HMGB1 is an ancient protein that predates speciation with extraordinary conservation among species, and acts as a nuclear protein that promotes transcriptional activation and access. Prototypic of other damage-associated molecular pattern molecules, it acts late as a downstream mediator of systemic inflammation in sepsis (9
). Macrophages that are stimulated with endotoxin secrete HMGB1 after a significant delay compared with the release of other cytokines, such as TNF and IL-1, which are much more immediate (23
). Release of HMGB1 also is released readily from necrotic or damaged cells, and serves as a signal for inflammation (11
). Although blockade of HMGB1 in a model of massive hepatocyte necrosis from acetaminophen in vivo did not protect against liver damage, inflammation was reduced as seen by reduced myeloperoxidase activity in total liver extracts (11
). The purpose of this study was to test the notion that HMGB1 acts as an early mediator of inflammation and injury in hepatic I/R injury. The major and novel findings of this investigation are: (a) HMGB1 levels are increased by hypoxia in cultured hepatocytes and by I/R in the liver in vivo; (b) blockade of HMGB1 protects against warm hepatic I/R injury; (c) the protection is associated with a decrease in MAP kinase activation, augmented NF-κB activation, and a decrease in local hepatic proinflammatory cytokine expression; and (d) evidence for HMGB1–TLR4 interaction in the mechanism of HMGB1-mediated damage in hepatic I/R.
Whereas HMGB1 is a late mediator of systemic inflammation in sepsis, our findings implicate HMGB1 as an early mediator following acute, local organ injury. This role for HMGB1 may include a rapid increase in HMGB1 levels in the liver. We showed that hepatic HMGB1 expression increases as early as 1 h after reperfusion of ischemic liver. That hypoxia alone is adequate to increase HMGB1 levels is shown by our studies with cultured hepatocytes. It is unknown whether the increases in HMGB1 levels are essential to the proinflammatory effects. Our observation that even early, proximal signaling events (i.e., MAP kinase activation) are partially HMGB1-dependent suggests that increases in HMGB1 levels are not essential. It is not known from our studies whether HMGB1 is secreted or passively released from damaged cells after I/R injury. It could be that severe ischemic stress leads to a regulated release or surface expression of HMGB1 as a danger signaling event. As the injury progresses and cells begin to die, the release may then be passive; however, this is yet to be proven. It is notable that circulating levels of HMGB1 did not increase; this is consistent with a local or paracrine-like action for HMGB1 in this organ-specific acute injury model.
To elucidate the molecular basis of protection in blocking HMGB1, we investigated its effect on the MAP kinase and NF-κB signaling pathways. HMGB1 activates one or more of these signaling pathways in endothelial cells, smooth muscle cells, enterocytes, and neutrophils (24
). The MAP kinase family represents a group of proteins that is involved in signal transduction of a variety of cellular stimuli. The JNK subgroup of MAP kinases, also known as stress-activated protein kinases, is activated in response to environmental stresses (28
). JNK phosphorylation occurs in cultured rat hepatocytes following hypoxia alone, with further increases upon reoxygenation (13
). JNK activation also has been found in the liver after I/R (17
) and hemorrhagic shock (13
). The reduction in JNK activation, as well as p38 and ERK, suggests that HMGB1 accounts, in part, for MAP kinase activation following I/R. Such is not the case for NF-κB, where neutralization increased NF-κB DNA binding activity. Thus, the actions of HMGB1 are selective in liver I/R. It is possible that the enhanced NF-κB activation contributed to the protection that was afforded by HMGB1 neutralization. Although NF-κB activation leads to proinflammatory gene expression in leukocytes and endothelial cells, it is known to protect hepatocytes from cell death (21
). In vivo, inhibition of NF-κB after partial hepatectomy results in massive hepatocyte apoptosis associated with impaired liver function and decreased survival (31
). Following I/R that is associated with liver transplantation, two peaks of NF-kB activation were observed, and blockade of NF-kB activation worsened the liver injury (22
). Further work is required to elucidate the mechanisms that are involved in the differential effects of HMGB1 on MAP kinase and NF-κB. Enhanced NF-κB activation is seen in mice that are protected from hepatic I/R following blockade of the receptor for advanced glycation end product (30
To study further the mechanism of HMGB1-mediated injury in hepatic I/R, we explored the TLR4 system as a possible signal transduction pathway for extracellular HMGB1. HMGB1, in vitro, interacts with TLR2, TLR4, and the receptor for advanced glycation end products for HMGB1-induced activation of inflammatory signaling (12
). The TLRs are one of the components by which the innate immune system senses the invasion of pathogenic microorganisms by recognizing specific molecular patterns that are present in microbial products (34
). TLR4 plays a critical role in endotoxin signaling and was shown to participate in the recognition of several endogenous ligands, such as hyaluronic acid, heparin sulfate, fibrinogen, and perhaps, heat shock proteins (35
). The release of these ligands stimulates inflammatory activity through TLR4 signaling. Our data suggest that the TLR4 system also may play a key role in HMGB1-mediated hepatic I/R injury; TLR4-defective mice who undergo hepatic I/R are not affected by the administration of rHMGB1 or neutralizing antibodies to HMGB1, compared with TLR4-intact mice. We hypothesize that the release of HMGB1 from damaged or necrotic cells may produce an early inflammatory response by activating the TLR4 system. Further, the protection from hepatic I/R in TLR4-defective mice may be due to their diminished ability to respond to HMGB1.
In summary, this study documents the contribution of HMGB1 to hepatic I/R injury. In this model of acute local tissue injury, HMGB1 seems to act as an early mediator of inflammation. This is in clear contrast to sepsis, in which its action is delayed. Our results also suggest that the protective effects of blocking HMGB1 and the detrimental effects of rHMGB1 in hepatic I/R are dependent on the activation of TLR4 signaling. Thus, interventions that inhibit HMGB1 activity may be effective in settings of ischemic liver injury to minimize organ damage and may be useful in other clinical settings that are associated with inflammation and cellular necrosis (39