title: Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.
abstract: BACKGROUND
  House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium.
  
  
  OBJECTIVE
  The present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1.
  
  
  METHODS
  Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation.
  
  
  RESULTS
  Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin.
  
  
  CONCLUSIONS
  Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.
authors.affiliation: Department of Pharmacology & Clinical Pharmacology, St George's Hospital Medical School, London, UK.
authors.forename: H
authors.initials: H
authors.lastname: Wan
authors.forename: H L
authors.initials: HL
authors.lastname: Winton
authors.forename: C
authors.initials: C
authors.lastname: Soeller
authors.forename: D C
authors.initials: DC
authors.lastname: Gruenert
authors.forename: P J
authors.initials: PJ
authors.lastname: Thompson
authors.forename: M B
authors.initials: MB
authors.lastname: Cannell
authors.forename: G A
authors.initials: GA
authors.lastname: Stewart
authors.forename: D R
authors.initials: DR
authors.lastname: Garrod
authors.forename: C
authors.initials: C
authors.lastname: Robinson
mesh_terms: D000818:Animals; D039741:Antigens, Dermatophagoides; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000494:pharmacology; D004285:Dogs; D004789:Enzyme Activation / Q000276:immunology; D004847:Epithelial Cells / Q000737:chemistry* / Q000276:immunology*; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000276:immunology; D013329:Structure-Activity Relationship; D019108:Tight Junctions / Q000737:chemistry* / Q000201:enzymology / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D003546:Cysteine Endopeptidases