IL-13 Activates STAT6 and Inhibits Liver Injury Induced by Ischemia/Reperfusion

Hepatic ischemia/reperfusion injury is initiated by the activation of Kupffer cells and their subsequent release of proinflammatory mediators, including tumor necrosis factor-alpha (TNFalpha). These mediators stimulate a cascade of events including up-regulation of CXC chemokines and vascular endothelial adhesion molecules, leading to hepatic neutrophil recruitment and tissue injury. Interleukin-13 (IL-13) is a cytokine that has been shown to suppress macrophage production of proinflammatory mediators. The objective of the current study was to determine whether IL-13 could regulate the liver inflammatory injury induced by ischemia and reperfusion. C57BL/6 mice underwent 90 minutes of partial hepatic ischemia followed by reperfusion with or without intravenous administration of recombinant murine IL-13. Hepatic ischemia/reperfusion increased expression of TNFalpha and macrophage inflammatory protein-2 (MIP-2), leading to hepatic neutrophil recruitment, hepatocellular injury, and liver edema. Administration of IL-13 reduced the production of TNFalpha and MIP-2 mRNA and protein. IL-13 suppressed liver neutrophil recruitment by up to 72% and hepatocellular injury and liver edema were each reduced by >60%. Administration of IL-13 had no effect on liver NFkappaB activation, but greatly increased the activation of STAT6. The data suggest that the hepatoprotective effects of IL-13 may be a result of STAT6 activation.     

In vivo vitamin E administration attenuates interleukin-6 and interleukin-1beta responses to an acute inflammatory insult in mouse skeletal and cardiac muscle

Antioxidants are associated with reduced pro-inflammatory cytokine expression in immune cells and isolated tissues; however, no studies have examined whether short-term vitamin E administration is associated with reduced lipopolysaccharide (LPS)-induced cytokine expression in mouse skeletal and cardiac muscle, in vivo. These experiments tested the hypothesis that vitamin E administration attenuates nuclear factor kappaB (NF-kappaB), IL-6, IL-1beta and tumour necrosis factor alpha (TNFalpha) responses in skeletal and cardiac muscle to an inflammatory challenge induced by systemic LPS. We compared IL-6, IL-1beta and TNFalpha mRNA and protein, activated NF-kappaB and total oxidized proteins in skeletal and cardiac muscle 4 or 24 h after saline or LPS injection in mice receiving vitamin E or placebo for 3 days prior to the insult. Skeletal and cardiac IL-6 mRNA and protein were significantly elevated by LPS in both groups, but responses were significantly lower in vitamin E- compared with placebo-treated mice. In skeletal and cardiac muscle, LPS increased IL-1beta mRNA and protein in placebo- but not vitamin E-treated mice. Lipopolysaccharide-induced levels of cardiac IL-1beta mRNA and protein and skeletal IL-1beta mRNA were lower with vitamin E than placebo. Lipopolysaccharide-induced NF-kappaB activation and increases in total oxidized proteins were attenuated with vitamin E compared with placebo in both tissues. Vitamin E decreased LPS-induced increases in plasma IL-1beta but not IL-6 compared with placebo. The major results provide the first in vivo evidence that short-term vitamin E administration reduces IL-6 and IL-1beta responses to LPS in skeletal and cardiac muscle and prevents LPS-induced increases in NF-kappaB activation and total oxidized proteins.     

Blockade of IL-6 signaling exacerbates liver injury and suppresses antiapoptotic gene expression in methionine choline-deficient diet-fed db/db mice

Our previous study revealed that blockade of interleukin-6 (IL-6)-STAT3 signaling ameliorated liver injury, although hepatic STAT3(-/-) or GP130(-/-) mice have been reported to develop severe liver injury, in a murine methionine choline deficient (MCD) diet-induced model of non-alcoholic steatohepatitis (NASH). In this study, to determine whether profound blockade of IL-6-STAT3 signaling may still ameliorate liver injury, we studied db/db mice, which have impaired leptin-mediated STAT3 activation, using the MCD diet-induced NASH model. Male lean and db/db mice (6 weeks old) were fed either control chow or an MCD diet for 8 or 12 weeks. Half of the mice were treated with 15 mg/kg rat anti-mouse IL-6 receptor neutralizing antibody (MR16-1) intraperitoneally twice weekly, the remainder were injected with 15 mg/kg rat IgG as a control. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress and antiapoptotic gene expression were evaluated. Plasma IL-6 levels were elevated in all groups of db/db mice. Although hepatic IL-6/ GP130 signaling was activated in chow-fed db/db mice, this was suppressed in MCD diet-fed db/db mice, accompanied by downregulation of hepatic IL-6 receptor and GP130 mRNA expression. MR16-1 treatment of MCD diet-fed db/db mice further repressed STAT3 activities and expression of STAT3-related antiapoptotic genes, such as Bcl-2 and Ref-1, but increased plasma-free fatty acid and hepatic markers of lipid peroxidation/oxidant stress, leading to increased liver injury, hepatocyte apoptosis and liver fibrosis. Although 'moderate' blockade of enhanced IL-6-STAT3 signaling may be beneficial in NASH, as we reported previously, these findings demonstrate that a profound defect in STAT3 activation is detrimental in terms of liver injury, hepatocyte apoptosis and liver fibrosis, indicating the hepato-protective role of IL-6 signaling in this severe NASH model.     

Carcinoembryonic antigen promotes tumor cell survival in liver through an IL-10-dependent pathway

Most circulating tumor cells die within 24 h of entering the hepatic microvasculature because their arrest initiates an ischemia-reperfusion (I/R) injury that is cytotoxic. Human colorectal carcinomas (CRC) produce the glycoprotein Carcinoembryonic Antigen (CEA) that increases experimental liver metastasis in nude mice. Since CEA induces release of IL-6 and IL-10, we hypothesized that CEA inhibits the I/R injury through a Kupffer cell-mediated cytokine-dependent pathway. We assessed cytokine effects in CRC co-cultured with liver and in vivo. Human CRC prelabeled with fluorescent dyes were incubated with a reoxygenated suspension of ischemic nude mouse liver fragments in a bioreactor. CEA, rhIL-6 or rhIL-10 were either administered to the donor mice prior to hepatic ischemia or during co-culture. Liver donors were athymic nude or iNOS, IL-6 or IL-10 knock out mice. Ischemic-reoxygenated liver kills Clone A CRC through production of nitric oxide (NO) and superoxide anion. Treatment of liver donors with CEA prior to hepatic ischemia inhibited this in vitro cytotoxicity through an IL-10 and Kupffer cell dependent pathway that inhibited NF-kappaB activation, NO production and iNOS upregulation. IL-10 but not IL-6 enhanced CRC survival in nude mouse liver in vivo. Thus, CEA enhanced metastasis by inducing IL-10 to inhibit iNOS upregulation in host liver.     

缺血-再灌注肝脏组织中ICAM-1基因的表达

目的：探讨肝脏缺血-再灌注过程中肝窦内皮细胞ICAM-1 mRNA的表达规律及其意义。方法：应用分子杂交技术,观察缺血时间分别15、30及45 min的3组兔肝脏于再灌注60 min时ICAM-1 mRNA的表达情况,并对肝窦腔内的白细胞数量进行计数。结果：3组肝脏缺血前及缺血末组织内仅有少量ICAM-1 mRNA表达于肝窦内皮细胞浆内,且肝窦腔内的白细胞数量也无明显改变;但于再灌注60 min时,ICAM-1 mRNA表达程度则显著增强,且缺血时间越长的肝脏,其表达强度越大。此外,组织内ICAM-1 mRNA含量越高的肝脏,肝窦腔内白细胞数量越多,两者呈显著的正相关关系。结论：肝脏的缺血能明显诱导再灌注期间肝窦内皮细胞表达ICAM-1,增强肝窦内皮细胞的粘附力,促进再灌注血流中的白细胞在肝窦内滞留,进而引发一系列病理生理改变。     

Enhanced liver regeneration in IL-10-deficient mice after partial hepatectomy via stimulating inflammatory response and activating hepatocyte STAT3

Emerging evidence suggests that proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a critical role in the initiation and progression of liver regeneration; however, relatively little is known about the role of anti-inflammatory cytokine IL-10 in liver regeneration after partial hepatectomy (PHx). Here, we examined the role of IL-10 in liver regeneration using a model of PHx in several strains of genetically modified mice. After PHx, expression of IL-10 mRNA in the liver and spleen was significantly elevated. Such elevation was diminished in TLR4 mutant mice. Compared with wild-type mice, IL-10(-/-) mice had higher levels of expression of proinflammatory cytokines (IL-6, TNF-α, and IFN-γ) and inflammatory markers (CCR2 and F4/80) in the liver, as well as higher serum levels of proinflammatory cytokines after PHx. The number of neutrophils and macrophages was also higher in the livers of IL-10(-/-) mice than in wild-type mice after PHx. Liver regeneration as determined by BrdU incorporation after PHx was higher in IL-10(-/-) mice than in wild-type mice, which was associated with higher levels of activation of IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes significantly reduced liver regeneration in IL-10(-/-) mice after PHx. Collectively, IL-10 plays an important role in negatively regulating liver regeneration via limiting inflammatory response and subsequently tempering hepatic STAT3 activation.     

