Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver.

BACKGROUND & AIMS: Apoptosis of hepatocytes is a central feature of ischemic injury in the liver. The aim of this study was to identify extracellular inducers of apoptosis in the murine ischemic liver. METHODS: Involvement of tumor necrosis factor (TNF)-alpha and Fas signaling was evaluated using various knockout mice (TNF-receptor 1 [TNF-R1]-/-, Fas[lpr]-/-, and Fas ligand[gld]-/-) and wild-type mice pretreated with pentoxifylline, an inhibitor of TNF-alpha synthesis. RESULTS: Expression of TNF-alpha was increased after ischemia and reperfusion in wild-type mice and TNF-R1-deficient mice when compared with sham-operated animals. Pentoxifylline prevented up-regulation of TNF-alpha expression. Inhibition of TNF-alpha resulted in significant decrease of serum aspartate aminotransferase levels and prolonged animal survival. Markers of apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, cytochrome C release, and caspase 3 activity) were consistently decreased, and animal survival was prolonged after blocking TNF-alpha. In contrast, inhibition of Fas signaling did not alter parameters of tissue injury or apoptosis, and animal survival remained unchanged. CONCLUSIONS: We identify TNF-alpha as a crucial inducer of apoptotic cell death in the ischemic liver. A role for Fas could not be identified. These findings may lead to novel strategies to prevent ischemic injury of the liver.     

肿瘤坏死因子相关凋亡诱导配体与肝细胞凋亡的相关性

肿瘤坏死因子相关的调亡诱导配体是TNF家族成员之一,通过其死亡受体诱导凋亡.研究显示TRAIL/死亡受体途径参与多种肝脏病理过程,本文就TRAIL/死亡受体途径的特性、致凋亡的机制及与肝细胞凋亡关系的最新进展作一综述.     

Enhanced tumor necrosis factor alpha in coronavirus but not in paracetamol-induced acute hepatic necrosis in mice

Abstract: Previous reports have demonstrated that tumor necrosis factor alpha (TNF-α) plays an important role in the pathogenesis of fulminant hepatic necrosis. The purpose of this experimental study was to measure TNF-α blood activity in paracetamol-induced liver necrosis and in coronavirus (MHV3)-induced fulminant hepatitis in mice. No elevation of TNF-α activity was found in hepatic failure complicating paracetamol poisoning. In contrast, TNF-α activity significantly increased in response to MHV3, reaching 16.3 ± 5.5 U/ml from 24 h post infection (P>0.01). This augmentation was observed even though the virus was not detectable in the liver. Serum alanine aminotransferase levels were low and no histological lesion was observed. In conclusion, our study further supports the implication of TNF-α in virus-induced hepatitis failure and confirms that paracetamol poisoning does not cause increased TNF-α activity in the circulation.     

TIA-1 regulates the production of tumor necrosis factor alpha in macrophages, but not in lymphocytes.

OBJECTIVE: To determine whether TIA-1 differentially regulates the production of tumor necrosis factor a (TNFalphalpha) in macrophages and lymphocytes. METHODS: Peritoneal macrophages derived from wild-type and TIA-1-/- mice were cultured in the absence or presence of lipopolysaccharide (LPS) before comparison of the production of TNFalpha protein by intracellular flow cytometry and the secretion of TNFalpha protein by enzyme-linked immunosorbent assay. In parallel experiments, splenocytes were cultured in the absence or presence of concanavalin A (Con A), phorbol myristate acetate (PMA)/ionomycin, or anti-CD3/anti-CD28 before comparing the production of TNFalpha protein. Finally, the relative expression of TIA-1 protein in macrophages and splenocytes was compared using immunoblotting analysis. RESULTS: LPS-activated peritoneal macrophages derived from TIA-1-/- mice produced significantly more TNFalpha than macrophages from wild-type controls. In contrast, splenic lymphocytes (CD3+, CD4+, or CD8+) derived from wild-type and TIA-1-/- mice produced similar amounts of TNFalpha in response to Con A, PMA/ionomycin, or anti-CD3/anti-CD28. Lymphocytes and macrophages expressed similar amounts of TIA-1 protein, indicating that differential expression of TIA-1 cannot account for these results. CONCLUSION: TIA-1 is the target of a regulatory pathway that operates in activated macrophages, but not in activated lymphocytes. Developing drugs that target this pathway might prevent the pathologic overexpression of TNFalpha without subverting the T lymphocyte response to microbial pathogens.     

Role for tumor necrosis factor alpha receptor 1 and interleukin-1 receptor in the suppression of mouse hepatocyte apoptosis by the peroxisome proliferator nafenopin

Peroxisome proliferators (PPs) cause rodent liver enlargement and tumors. In vitro, PPs induce rat and mouse hepatocyte DNA synthesis and suppress apoptosis, a response mimicked by exogenous tumor necrosis factor alpha (TNFalpha). Here, we determine the role of TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), and nuclear factor kappa beta (NFkappaB) in the response of mouse hepatocytes to the PP, nafenopin. Nafenopin (50 micromol/L) induced DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, suppressed cell death as measured by Hoechst 33258 staining, induced peroxisomal beta-oxidation as measured by cyanide insensitive palmitoyl CoA oxidation (PCO) and caused activation of nuclear factor kappa beta (NFkappaB) as determined by electrophoretic mobility gel shift assay (EMSA). The induction of DNA synthesis and the suppression of apoptosis in response to nafenopin was abrogated completely by blocking antibodies to TNFR1 but not to TNFR2. In contrast, the induction of peroxisomal beta-oxidation by nafenopin was not blocked by the anti-TNFR1 antibody. Next, we evaluated the response of hepatocytes to interleukin-1 (IL-1), another proinflammatory cytokine. IL-1alpha (2.5 ng/mL) and, to a lesser extent, IL-1beta (5 ng/mL), shared the ability of TNFalpha to induce DNA synthesis and suppress apoptosis. In addition, anti-IL-1 receptor, type 1/p80 (IL-1R) antibodies were able to abrogate the response to nafenopin. IL-1alpha was still able to perturb hepatocyte growth in the presence of the anti-TNFR1 antibody suggesting that IL-1alpha acts independently rather than by elaborating TNFalpha. In summary, these data provide additional evidence for a role for hepatic cytokines in the perturbation of hepatocyte growth by PPs such as nafenopin.     

Tumor necrosis factor alpha blockade reduces the synovial cell infiltrate early after initiation of treatment,but apparently not by induction of apoptosis in synovial tissue

OBJECTIVE: To determine whether treatment with the chimeric anti-tumor necrosis factor alpha antibody infliximab could reduce cellularity by the induction of apoptosis in synovial tissue. METHODS: Twenty-four rheumatoid arthritis patients with active disease were randomized to receive either infliximab (3 mg/kg) (n = 12) or placebo (n = 12) intravenously. All patients were subjected to arthroscopic synovial biopsy directly before initiation of treatment. A second arthroscopic synovial biopsy of the same index joint was performed 48 hours after the first arthroscopy. After the second arthroscopy, the patients who had initially received placebo were also treated with infliximab in an extension study. A third arthroscopy was performed in all patients on day 28. Immunohistologic analysis was performed to characterize the cell infiltrate. In situ detection of apoptotic cells was performed by TUNEL assay and electron microscopy. RESULTS: At 48 hours after initiation of infliximab treatment, there was a significant reduction in the number of intimal macrophages; this was not observed in the placebo group. The number of sublining macrophages, T cells, and plasma cells also tended to be decreased in infliximab-treated patients, but not in the placebo group. Of interest, we did not detect any increase in the number of apoptotic cells after infliximab treatment. CONCLUSION: Infliximab therapy may reduce the number of inflammatory cells in rheumatoid synovial tissue as soon as 48 hours after initiation of treatment, but apparently not by induction of apoptosis. Conceivably, decreased cell infiltration primarily results from early inhibition of cell migration.     

Tumor necrosis factor α antibody prevents brain damage of rats with acute necrotizing pancreatitis

AIM: To study the protective effects of tumor necrosis factor α (TNFα) antibody on pancreatic encephalopathy in rats.METHODS: One hundred and twenty SD rats were randomly divided into normal control group, acute necrotizing pancreatitis group and TNFα antibody treated group. Acute hemorrhage necrotizing pancreatitis model in rats was induced by retrograde injection of 50 g/L sodium taurocholate into the pancreatobiliary duct. Serum TNFα was detected and animals were killed 12 h after drug administration. Changes in content of brain water, MDA and SOD as well as leucocyte adhesion of brain microvessels were measured.RESULTS: In TNFα antibody treated group, serum TNFα level was decreased. Content of brain water, MDA and SOD as well as leucocyte adhesion were decreased significantly in comparison with those of acute necrotizing pancreatitis group (P<0.05).CONCLUSION: TNFα antibody can alleviate the brain damage of rats with acute hemorrhage necrotizing pancreatitis.     

Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice

Background/Aims: Tumor necrosis factor α (TNF-α) and Fas ligand are apoptotic cell-death mediators that act by binding to their responsive receptors. The aims of this study were to assess the differences between liver cell deaths induced by TNF-α and anti-Fas antibody, and to investigate the mechanism by which GalN sensitizes the hepatocyte to injury by TNF-α.Methods: TNF-α or anti-Fas antibody was injected into BALB/c mice sensitized or unsensitized by D-galactosamine (GalN). Liver injury was assessed biochemically and histologically. The expressions of TNF receptor (TNFR)1 and TNFR2 mRNA in the liver were determined by Northern blot analysis. Nuclear factor-κB (NF-κB) DNA binding activity was determined by gel shift assay.Results: In GalN-sensitized mice, hepatocyte apoptosis and liver failure were observed after TNF-α injection, but neither occurred in unsensitized mice. Microscopically, GalN preceding TNF-α caused massive hemorrhagic liver damage with fragmented hepatocyte nuclei resembling effects of anti-Fas antibody, but GalN largely failed to sensitize to injury by this antibody. TNFR1 mRNA expression in the liver was upregulated within 3 h after GalN administration, and anti-TNFR1 antibody protected GalN-sensitized mice from hepatotoxic effects of TNF-α. GalN treatment failed to affect TNF-α-induced NF-κB activation.Conclusions: Unlike Fas-related apoptosis, TNFR-mediated apoptosis requires hepatocyte sensitization involving TNFR1 upregulation.     

Association between rheumatoid arthritis and polymorphism of tumor necrosis factor receptor II, but not tumor necrosis factor receptor I, in Caucasians

OBJECTIVE: Tumor necrosis factor (TNF) is a powerful mediator of inflammation in rheumatoid arthritis (RA). In vivo, its acute effects are limited by binding to soluble receptors (TNFR), suggesting that TNFR genes could be important candidate risk factors. The present study was undertaken to investigate association of polymorphisms of TNFRI and TNFRII with RA in subjects in the UK. METHODS: Unrelated Caucasian RA patients (n = 291) and healthy Caucasian controls (n = 143) were genotyped for A/G polymorphism in exon 1 of TNFRI. From this sample, 240 of the patients and 137 controls were also typed for a single-nucleotide polymorphism (SNP) in exon 6 of the TNFRII gene. In followup studies, DNA samples from UK Caucasian RA patients with a positive family history (n = 149) and UK Caucasian patients with sporadic RA (n = 208) were also typed for the exon 6 TNFRII polymorphism. RESULTS: TNFRI polymorphism was not associated with RA (odds ratio [OR] for GG genotype 0.93, 95% confidence interval [95% CI] 0.54-1.60). For TNFRII, in the initial study group, patients with RA were significantly more likely to be positive for both the G allele and GG genotype than were controls (OR for GG genotype 2.55, 95% CI 1.11-5.86). The association appeared to be confined to those with a family history of RA. This finding was replicated in an independent cohort of patients with familial RA. CONCLUSION: The results of this study provide evidence of association between an SNP in the TNFRII gene and RA, the strongest association being observed in patients with a family history. No evidence of association between RA and TNFRI was demonstrated.     

Clinical responses to tumor necrosis factor alpha antagonists do not show a bimodal distribution: data from the Stockholm tumor necrosis factor alpha followup registry

OBJECTIVE: To study the distribution of clinical responses to treatment with the tumor necrosis factor alpha (TNFalpha) antagonists etanercept and infliximab, and in particular, to determine whether there is a biologically meaningful distinction between responders and nonresponders. METHODS: Among patients in the Stockholm TNFalpha Followup Registry, we analyzed the clinical responses to etanercept and infliximab, using the American College of Rheumatology (ACR) core set of outcome measures. For each parameter, the absolute change (value at baseline - current value) and the percentage change ([absolute change]/[value at baseline] x 100) from baseline were calculated. The results were plotted as histograms and inspected visually, and the distributions were statistically compared with computer-generated normal distributions. RESULTS: Absolute and relative changes in outcomes on the ACR core set of measures in 406 patients receiving etanercept or infliximab were studied. All but a few of these analyses yielded normal or somewhat skewed distributions. The statistical analyses did not detect any non-normal distributions, and visually, the distributions did not appear to be bimodal. CONCLUSION: The clinical response to TNFalpha blockade displays a normal or skewed, but not bimodal, distribution. The frequently encountered perception that a clear distinction can be made between responders and nonresponders is not borne out. These relatively straightforward findings imply that the biologic mechanisms determining responsiveness to TNFalpha blockade are multifactorial and may also have important implications for regulatory guidelines pertaining to treatment with these biologic agents.     

Soluble TNF-R1, but not tumor necrosis factor alpha, predicts the 3-month mortality in patients with alcoholic hepatitis

BACKGROUND/AIMS: In alcoholic hepatitis (AH), soluble TNF alpha receptor-1 (sTNF-R1) is increased. Elevated TNF alpha predicts mortality, but infection influences TNF alpha values. In patients with AH, we determined the prognostic value of TNF alpha, sTNF-R1, and lipopolysaccharide binding protein (LBP) and CD14, both involved in endotoxemia-associated inflammation. METHODS: One hundred and eight cirrhotic patients (Pugh score 10 [6-13]) and biopsy-proven AH (Maddrey's DF <32: n=46; > or =32: n=62) without associated infection were included within 8 days of admission and followed-up for 3 months. Cytokines were measured using specific immunoassays. Patients with severe AH received steroids. RESULTS: Twenty four patients died at a median time of 35 days (range: 3-89). The overall survival was 78%. Multivariate Cox regression analysis showed that sTNF-R1 was an independent predictor of mortality, (OR 4.33: 95% CI [1.12-16.75]). Pugh's score (P=0.618), Maddrey's DF (P=0.182), creatinine (P=0.197), TNF alpha (P=0.319), LBP (P=0.362), and CD14 (P=0.347) were not related to survival. CONCLUSIONS: In patients with AH, sTNF-R1 measured at admission is an independent predictor of survival at 3 months. Provided that TNF-R1 mediates the cytotoxic actions of TNFalpha, these results support the concept of dysregulated TNF alpha metabolism in AH.     

Alcohol increases tumor necrosis factor alpha and decreases nuclear factor-kappab to activate hepatic apoptosis in genetically obese mice

Both obesity and alcohol can cause oxidative stress, cytokine induction, and steatohepatitis. To determine the consequences of their combination, we compared the hepatic effects of moderate ethanol binges in lean and obese ob/ob mice. Mice received water or ethanol (2.5 g/kg) by gastric intubation daily for 4 days, and were killed 2 hours after the last administration. Some obese mice also received pentoxifylline, an inhibitor of tumor necrosis factor-alpha (TNF-alpha) production, before each ethanol administration. In lean mice, these moderate ethanol doses did not increase plasma TNF-alpha and hepatic caspase-3 activity, but triggered some apoptotic hepatocytes. Naive ob/ob mice had a few necrotic and apoptotic hepatocytes, but exhibited little oxidative stress, possibly because of adaptive increases in manganese superoxide dismutase, heat shock protein 70 (Hsp70), mitochondrial cytochrome c, and mitochondrial DNA. Alcohol administration to ob/ob mice did not increase oxidative stress despite increased CYP2E1, but increased plasma TNF-alpha, further increased Hsp70, and profoundly decreased p65 nuclear factor kappaB (NF-kappaB) protein and DNA-binding activity in nuclear extracts. Caspase-3 was activated, and more apoptotic hepatocytes were found in intoxicated obese mice than naive obese mice. In intoxicated obese mice, pentoxifylline fully prevented the increase in plasma TNF-alpha the decrease in nuclear NF-kappaB activity, and the increase in hepatic caspase-3, and it also decreased hepatic triglycerides. In conclusion, obese mice develop adaptations that may limit oxidative stress. Moderate ethanol intoxication does not increase oxidative stress in obese mice, but increases TNF-alpha and also decreases nuclear NF-kappaB activity, thus unleashing the apoptotic effects of TNF-alpha.     

肿瘤坏死因子α致暴发性肝功能衰竭小鼠肠上皮细胞凋亡的作用

目的研究肿瘤坏死因子α(TNFα)对暴发性肝功能衰竭(FHF)小鼠肠黏膜上皮细胞凋亡的作用。方法用D-氨基半乳糖(GalN) 脂多糖(LPS)或TNFα造FHF小鼠模型;酶联免疫吸附法测定血清TNFα含量;光学显微镜和电子显微镜观察肠组织;末端转移酶介导的dUTP缺口末端标记法检测肠组织细胞凋亡情况;免疫组织化学法检测肿瘤坏死因子受体I(TNFRⅠ)蛋白在肠组织的表达与分布。结果实验各组动物各时间点光学显微镜下肠黏膜上皮细胞结构均保持完整,电镜下可见肝功能衰竭组有典型的凋亡细胞;对照组、LPS组、GalN组血清TNFα水平几乎正常,凋亡率低且基本没有TNFRⅠ蛋白表达;GalN LPS组血清TNFα水平12h明显升高(P<0.01),且TNFRⅠ蛋白在6h(大肠:2.82e 4±4.60e 3;小肠:1.14e 4±2.13e 3)即有表达,此时肠上皮细胞凋亡不明显,9h和12hTNFRⅠ蛋白表达明显增加,同时肠上皮细胞凋亡也明显。用GalN TNFα造FHF模型,结果也与GalN LPS组类似。应用抗TNFα抗体,可降低TNFRⅠ蛋白表达和减少肠上皮细胞凋亡的发生。结论TNFα能诱导肝功能衰竭小鼠肠上皮细胞凋亡,抗TNFα抗体能阻断这一作用;在FHF的模型动物中,TNFRⅠ蛋白表达增多,TNFRⅠ蛋白表达与肠上皮细胞凋亡在一定程度上呈正相关。