CXC chemokines and their receptor, CXC chemokine receptor-2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild-type and CXCR2 ؊/؊ mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2 ؊/؊ mice had significantly less liver injury at all reperfusion times compared with wild-type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2 ؊/؊ mice, but within 24 hours it was the same as that of wild-type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2 ؊/؊ mice compared with wild-type mice. These effects were associated with increased activation of nuclear factor B and signal transducers and activators of transcription-3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor ␣, interleukin-6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2 ؊/؊ mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2 ؊/؊ mice. Treatment of primary wild-type hepatocytes with macrophage inflammatory protein-2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2 ؊/؊ mice. Conclusion: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration. (HEPATOLOGY 2008;48:1213-1223

Hepatic ischemia-reperfusion (I/R) injury is an important complication of liver surgery and transplantation. Mitochondrial function is central to this injury. To examine alterations in mitochondrial function during I/R, we assessed the mitochondrial proteome in C57Bl/6 mice. Proteomic analysis of liver mitochondria revealed 234 proteins with significantly altered expression after I/R. From these, 13 proteins with the greatest expression differences were identified. One of these proteins, peroxiredoxin-6 (Prdx6), has never before been described in mitochondria. In hepatocytes from sham-operated mice, Prdx6 expression was found exclusively in the cytoplasm. After ischemia or I/R, Prdx6 expression disappeared from the cytoplasm and appeared in the mitochondria, suggesting mitochondrial trafficking. To explore the functional role of Prdx6 in hepatic I/R injury, wild-type and Prdx6-knockout mice were subjected to I/R injury. Prdx6-knockout mice had significantly more hepatocellular injury compared with wild-type mice. Interestingly, the increased injury in Prdx6-knockout mice occurred despite reduced inflammation and was associated with increased mitochondrial generation of H2O2 and dysfunction. The mitochondrial dysfunction appeared to be related to complex I of the electron transport chain. These data suggest that hepatocyte Prdx6 traffics to the mitochondria during I/R to limit mitochondrial dysfunction as a protective mechanism against hepatocellular injury.

The function of peroxisome proliferator-activated receptor-␥ (PPAR␥) in hepatic inflammation and injury is unclear. In this study, we sought to determine the role of PPAR␥ in hepatic ischemia/reperfusion injury in mice. Male mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 8 hours of reperfusion. PPAR␥ was found to be constitutively activated in hepatocytes but not in nonparenchymal cells. Upon induction of ischemia, hepatic PPAR␥ activation rapidly decreased and remained suppressed throughout the 8-hour reperfusion period. This reduced activation was not a result of decreased protein availability as hepatic nuclear PPAR␥, retinoid X receptor-␣ (RXR␣), and PPAR␥/RXR␣ heterodimer expression was maintained. Accompanying the decrease in PPAR␥ activation was a decrease in the expression of the natural ligand 15-deoxy-Delta 12,14 -prostaglandin J 2 . This was associated with reduced interaction of PPAR␥ and the coactivator, p300. To determine whether PPAR␥ activation is hepatoprotective during hepatic ischemia/reperfusion injury, mice were treated with the PPAR␥ agonists, rosiglitazone and connecting peptide. These treatments increased PPAR␥ activation and reduced liver injury compared to untreated mice. Furthermore, PPAR␥-deficient mice had more liver injury after ischemia/reperfusion than their wildtype counterparts. Conclusion: These data suggest that PPAR␥ is an important endogenous regulator of, and potential therapeutic target for, ischemic liver injury. I schemia and reperfusion of the liver is a primary complication of liver resection surgery, transplantation, and trauma. 1 This insult can lead to hepatocellular damage and organ dysfunction through the initiation of a biphasic inflammatory response. 2 The initial phase of this response is characterized by activation of Kupffer cells and their subsequent production and release of reactive oxygen species. This causes mild injury to the hepatic parenchyma, but the oxidative stress activates redox-sensitive transcription factors, such as nuclear factor B and activator protein-1, that regulate the expression of many proinflammatory mediators. [3][4][5] Proximal cytokines, such as interleukin-12 (IL-12), tumor necrosis factor-␣ (TNF␣), and IL-1 are critically involved in promoting the second phase of liver injury by inducing the hepatic expression of neutrophil-attracting CXC chemokines and endothelial cell adhesion molecules. 6-11 CD4 T lymphocytes are also recruited to the liver early during reperfusion, and these cells facilitate the subsequent recruitment of neutrophils by modulating chemokine expression. 12,13 The cooperative effects of CXC chemokines and adhesion molecules result in adherence of neutrophils in the hepatic microcirculation and their subsequent transmigration into the hepatic parenchyma. Accumulated neutrophils release oxidants and proteases that directly injure hepatocytes and vascular endothelial cells. 14

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.