TLR-2 is upregulated and mobilized to the hepatocyte plasma membrane in the space of Disse and to the Kupffer cells TLR-4 dependently during acute endotoxemia in mice

Membrane components of bacteria and fungi are recognized by Toll-like receptors (TLRs) which, when activated, induce several inflammatory mediators important in the host defense. As the liver is constantly exposed to ingested bacteria, hepatic TLRs must be broadly responsive and highly regulated to prevent uncontrolled inflammatory activation. Although several hepatic cells express microbe recognition molecules and inflammatory mediators in vitro, the regulation and cellular localization of these proteins in vivo remain uncertain. The expression and regulation of TLR-2 and TLR-4, and the cytokine expression patterns were evaluated in mouse tissues using a model of acute inflammation induced by intraperitoneal injection of LPS. Five hours after intraperitoneal LPS, induction of TLR-4 was evident in lung, while the low hepatic TLR-4 expression was non-inducible. TLR-2 mRNA and protein were induced both in lung and liver TLR-4 dependently. However, IL-1alpha also contributed to this induction, and IL-1R1 antibody attenuated the TLR-2 increase. Immunoelectron microscopy showed accumulation of cytoplasmic TLR-2 to vesicles near the hepatocyte plasma membrane in the space of Disse, to the sinusoidal endothelium and to the Kupffer cells. NF-kappaB activation was clear in Kupffer cells and hepatocytes during LPS-challenge, suggesting these cells to be the main source of in vivo cytokine production. Hepatic cytokine response to LPS was remarkably rapid in liver, whereas lung responded less acutely. Secondary inflammatory challenge attenuated the TLR-2 response. The innate immune system of the liver is rapidly and transiently activated during endotoxemia by mechanism involving both TLR-4 and TLR-2.     

辣椒素受体拮抗剂AMG517在小鼠脑缺血/再灌注损伤中通过调节炎症因子释放发挥神经保护作用#br#

目的 观察辣椒素受体(TRPV1)拮抗剂AMG517在小鼠脑缺血/再灌注损伤中的作用及相关机制。方法 40只雄性C57BL/6小鼠随机分为假手术组(sham,n=10)、赋形剂（vehicle）+缺血/再注灌注组(vehicle,n=10)、辣椒素+缺血/再注灌注组(capsaicin,n=10)和AMG517+缺血/再注灌组(AMG517,n=10)。采用大脑中动脉闭塞(MCAO)诱导缺血/再灌注损伤,再灌注72 h进行神经行为学评分;同时检测各组小鼠梗死体积、脑水肿、TRPV1 mRNA表达和血清肿瘤坏死因子-α(TNF-α)浓度及白细胞介素-10(IL-10)浓度。结果 与vehicle组相比,AMG517显著减小鼠脑梗死体积(P<0.01)。AMG517给药后也可显著降低小鼠神经行为学评分(P<0.01)。与sham组比较,vehicle组TRPV1 mRNA表达显著增加(P<0.01)。AMG517给药后可显著增加抗炎性细胞因子IL-10浓度,并降低炎性细胞因子TNF-α浓度(P<0.05)。结论 AMG517可改善小鼠缺血/再灌注损伤,可能是通过缓解炎症发挥神经保护作用。     

IL-8单抗保护兔肝脏免受中性粒细胞介导的再灌注损伤

通过建立新西兰家兔的肝缺血再灌注损伤模型 ,观察抗IL 8中和性单克隆抗体对缺血再灌注 (IR )损伤肝脏的保护作用。肝缺血再灌注 4h后 ,外周血中各种肝功能指标酶活性与假手术组相比显著升高 (P <0 0 5 ) ,同时肝脏出现大量散点状中性粒细胞的浸润 ,此时肝细胞明显肿胀 ,细胞呈散乱状排列 ,细胞壁结构不完整并出现液化现象。而使用抗IL 8抗体后可明显阻断再灌注过程中中性粒细胞对肝脏的浸润 ,肝细胞虽仍有肿胀现象 ,但细胞呈正常的规则排列 ,细胞壁结构完整。其外周血肝功能指标酶活性与对照组相比无显著差别。该结果显示在肝再灌注损伤过程中 ,IL 8是趋化中性粒细胞浸润的关键因子 ,抗IL 8单克隆抗体对肝缺血再灌损伤肝脏有保护作用。     

The pancreatitis-associated protein induces lung inflammation in the rat through activation of TNFalpha expression in hepatocytes

