title: Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions.
abstract: OBJECTIVE
  Blood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.
  
  
  METHODS
  The present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.
  
  
  RESULTS
  In response to treatment with either TNF-α or IL-6 (0-100 ng/ml, 0-24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766).
  
  
  CONCLUSIONS
  A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Rochfort
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Collins
authors.affiliation: School of Health and Human Performance, Dublin City University, Dublin, Ireland; Centre for Preventive Medicine, Dublin City University, Dublin, Ireland.
authors.forename: Ronan P
authors.initials: RP
authors.lastname: Murphy
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland; Centre for Preventive Medicine, Dublin City University, Dublin, Ireland.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Cummins
mesh_terms: D022005:Adherens Junctions / Q000187:drug effects*; D001812:Blood-Brain Barrier / Q000187:drug effects*; D002478:Cells, Cultured; D015536:Down-Regulation; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D015850:Interleukin-6; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019255:NADPH Oxidases