title: TNF-α-mediated bronchial barrier disruption and regulation by src-family kinase activation.
abstract: BACKGROUND
  Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted.
  
  
  OBJECTIVE
  We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma.
  
  
  METHODS
  Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors.
  
  
  RESULTS
  TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways.
  
  
  CONCLUSIONS
  Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Michelle A
authors: MA
authors: Hardyman
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Emily
authors: E
authors: Wilkinson
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Emma
authors: E
authors: Martin
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Nivenka P
authors: NP
authors: Jayasekera
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Cornelia
authors: C
authors: Blume
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Emily J
authors: EJ
authors: Swindle
authors: UCB Celltech, Slough, United Kingdom.
authors: Neil
authors: N
authors: Gozzard
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Stephen T
authors: ST
authors: Holgate
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Peter H
authors: PH
authors: Howarth
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.
authors: Donna E
authors: DE
authors: Davies
authors: Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom. Electronic address: J.E.Collins@soton.ac.uk.
authors: Jane E
authors: JE
authors: Collins
mesh_terms: D022005:Adherens Junctions / Q000187:drug effects / Q000378:metabolism; D001249:Asthma / Q000378:metabolism*; D001980:Bronchi / Q000166:cytology / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D051177:Catenins / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000095002:Delta Catenin
chemical_list: D015820:Cadherins; D051177:Catenins; D016207:Cytokines; D007211:Indoles; D047428:Protein Kinase Inhibitors; C416927:SU 6656; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D019061:src-Family Kinases; D000095002:Delta Catenin; C000722347:CTNND1 protein, human
keywords: AJ; ALI; Adherens junctions; Air-liquid interface; Airway; ECL; EVOM; Electrochemiluminescence; Epithelial volt ohm meter; FITC; Fluorescein isothiocyanate; HBEC; HRP; Horseradish peroxidase; Human bronchial epithelial cell; LDH; Lactate dehydrogenase; MDCK; MMP; MSD; Madin-Darby canine kidney epithelial; Matrix metalloprotease; Meso Scale Discovery; Monoclonal antibody; SFK; SU6656; Src family kinase; TER; TIMP; TJ; TNF-α; TSLP; Thymic stromal lymphoprotein; Tight junction; Tissue inhibitor of metalloproteinase; Transepithelial electrical resistance; ZO; Zonula occluden; barrier; bronchial; cytokines; epithelial; mAb; proMMP-9; src kinase