTRACT
High mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. In contrast to the proinflammatory role of HMGB1, recent evidence suggests beneficial applications of HMGB1 in injury states. One such application is the use of HMGB1 as a preconditioning stimulus. Preconditioning is a phenomenon whereby a low level of stressful stimuli confers protection against subsequent injury. Preconditioning has been demonstrated in multiple species, can be induced by various stimuli, and is applicable in different organ systems. Only with the recent introduction of the concept of endogenous molecules, such as HMGB1, as signals and mediators for inflammation during injury states has the use of endogenous molecules been investigated for this use. This review will focus on the use of endogenous molecules, specifically HMGB1, as a preconditioning stimulus and its mechanism of protection, as well as other protective applications for HMGB1.
Key Words: ischemia/reperfusion • endogenous danger molecules • Toll-like receptor • endotoxin
INTRODUCTION
The preconditioning effect is observed when the delivery of a known hazard to the body results in the reduction of injury from a larger insult to follow. Preconditioning can be induced by various stimuli and is applicable in different organ systems. Although early work about preconditioning focused on using similar insults, such as a brief period of ischemia delivered prior to a much longer one, it has been shown that one form of insult can induce cross-tolerance against different forms of injury. This allows the possibility of using molecules known to be involved in the pathophysiologic process of the injury as potential preconditioning stimuli. The use of high mobility group box 1 (HMGB1), a nuclear protein involved in the pathophysiology of many disease states, as a preconditioning stimuli is a protective technique potentially useful in a wider variety of clinical scenarios. This review will describe preconditioning and its applications, focusing on endogenous danger molecules, such as HMGB1, as a protective strategy in various disease states.
PRECONDITIONING
Preconditioning is a phenomenon that has been observed for a number of years, in which delivery of a minor insult prepares the body to better withstand a more significant insult to follow. The phenomenon was first described in an infection model in 1946, in which multiple injections of a typhoid vaccine would reduce fevers [1 ]. Soon after, preconditioning was demonstrated in other infectious settings, such as endotoxemia, to prevent death from lethal injections of endotoxin [2 ]. In addition to infectious settings, this technique has been applied to other clinical models including ischemia/reperfusion (I/R) injury, which is a pathophysiologic process, whereby hypoxic organ damage is accentuated following return of blood flow and oxygen delivery. Transient episodes of ischemia are encountered during various surgical resections, solid organ transplantation, trauma, and hypovolemic shock. Ischemic preconditioning was described first in 1986 using a canine model of cardiac infarction, in which they found that subjection of hearts to several brief periods of ischemia prior to an infarct model reduced the injury by as much as 75% [3 ]. Since this work, additional techniques of preconditioning for protection against ischemic injury have been described. These can include using brief periods of heat shock or hyperthermia prior to the ischemic insult [4 , 5 ], using ischemic preconditioning in organs remote from the organ being prepared for ischemia [6 , 7 ], and exogenous applications of known, harmful molecules or pharmacologic analogs to endotoxin to induce a preconditioning protection against ischemia [8 9 10 ].
Preconditioning can be divided into early and late events. Ischemic preconditioning is one example of early preconditioning, where protection is conferred within minutes to hours of the preconditioning stimulus and appears to be mediated by immediate changes within the cell, such as phosphorylation of signaling molecules and changes in intracellular signaling systems [11 ]. Late preconditioning protection can be induced by hyperthermia, endotoxin administration, and other tissue-specific stimuli and is protection that occurs >24 h after the preconditioning stimulus, likely mediated by changes in cell signaling, which results in alterations in cellular protein expression with up-regulation of expression of the cell’s stress molecules [11 ]. In a study using endotoxin preconditioning at 72 h and ischemic preconditioning immediately prior to an ischemic insult, animals subjected to both methods of preconditioning had additional protection beyond either method individually, thus further demonstrating different protective mechanisms [11 ].