The pancreatitis-associated protein (PAP) is a pancreatic stress protein overexpressed during acute pancreatitis, a disease often accompanied by lung inflammation. We investigated whether PAP was involved in the occurrence of this remote complication of pancreatitis and whether the liver might be implicated in the process. PAP was injected into the vena cava of rats (40 or 400 micro g/kg body weight). For comparison, pancreatitis was induced in rats by intraductal administration of sodium taurocholate. Three hours later, parameters of inflammation and mRNA concentrations of TNFalpha, P-selectin, heat shock protein (HSP)-70, and extracellular superoxide dismutase (EC-SOD) were monitored in lung and liver. Significant increases in P-selectin expression, neutrophil infiltration, and oxidative stress revealed that PAP treatment induced lung inflammation in rats and exacerbated inflammation in animals with pancreatitis. Plasma TNFalpha level was increased and TNFalpha mRNA was strongly overexpressed in liver, with concomitant activation of NF-kappaB; in situ hybridization revealed that TNFalpha overexpression was mainly located to hepatocytes. Lung inflammation induced by PAP could be prevented by injection of anti-TNFalpha antibodies. It was concluded that, during pancreatitis, PAP released by the pancreas could mediate lung inflammation through induction of hepatic TNFalpha expression and subsequent increase in circulating TNFalpha.     

Potential role for IL-23 in hepatic ischemia/reperfusion injury

Summary. IL-12 and IL-23 are related cytokines that share a p40 subunit. Our previous studies identified IL-12 as a primary initiator of the cytokine cascade induced after hepatic ischemia/reperfusion. Because those studies were conducted prior to the discovery of IL-23, it is not clear whether IL-12 or IL-23 is the relevant cytokine in this response. The current studies show that the antibodies used in our original study cross-react with IL-23. We also found that both IL-12 p35 and IL-23 p19 mRNA are expressed rapidly in the liver after ischemia/reperfusion. Finally, isolated Kupffer cells produced TNFα in response to IL-23, but not IL-12, suggesting that IL-23 may be the relevant initiator of the hepatic inflammatory response to ischemia/reperfusion. Received 19 August 2005; returned for revision 5 December 2005; accepted by K. Visvanathan 27 January 2006     

Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.

Ischemia in rat hind limbs followed by reperfusion results in local as well as remote organ (lung) injury characterized by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (51Cr-labeled rat erythrocytes extravasation) in skeletal muscle and lung, together with an associated increased tissue content of myeloperoxidase, reflecting neutrophil accumulation. Within 60 minutes of reperfusion following ischemia, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 plasma levels increased significantly, reaching maximum levels after 2 hours of reperfusion. Polyclonal antibodies to TNF-alpha and IL-1 provided significant protection against vascular injury in both muscle and lung. These results were confirmed by the use of soluble TNF-alpha receptor and IL-1 receptor antagonist. In rat lungs following ischemia and reperfusion, there was immunohistochemical evidence of E-selectin expression in the lung vasculature; this expression was blocked by treatment of animals with anti-TNF-alpha. These data indicate that both local (hind limb) and remote (lung) organ injury after ischemia/reperfusion requires participation of TNF-alpha and IL-1. The cytokines may, in part, be involved in the up-regulation of endothelial adhesion molecules.     

高渗盐水预处理可减轻中性粒细胞介导的肝脏缺血再灌注损伤

目的：探讨高渗盐水预处理对肝脏缺血再灌注损伤的拮抗作用及其机制。方法：建立大鼠局部肝脏缺血再灌注模型。设假手术组（sham组）、缺血再灌注组（IR组）和高渗盐水预处理组（HTS组），分别于再灌注后1 h、3 h、6 h、12 h和24 h处死大鼠，测定谷丙转氨酶（ALT）；抗凝血流式细胞仪测定中性粒细胞CD11b/CD18(Mac-1)的阳性率；RT-PCR和Western blotting分别测定肝内细胞间黏附分子1（ICAM-1）的mRNA和蛋白表达；比色法测定肝脏内髓过氧化物酶（MPO）活性；常规病理及电镜观察肝脏的病理学改变及肝脏内中性粒细胞的浸润情况。结果： ① HTS组在3 h、6 h和12 h血清ALT水平明显低于IR组（P<0.05）。②HTS组在6 h和12 h中性粒细胞Mac-1表达显著弱于IR组（P<0.05）。③HTS组肝脏MPO活性在再灌注后6 h、12 h和24 h明显低于IR组（P<0.05）。④HTS组大鼠肝脏内ICAM-1mRNA及蛋白表达明显低于IR组。⑤HTS组肝内中性粒细胞浸润、肝细胞浊肿和肝窦狭窄程度轻于IR组。结论： HTS预处理能够通过抑制中性粒细胞Mac-1和肝内ICAM-1的表达，明显抑制肝内中性粒细胞的黏附和聚集，起到拮抗肝脏缺血再灌注损伤的作用。