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Helper T cells are known to mediate hepatic ischemia/reperfusion (I/R) injury. However, the precise mechanisms and subsets of [CD4.sup.+] T cells that contribute to this injury are still controversial. Therefore, we sought to determine the contributions of different [CD4.sup.+] T cell subsets during hepatic I/R injury. Wild-type, OT-II, or T cell receptor (TCR)-[delta]-deficient mice were subjected to 90 min of partial hepatic ischemia followed by 8 h of reperfusion. Additionally, wild-type mice were pretreated with anti-CD1d, -NK1.1, or -IL-2R-[alpha] antibodies before I/R injury. OT-II mice had diminished liver injury compared with wild-type mice, implicating that antigen-dependent activation of [CD4.sup.+] T cells through TCRs is involved in hepatic I/R injury. TCR-[delta] knockout mice had decreased hepatic neutrophil accumulation, suggesting that [gamma][delta] T cells regulate neutrophil recruitment. We found that natural killer T (NKT) cells, but not NK cells, contribute to hepatic I/R injury via CD1d-dependent activation of their TCRs, as depletion of NKT cells by anti-CD1d antibody or depletion of both NKT cells and NK cells by anti-NK1.1 attenuated liver injury. Although regulatory T ceils (Treg) are known to suppress T cell-dependent inflammation, depletion of Treg cells had little effect on hepatic I/R injury. The data suggest that antigen-dependent activation of [CD4.sup.+] T cells contributes to hepatic I/R injury. Among the subsets of [CD4.sup.+] T cells, it appears that [gamma][delta] T cells contribute to neutrophil recruitment and that NKT cells directly injure the liver. In contrast, NK cells and Treg have little effects on hepatic I/R injury. T cell receptor; [gamma][delta] T cell; natural killer T cell; OT-II

Expression of spermine/spermidine-[N.sup.1]-acetyltransferase (SSAT), the rate-limiting enzyme of polyamine backconversion cascade, increases after ischemia-reperfusion injuries (IRI). We hypothesized that SSAT plays an important role in the mediation of IRI. To test our hypothesis, wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice were subjected to liver or kidney IRI by ligation of hepatic or renal arteries. The liver and kidney content of putrescine (Put), a downstream by-product of SSAT activity, increased in SSAT-wt animals but not in SSAT-ko animals after IRI, indicating that polyamine backconversion is not functional in SSAT-deficient mice. When subjected to hepatic IRI, SSAT-ko mice were significantly protected against liver damage compared with SSAT-wt mice. Similarly, SSAT-ko animals subjected to renal IRI showed significantly greater protection against damage to kidney tubules than SSAT-wt mice. These studies indicate that SSAT-deficient animals are protected against IRI and suggest that SSAT is an important mediator of the tissue damage in IRI. polyamine catabolism; polyamine backconversion; acute liver failure; acute kidney injury

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 C57B1/6 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 [H.sub.2][O.sub.2] 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. mitochondrial respiration; hepatocytes

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 TNF[alpha] 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-[kappa]B because inhibition of NF-K[kappa] with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun [NH.sub.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-[kappa]B 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. liver; chemokines; Toll-like receptors; nuclear factor-[kappa]B; inflammation

It is well established that liver ischemia-reperfusion induces the expression of heat shock protein (HSP) 70. However, the biological function of HSP70 in this injury is unclear. In this study, we sought to determine the role of HSP70 in hepatic ischemia-reperfusion injury in mice. Male mice were subjected to 90 rain of partial hepatic ischemia followed by up to 8 h of reperfusion. HSP70 was rapidly upregulated after reperfusion. To explore the function of HSP70, sodium arsenite (8 mg/kg iv) was injected before surgery. We found that this dose induced HSP70 expression within 6 h of treatment. Induction of HSP70 with arsenite resulted in a >50% reduction in liver injury as determined by serum transaminases and histology. In addition, arsenite similarly reduced liver neutrophil recruitment and liver nuclear factor-[kappa]B activation, and attenuated serum levels of tumor necrosis factor-[alpha] and macrophage inflammatory protein-2, but increased levels of interleukin (IL)-6. In HSP70 knockout mice, arsenite did not protect against liver injury but did reduce liver neutrophil accumulation. Arsenite-induced reductions in neutrophil accumulation in HSP70 knockout mice were found to be mediated by IL-6. To determine whether extracelullar HSP70 contributed to the injury, recombinant HSP70 was injected before surgery. Intravenous injection of 10 [micro]g of recombinant HSP70 had no effect on liver injury after ischemia-reperfusion. The data suggest that intracellular HSP70 is directly hepatoprotective during ischemia-reperfusion injury and that extracellular HSP70 is not a significant contributor to the injury response in this model. Targeted induction of HSP70 may represent a potential therapeutic option for postischemic liver injury. liver; inflammation; neutrophils; cytokines

The present study examined the role of hepatocyte NF-[kappa]B activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-[kappa]B activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37[degrees]C, 33 and 35[degrees]C, 29 and 33[degrees]C or unregulated, where temperature fell to liver inflammation; nuclear factor-[kappa]B p65; Pin1

Hepatic ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence suggests that CD[4.sup.+] lymphocytes may regulate this neutrophil-dependent injury, but the mechanisms by which this occurs remain to be elucidated. In the present study, we sought to determine the type of CD[4.sup.+] lymphocytes recruited to the liver after ischemia-reperfusion and the manner in which these cells regulated neutrophil recruitment and tissue injury. Wild-type and CD4 knockout (CD[4.sup.-/-]) mice were subjected to hepatic ischemia-reperfusion. CD4+ lymphocytes were recruited in the liver within 1 h of reperfusion and remained for at least 4 h. These cells were comprised of conventional ([alpha][beta]TCR-expressing), unconventional ([gamma][delta]STCR-expressing), and natural killer T cells. CD[4.sup.-/-] mice were then used to determine the functional role of CD[4.sup.+] lymphocytes in hepatic ischemia-reperfusion injury. Compared with wild-type mice, CD[4.sup.-/-] mice had significantly greater liver injury, yet far less neutrophil accumulation. Adoptive transfer of CD[4.sup.+] lymphocytes to CD[4.sup.-/-] mice recapitulated the wild-type response. In wild-type mice, neutralization of interleukin (IL)-17, a cytokine released by activated CD[4.sup.+] lymphocytes, significantly reduced neutrophil recruitment in association with suppression of MIP-2 expression. Finally, oxidative burst activity of liver-recruited neutrophils was higher in CD[4.sup.-/-] mice compared with those from wild-type mice. These data suggest that CD[4.sup.+] lymphocytes are rapidly recruited to the liver after ischemia-reperfusion and facilitate subsequent neutrophil recruitment via an IL-17-dependent mechanism. However, these cells also appear to attenuate neutrophil activation. Thus the data suggest that CD[4.sup.+] lymphocytes have dual, opposing roles in the hepatic inflammatory response to ischemia-reperfusion. liver injury; cell trafficking; neutrophils; T cells; interleukin-17

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI. liver failure; heme oxygenase-1; polyamines

Okaya, Tomohisa, and Alex B. Lentsch. Peroxisome proliferator-activated receptor-[alpha] regulates postischemic liver injury. Am J Physiol Gastrointest Liver Physiol 286: G606-G612, 2004. First published November 13, 2003; 10.1152/ajpgi.00191.2003.--Peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]) is a transcription factor that in some in vitro systems has been linked with downregulation of proinflammatory mediators, thus implicating a potential role for PPAR[alpha] in the regulation of inflammatory processes. Hepatic ischemia-reperfusion injury is characterized by an intense acute inflammatory response that is dependent on a number of proinflammatory mediators. PPAR[alpha] is abundantly expressed in hepatic parenchymal cells but not in Kupffer cells. This study examined whether PPAR[alpha] is involved in regulation of the hepatic inflammatory response to ischemia-reperfusion. Mice nullizygous for PPAR[alpha] had significantly greater liver injury than did their wild-type counterparts. Consistent with these findings, C57BL/6 mice treated with the PPAR[alpha] agonist, WY-14643, had significantly less liver injury than mice receiving vehicle. PPAR[alpha]-knockout mice also had greatly augmented liver neutrophil accumulation and modest increases in activation of the transcription factors NF-[kappa]B and activator protein-l. However, these effects were not associated with increased expression of proinflammatory cytokines or chemokines. In addition, PPAR[alpha]-knockout mice expressed far less inducible nitric oxide synthase in liver than did wild-type mice after ischemia-reperfusion. Finally, treatment of cultured murine hepatocytes with WY-14643, a specific agonist of PPAR[alpha], protected cells against oxidant-induced injury. The data suggest that PPAR[alpha] is an important regulator of the hepatic inflammatory response to ischemia-reperfusion in a manner that is independent of proinflammatory cytokines. repeffusion injury; neutrophils; inflammation

Shen, Hui, and Alex B. Lentsch. Progressive dysregulation of transcription factors NF-[kappa]B and STAT1 in prostate cancer cells causes proangiogenic production of CXC chemokines. Am J Physiol Cell Physiol 286: C840-C847, 2004. First published December 3, 2003; 10.1152/ajpcell.00335.2003.--The CXC chemokine family includes members that possess angiogenic and angiostatic properties. Angiogenic CXC chemokines are produced by prostate cancer cells and contribute to prostate tumor growth. Production of angiostatic CXC chemokines by prostatic cells has not been previously studied. Here we show that normal prostate epithelial (PZ-HPV-7) cells produce low amounts of angiogenic CXC chemokines, whereas prostate cancer cells from primary (CA-HPV-10) and metastatic (PC-3) tumors produce progressively greater amounts. These effects were caused by progressive increases in activation of the transcription factor nuclear factor-[kappa]B in prostate cancer cells. Conversely, PZ-HPV-7 cells produced relatively high levels of angiostatic CXC chemokines, whereas CA-HPV-10 and PC-3 cells produced stepwise lower amounts. These effects were dependent on reduced activation of signal transduction and activator of transcription 1 (STAT1) in prostate cancer cells. These data suggest that there is progressive dysregulation of nuclear factor-[kappa]B and STAT1 in prostate cancer cells that leads to proangiogenic production of CXC chemokines. epithelial; angiogenesis; angiostatic; oncogene; tumor