The finding that use of endotoxin as a preconditioning stimulus can protect against future ischemic insults in cardiac [8 , 10 ] and hepatic [12 ] I/R models provides evidence that one form of insult can cause cross-tolerance against different injuries. This allows the possibility of using molecules known to be involved in the pathophysiologic process of the injury as potential preconditioning stimuli. Activation of the innate immune system by pathogen-associated molecular pattern (PAMP) molecules, such as endotoxin, has long been recognized as a necessary step in mounting an antimicrobial response to pathogens. These PAMPs, which are restricted to patterns associated with infection, are one class of a larger group of signals that all act to activate the immune system. Another group of signals emanate from stressed or damaged tissues and are known as endogenous danger molecules or more recently, referred to as alarmins [13 ]. PAMPs, associated with infection, and alarmins, associated with tissue damage, are subgroups within the larger category of damage-associated molecular patterns. The alarmins, or endogenous danger molecules, can be classified as normal cell constituents released by damaged or dying cells or components of the extracellular matrix, released by the action of proteases at the site of tissue damage. They have been shown to play a role in a variety of sterile inflammatory insults such as trauma or ischemia and can activate similarly the innate immune system inflammatory pathways as seen in infectious settings. At the molecular level, these observations may be explained, at least in part, by the capacity of PAMP molecules and endogenous danger molecules to activate cell signaling through common pattern recognition receptors of the innate immune system. As such, these endogenous danger molecules have been successful as stimuli for preconditioning, as will be discussed below.
HMGB1 PRECONDITIONING
HMGB1 is a nuclear DNA-binding protein with multiple functions. Evolutionarily ancient, HMGB1 predates speciation and is highly conserved across species [14 ]. Initially identified in 1973, early studies focused on its role as a DNA-binding, nuclear protein that copurified with chromosomal DNA, as HMGB1 is loosely bound to chromatin (unlike the more tightly bound histones) [15 , 16 ]. HMGB1 is present in almost all eukaryotic cells, functions to stabilize nucleosomes, and acts as a transcription factor that regulates the expression of several genes [17 , 18 ]. During the course of experiments to identify late-acting mediators of endotoxemia and sepsis, HMGB1 was discovered to be secreted by activated macrophages [19 ]. HMGB1 release occurred significantly later than macrophage secretion of the classical, early proinflammatory mediators TNF and IL-1 [19 ] and has also been shown to be an independent mediator of cytokine release, stimulating release of TNF, IL-1, IL-6, and IL-8 in human monocytes [20 ]. In a murine model of sepsis (induced by cecal ligation and puncture), serum HMGB1 levels begin to increase 12–18 h after the peak of TNF, which occurs at 2 h, and of IL-1 (4–6 h) [21 ]. Neutralizing HMGB1 markedly improved survival in these septic mice. HMGB1 is also readily released from necrotic or damaged cells and serves as a signal for inflammation [22 , 23 ]. Thus, HMGB1, in addition to its nuclear role, is a critical mediator of the response to infection, injury, and inflammation.
Recently, the use of HMGB1 as a preconditioning stimulus has been explored. Exogenous HMGB1, given as a pharmacologic pretreatment, has been shown to protect against hepatic I/R injury [24 ]. Mice were pretreated with a single HMGB1 injection ranging from 2 to 20 µg, administered systemically 1 h prior to 60 min of ischemia and 6 h of reperfusion of the liver. Injury was measured by circulating alanine transaminase (ALT) and cytokine levels. Protection from HMGB1 preconditioning was demonstrated by decreased circulating ALT levels, which were decreased in a dose-dependent manner. In addition, levels of circulating TNF and IL-6, inflammatory cytokines shown to play key roles in the pathophysiology of hepatic I/R injury, were also decreased. The sources of HMGB1 used were bacterial-derived recombinant HMGB1 and HMGB1 purified from HeLa cells, both tested by Limulus assay and demonstrated to have undetectable amounts of endotoxin. In addition, heat inactivation of the recombinant HMGB1 reversed the protective effects further, demonstrating that this was an endotoxin-independent phenomenon.
