title: Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation.
abstract: BACKGROUND
  Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.
  
  
  METHODS
  Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2',7'-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.
  
  
  RESULTS
  Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.
  
  
  CONCLUSIONS
  CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.
authors.affiliation: Centre for Liver Research, School of Infection and Immunity, Institute of Biomedical Research, The Medical School, The University of Birmingham, Edgbaston, Birmingham, United Kingdom. balsin@hotmail.com
authors.forename: Ricky H
authors.initials: RH
authors.lastname: Bhogal
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Weston
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Curbishley
authors.forename: David H
authors.initials: DH
authors.lastname: Adams
authors.forename: Simon C
authors.initials: SC
authors.lastname: Afford
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism*; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000737:chemistry / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D053222:Fas Ligand Protein / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009336:Necrosis / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D012995:Solubility; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D019013:CD40 Antigens; D053222:Fas Ligand Protein; D017382:Reactive Oxygen Species; D023201:CD40 Ligand; D009249:NADP; D019255:NADPH Oxidases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen