The results of this study demonstrate for the first time that: (a) HMGB1, a known early mediator of sterile inflammation, is released in the plasma within 45 minutes after severe trauma in humans; (b) the release of HMGB1 in the plasma requires severe tissue injury and tissue hypoperfusion; and (c) HMGB1 is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.
Severe trauma is associated with an early SIRS seen within 30 to 60 minutes after injury followed by a CARS observed 24 to 48 hours after injury, although the molecular mechanisms responsible for this altered host defense are not well understood [2
]. Recent studies have provided new information on the molecular mechanisms that lead to the early inflammatory response. Complement and alarmins have been shown in experimental studies to play an important role as endogenous triggers of trauma-associated inflammation. Among the alarmins, HMGB1 appears to be one of the important mediators in triggering this posttraumatic sterile inflammation via receptors, such as TLR4 and RAGE [12
] (Figure ). However, whether HMGB1 is an early mediator of the early inflammatory response induced by severe trauma in humans is unknown. Only one previous study had described HMGB1 release in plasma in a small group of patients several hours after trauma [17
]. We present here for the first time evidence that HMGB1 is released within 30 minutes after trauma in patients with severe injury and tissue hypoperfusion. There was no significant fluid resuscitation or other potentially confounding treatment prior to blood sampling and therefore our findings represent the direct effects of the injury and shock on the release of HMGB1 into the bloodstream.
Figure 6 Schematic diagram: relation between the release of HMGB1, complement activation and induction of an inflammatory response in the vascular endothelium early after trauma. HMGB1 = high mobility group box nuclear protein 1; RAGE: receptor for the advanced (more ...)
Initial interest in HMGB1 as a biomarker of inflammation came from the work of Tracey and colleagues [17
] who showed that HMGB1 was released in response to lipopolysaccharide (LPS) in mice. Significantly HMGB1 was released at a later time point (peak at 16 hours) as compared with the nearly immediate release of TNF-α and IL-1β after exposure to LPS. These findings were extended by the same research group who showed that HMGB1 is a factor of lethality in mice rendered septic by the induction of a polymicrobial bacterial peritonitis. Further studies reported that HMGB1 could induce the release of proinflammatory cytokines and induce an increase in permeability across intestinal cell monolayers [14
]. The interest for this late release of HMGB1 after exposure to LPS was related to the fact that an anti-HMGB1 blocking antibody could rescue mice from lethality after cecal ligation and puncture as late as 24 hours after the beginning of sepsis [30
]. In humans, plasma levels of HMGB1 have been shown to be elevated in ICU patients with sepsis and patients after major surgery (esophagectomy) [32
]. Both Wang and colleagues and Sunden-Cullberg and colleagues reported a prolonged elevation of plasma levels of HMGB1 in septic patients [33
]. Interestingly in these studies, there was no correlation between elevation in HMGB1 levels and severity of infection. In a more recent study, Gibot and colleagues reported that plasma levels of HMGB1 measured at day three after onset of severe sepsis discriminated survivors from non-survivors [35
]. Taken together, these results indicate that HMGB1 is a late mediator of sepsis that has an important mechanistic role in that disease, because the inhibition of HMGB1 activity significantly ameliorates the survival in experimental animal models of septic shock.
In contrast to the data reported for sepsis, we found a significant difference in plasma levels of HMGB1 between survivors and non-survivors from severe trauma. This major difference in the plasma level profile of HMGB1 between septic and hemorrhagic shock may be explained by the fact that experimental studies have shown that HMGB1 is one of the alarmins, proteins that play a critical role in initiating the sterile inflammatory response after onset of ischemia-reperfusion injury [16
]. The results of these experimental studies are supported by the correlation we found between plasma levels of HMGB1 and several inflammatory mediators, such as IL-6 and TNF-α, as well as markers of endothelial cell activation, such as Ang-2 and vWF antigen. Taken together, previous studies and our results indicate different kinetics for the release of HMGB1 during the two major causes of shock: sepsis and hemorrhage. HMGB1 appears to be an early mediator of the sterile inflammation induced by trauma-hemorrhage; in contrast, the kinetics of HMGB1 release due to sepsis may differ depending on the primary source of infection [34
The second important result of our study is the relation between the plasma levels of HMGB1 and the activation of the protein C pathway that we have previously shown to be induced by tissue injury and hypoperfusion. This relation is particularly interesting in light of the recent discovery that HMGB1 binds in vitro
to the lectin domain of TM. Abeyama and colleagues reported that TM could bind HMGB1 and serves thus as a sink for active HMGB1 in the plasma [36
]. These results add to the concept that TM is an anti-inflammatory protein via its sequestration of thrombin, and its activation of protein C and Thrombin activated fibrinogen inhibitor (TAFI)[29
]. Whether TM after binding HMGB1 would still maintain its ability to activate protein C is unclear, although protein C activation is dependent on the Gla domain of TM while HMGB1 is bound to its lectin domain. Ito and colleagues recently reported that administration of HMGB1 caused fibrin deposition and prolonged clotting times in healthy rats [19
]. These investigators also showed that HMGB1-bound TM and thereby reduced the ability of thrombomodulin to activate protein C in vitro
. In contrast to the results of these experimental studies, our current data show a simultaneous release of HMGB1 in the plasma and an activation of the protein C pathway by tissue injury and hypoperfusion suggesting that the release of HMGB1 in the plasma is not sufficient to inhibit the activation of the protein C pathway and the development of coagulopathy within 45 minutes after severe trauma-hemorrhage. However, these clinical results do not exclude that, in addition to the cytokine-like effect of HMGB1 via the TLR4 and RAGE receptors, extracellular HMGB1 could also attenuate the maladaptive activation of the protein C observed after severe trauma. Additional studies with a mouse model of trauma-hemorrhage that mimics the findings in trauma patients are needed to demonstrate this new function of extracellular HMGB1 after severe trauma and are currently being performed in our laboratory.