MECHANISMS OF HMGB1 PRECONDITIONING
The mechanisms leading to protection in preconditioning are complex, and many different theories have been proposed. As discussed earlier, preconditioning can be delineated between early and late protection, and these occur by different mechanisms. Early protection (within minutes to hours of the preconditioning stimulus) may be mediated by immediate phosphorylation of signaling molecules and changes in intracellular signaling systems such as protein kinase C [25 26 27 ] and mitochondrial ATP-sensitive potassium channels [28 , 29 ]. Delayed protection from preconditioning (>24 h after preconditioning stimulus) appears to involve the induction of synthesis of various proteins including manganese-containing superoxide dismutase (SOD) [30 , 31 ], catalase [31 ], inducible NO synthase [32 , 33 ], and heme oxygenase 1 (HO-1) [34 ], each of which has known to function to reduce injury in cell stress and related states. Other work has highlighted the relationships among these molecules [35 ] and has investigated each of these molecules for its independent importance for decreasing I/R injury [36 , 37 ].
Another physiologic mechanism that lies central in the pathophysiology of endotoxemia and I/R injury and may be involved in preconditioning is the involvement of TLRs. The TLR family is a family of receptors that includes cell surface and intracellular members, well known for their involvement in induction of NF-
B and inflammation in response to a variety of stimuli. TLR4, specifically, has been shown to respond to extracellular pathogens such as endotoxin [38
] as well as endogenous danger molecules such as some of the heat shock proteins (HSP) [39
] to elicit its strong NF-B and proinflammatory response. Studies have shown that TLR4 is involved in ischemic injury [40
, 41
], and further, this response includes the production of some of the enzymes discussed previously as being involved in preconditioning mechanisms, such as HO-1 [42
]. With its known involvement in ischemic injury and its relationship to molecules known to be involved in preconditioning, the involvement of TLR4 in ischemic preconditioning has been investigated, but the mechanism in which TLR4 contributes to preconditioning protection remains unclear with multiple hypotheses advanced [43
].
There are several known, negative regulators of TLR4 that function to inhibit the inflammatory response [44
]. Some are constitutively expressed, and others are inducible factors. One such molecule that has been known to be an important negative regulator of TLR4 signaling in endotoxin tolerance is IL-1R-associated kinase (IRAK)-M [45
, 46
]. The IRAK family consists of four distinct molecules that have been known to be involved in downstream signaling from the TLR family as well as IL-1R and are intermediates in the activation of NF-B and MAPK pathways [45
]. There are four distinct molecules, and although IRAK-1, IRAK-2, and IRAK-4 have all been demonstrated to have different proinflammatory roles in downstream signaling from TLR/IL-1R stimulation, IRAK-M has been demonstrated to be a negative regulator of the signaling pathways [45
].
Regarding HMGB1, much of the recent work about its signaling functions has focused on its proinflammatory properties and relationship to known inflammatory receptors such as TLR4. It has been demonstrated that HMGB1 can interact with multiple receptors known to contribute to inflammation, including TLR2, TLR4, and receptor for advanced glycation end products (RAGE) [47
]. When HMGB1 is cultured with neutrophils or macrophages, it appears that it stimulates an inflammatory response similar to that of endotoxin by activation of IB kinase (IKK)-
and IKK-β, leading to NF-B activation [48
]. It is interesting that although HMGB1 is known to interact with the RAGE receptor, it appeared that this phenomenon of NF-B activation was largely dependent on signaling through TLR2 and TLR4, with only minimal contribution from RAGE [48
]. Although endotoxin and HMGB1 are known to stimulate inflammation through TLR4 signaling mechanisms, a recent study using gene arrays on neutrophils isolated from adult patients with sepsis-induced lung injury has demonstrated that culturing these cells with HMGB1 or endotoxin results in gene expression patterns that have some overlap but are significantly distinct [49
]. It is important that HMGB1 has been demonstrated to be a significant component of hepatic I/R injury through a TLR4-dependent mechanism [50
]. It was a result of this involvement of HMGB1 through TLR4 signaling mechanisms as a contributor to hepatic I/R injury that use of exogenous HMGB1 as a pharmacologic pretreatment for I/R injury was proposed.
