Ischemic tissues require mechanisms to notify the immune system of the impending cell damage and possible breech of tissue integrity. Cells of the innate immune system are activated after ischemic injury to initiate tissue repair processes and to provide defense against microbial invasion. Excessive activation can lead to exaggerated local and systemic inflammation that may extend the tissue damage. In this study, we sought to determine how one of the key alarm molecules released during liver I/R, HMGB1, was mobilized in response to hypoxia. We show that hypoxic hepatocytes release HMGB1 through an active process facilitated by TLR4-dependent ROS production. ROS, in turn, induce HMGB1 release through a CaMK-dependent mechanism. That these events may be important in vivo was shown by experiments in a mouse liver I/R model in which both CaMK inhibitors and antioxidants reduce HMGB1 release and liver damage to the level seen in mice deficient in TLR4 signaling.
HMGB1 is becoming increasingly recognized as the prototypic alarmin (28
). Initially identified as a chromatin-binding protein in the nucleus, its cytokine-like properties were revealed in experiments showing that HMGB1 is released by activated macrophages and that it acts as a late mediator of lethality in sepsis models (16
). Ample evidence now exists to indicate the HMGB1 also acts as an early inflammatory mediator in ischemia (2
), hemorrhagic shock (10
), noninfectious hepatitis (19
), and peripheral tissue trauma (unpublished data). Both passive (19
) and active (30
) pathways for HMGB1 release have been described. The passive pathway requires the loss of cell membrane integrity, as seen in necrosis. The active pathway, best described in LPS- or TNF-stimulated macrophage cell lines (30
) and cytokine-treated enterocyte cell lines (32
), appears to involve HMGB1 hyperacetylation and packaging into secretory vesicles, at least for enterocytes. Recent evidence suggests that phosphorylation on serine residues within the two nuclear localization sequences prevents return of the mobilized HMGB1 to the nucleus (31
). We postulated that the initial HMGB1 release in ischemia would be an active process triggered by redox signaling. Indeed, antioxidants prevented HMGB1 release in hypoxic hepatocytes in vitro and systemic release of HMGB1 in liver I/R. 250–500 μM H2
also induced hepatocyte HMGB1 release without causing measurable cell death. Interestingly, exogenous H2
was also recently shown to induce HMGB1 release from a macrophage cell line in the absence of cell death (33
). Not previously shown was the requirement for intact TLR4 signaling for hypoxia-induced ROS production and HMGB1 release, as well as the involvement of CaMK signaling in HMGB1 release.
TLR4 has been shown to be involved in I/R-induced inflammation in several organs in addition to the liver (8
). The only molecule known to stimulate TLR4 signaling shown to be involved in I/R-induced injury is HMGB1 (2
). We previously reported that hepatocytes express functional TLR4 (34
). Furthermore, others have shown that oxidant stress up-regulates TLR4 expression (35
). However, the requirement for intact TLR4 signaling for hypoxia-induced ROS production is novel. Whether a TLR4-activating ligand is required for this response is unclear. A neutralizing anti-HMGB1 antibody had no effect on the production of ROS by hypoxic hepatocytes. Therefore, at least the initial signaling is not dependent on the release of HMGB1 into the extracellular space. In HEK294 T cells, TLR4 has been linked to the activation of the NADPH oxidase (36
). However, the source of the hypoxia-induced ROS production is unknown. Some HMGB1 release was observed in the absence of TLR4 signaling. This may be caused by production of ROS through other pathways or by the involvement of other signaling events. The suppression of even this lower level of HMGB1 release by CaMK inhibitors suggests that TLR4 serves primarily to amplify ROS production. This reduction but not inhibition of HMGB1 in the absence of hepatocyte TLR4 signaling is consistent with our observations in TLR4 chimeric mice where the absence of TLR4 on only parenchymal cells caused only a minor reduction in I/R-induced inflammatory signaling compared with the reduction seen in an absence of TLR4 signaling on nonparenchymal cells (20
). The possibility that even very low levels (<1%) of contaminating nonparenchymal cells in our hepatocyte preparation could contribute to the ROS generation and HMGB1 release remains a possibility. However, hepatocyte purity exceeded 99%, and we have previously shown that hepatocytes express a functional TLR4 (34
The capacity of redox stress to mobilize calcium in hepatocytes was recognized more than two decades ago (37
); however, the involvement of CaMKs in I/R injury has not been previously shown. CaMKs are a family of proteins composed of CaMK I–IV, myosin light chain kinase, and phosphorylase kinase (38
). Activation typically requires Ca–calmodulin binding and can be augmented or sustained by phosphorylation. H2
treatment has been shown to activate CaMKs II and IV in Jurkat T cells (40
), and redox activation of CaMKs may occur through both calcium-dependent and -independent pathways (41
). Our observation that calcium chelation abrogates hypoxia-induced HMGB1 release suggests that calcium-dependent CaMK activity is required. The specific CaMK family members involved in hypoxia-induced HMGB1 release is uncertain, and we show that hepatocytes express CaMKs I, II, and IV. Based on experiments in other cells, it is possible that more than one isoform could be involved.
The downstream targets for CaMK in triggering HMGB1 release are not known. The greater accumulation of HMGB1 in the cytosolic compartment in the presence of CaMK inhibitors during I/R suggests that HMGB1 secretion and not nuclear mobilization is the process regulated by CaMK activity. Whether CaMKs directly modify HMGB1 is uncertain.
Models of organ I/R have revealed unique insights into how tissues respond to ischemic stress to activate the resident cells of the innate immune system. That the pattern recognition receptors involved in the recognition of pathogens are also used to activate innate immune pathways in response to ischemia or tissue damage is now clear. From this and previous studies, it is important to appreciate that TLR4-dependent signaling is among the most proximal events in I/R-induced inflammation and may occur in response to ischemia alone to trigger downstream signaling events. CaMKs now enter the equation as a potential therapeutic target in acute ischemic events, along with TLR4 and HMGB1. A recent study also points to the importance of resident NKT cells in the early inflammatory response after I/R (42
). Whether NKT cell involvement is dependent on TLR4 signaling is not yet known; however, the central role of nonparenchymal cells and, more specifically, immature dendritic cells (43
) in the TLR4-dependent responses seems more certain. A scheme whereby I/R induces inflammation through the activation of TLR4 signaling by an endogenously released cell constituent supports tenants of the “Danger Theory” initially proposed by Matzinger (44