The study using HMGB1 as a preconditioning agent also investigated the role of TLR4 in preconditioning. When applied to TLR4 wild-type and mutant mice, the pretreatment with HMGB1 did not confer any additional protection in the TLR4 mutants beyond the baseline decrease demonstrated previously in injury in the mutant mice, a finding that was also paralleled by levels of TNF and IL-6. Therefore, the mechanism by which HMGB1 preconditioning results in protection from ischemic insults is a TLR4-dependent phenomenon, confirming the previous findings that extracellular HMGB1 signals through TLR4 signaling pathways.
The dependence on TLR4 signaling for HMGB1 preconditioning suggested that inhibition of downstream signaling from TLR4 was involved. As known signaling molecules and inhibitory molecules that occur early in TLR4 signaling, the IRAK family, specifically IRAK-M, was hypothesized to be involved in the mechanism of protection. Western blotting confirmed significantly increased levels of IRAK-M in the HMGB1-pretreated mice, whereas these mice demonstrated decreased levels of activated phospho-IRAK1 simultaneously [24 ]. These findings suggest that the HMGB1 pretreatments activate TLR4 signaling and result in increased levels of IRAK-M in the cells, thereby inhibiting the TLR4-dependent I/R injury demonstrated previously during the later ischemic insult.
POTENTIAL USE OF ENDOGENOUS DANGER MOLECULES FOR PRECONDITIONING
Recent work has also explored the involvement of other endogenous danger molecules in I/R injury and preconditioning, including the induction of the HSP as a class of molecules involved in the preconditioning response. First proposed in 1992 in a cardiac hyperthermia-preconditioning model [51 ], the role of HSP in other preconditioning models has also been explored [31 , 52 53 54 ]. HSP, along with HMGB1, are in the class of endogenous danger molecules or alarmins. HSP are one of the most extensively studied endogenous danger molecules and can serve as constitutive and inducible danger signals. Various forms of constitutively active or stress-induced HSP are released from different cellular compartments during necrotic cell death and activate the immune response [55 ]. Although the heat shock model for preconditioning appears to be applicable to multiple models [4 , 5 ], the mechanism and the role of HSP are still debated [5 , 56 ].
The function of these intracellular molecules up-regulated in preconditioning, such as HSP, has been pursued, and their use toward decreasing I/R injury has been studied. It has been found that the overexpression by adenoviral gene transfer of certain HSP genes can attenuate I/R injury [57 ], and other models demonstrate a correlation between ischemic or thermal preconditioning with decreased I/R injury and induction of HSP expression [58 ]. In addition, it has been demonstrated that the degree of myocardial protection in a heat shock-preconditioning model for myocardial ischemia was correlated directly with the levels of induction of HSP expression [59 ].
Recently, the use of exogenous administration of HSP proteins as another endogenous danger molecule has been used as a method of preconditioning against future ischemic or endotoxemic insults. Exogenous administration of HSP-70 18 h before endotoxin stimulation resulted in an increased tolerance to endotoxin challenge, as measured by NF-B activation [60
]. In addition, HSP-72 has been demonstrated to have protective effects in models of oxidative stress as well as LPS or TNF stimulation in murine macrophages [61
, 62
]. In addition to HSP, other endogenous molecules have been used, including -lipoic acid (an essential cofactor of mitochondrial dehydrogenase enzymes) [63
, 64
], IL-1 (an endogenous cytokine that induces SOD activity) [65
], peroxynitrite (a resultant molecule of the interaction of NO with superoxide) [66
], and most recently, the study using HMGB1 as described above [24
].
It is interesting that the protective function of HSP-72 in oxidative stress and LPS or TNF stimulation seems to be through inhibitory interactions with the release and the function of HMGB1. It was demonstrated that HSP-72, whether induced by heat shock or gene transfer, localized to the nucleus upon increased oxidative stress and further, that this inhibited the translocation and release of HMGB1 from macrophages [61
]. Modification of the nuclear localization sequence or the protein-binding sequence of HSP eliminated this effect. The ability of HSP-72 to inhibit the release of HMGB1 from macrophages was confirmed in LPS and TNF stimulation models [62
]. In addition, overexpression of HSP-72 in macrophages was demonstrated to inhibit the HMGB1-induced cytokine production and release through inhibition of the MAPK and NF-B pathways [62
]. These studies demonstrate the complexity inherent in endogenous danger molecule signaling pathways and elucidate further the possibilities that may exist for using these endogenous signals to minimize, rather than exacerbate, organ injury in states of stress.
PROTECTIVE EFFECTS OF HMGB1
The findings that HMGB1, known best for its proinflammatory properties, may be used as a preconditioning stimulus suggest that HMGB1 may have potential beneficial effects. Recent work in cardiac and vascular models has demonstrated the positive effects of HMGB1 on tissue remodeling and repair. In addition, there has been some evidence that as a nuclear-binding protein, HMGB1 is involved in the recognition and repair of human DNA mismatches, promoting mismatch excision by direct, physical interaction with MutS (an enzyme involved in DNA repair) [67
].
HMGB1 was first shown to function as a chemoattractant for rat vascular smooth muscle cells by inducing cytoskeleton reorganization and cell migration in 2001 [68 ]. Work in 2004 highlighted the use of HMGB1 in promoting migration and proliferation of regenerative cells to the sites of inflammation and injury [69 ]. Cell proliferation with HMGB1 stimulation was noted to increase in a dose-dependent manner, and in vitro and in vivo experiments appeared to show that migration of mesoangioblasts functioned in a dose-dependent manner through interactions with the RAGE receptor. More recent evidence has suggested that the HMGB1 activation of homing of endothelial progenitor cells to ischemic tissues to increase neovascularization involves an integrin-dependent mechanism [70 ]. This study also confirmed findings that migration of these cells was RAGE-dependent but in contrast to earlier studies, found that HMGB1 did not have an effect on cell proliferation on this line of cells.
In a model of cardiac infarction, exogenous HMGB1 has been shown to be beneficial in decreasing promoting left-ventricular function and myocyte regeneration [71
]. In this study, purified HMGB1 was injected directly into the peri-infarcted myocardium 4 h after an infarction event, and structural and functional endpoints were assessed in the days and weeks following. Structurally, HMGB1-treated hearts were seen to have increased tissue repair, evidenced by regeneration of -sarcomeric-expressing cells in the treated hearts. Cardiac stem cells, c-kit+ cells, were found to be affected by the HMGB1 treatments, and increased migration and proliferation were at the site of damage. Functional studies about the left-ventricular function performed 1, 2, and 4 weeks postinfarction demonstrated that there was consistently improved function in the hearts that were injected with exogenous HMGB1 following infarction as compared with control animals.
CONCLUSIONS
Significant insight into the physiology of preconditioning has been obtained over the years. This has led to the novel concept about the use of endogenous danger molecules as preconditioning agents. However, this problem remains complex, and clearly much remains to be elucidated in the complex interactions of these molecules in states of stress. Evidence has emerged that use of endogenous danger molecules, such as HMGB1, as preconditioning tools can reduce injury significantly in various disease states. Clearly, what was once highlighted for its proinflammatory properties and role in propagation of injury has now been shown also to be effective in promoting tissue repair and regeneration following insult to the body. There remains potential for the biology of HMGB1 to serve as a clinical tool to improve clinical outcomes in patients suffering from different disease states.
Received June 15, 2007; revised August 5, 2007; accepted August 22, 2007.
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