title: Pollen proteolytic enzymes degrade tight junctions.
abstract: OBJECTIVE
  Asthma and allergic rhinitis are significant, increasing causes of morbidity worldwide. Pollen, a major cause of seasonal rhinitis/conjunctivitis, carries proteolytic enzymes on its surface. We showed previously that peptidase allergens from house dust mites compromise epithelial barrier function by degrading the extracellular domains of the tight junction proteins, occludin and claudin, thus facilitating allergen delivery across epithelial layers. In this study, we aimed to determine whether peptidases from allergenic pollens should similarly be considered to have a role in disrupting tight junctions.
  
  
  METHODS
  Diffusates from stored pollen of Giant Ragweed, White Birch and Kentucky Blue Grass, and fresh pollen from Easter Lily were applied to confluent monolayers of Madin-Darby canine kidney (MDCK) and Calu-3 cells in serum-free medium. Immunofluorescence was performed for the tight junction proteins, occludin, claudin-1 and ZO-1. The effect of pollen diffusate on occludin was studied by Western blotting, and enzymatic activity in the diffusates was demonstrated by zymography. The ability of protease inhibitors to block the action of the diffusate on tight junctions was investigated.
  
  
  RESULTS
  Diffusates from all four allergenic pollens caused loss of immunofluorescence labelling for tight junction proteins on MDCK and Calu-3 cells. The effect was blocked by inhibitors of serine and cysteine proteases. Degradation of occludin was demonstrated by Western blotting and zymography indicated that diffusates contain proteolytic activity.
  
  
  CONCLUSIONS
  Pollen peptidases directly or indirectly disrupt epithelial tight junctions, and this activity should be considered as a possible mechanism for facilitating allergen delivery across epithelia.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK. david.garrod@manchester.ac.uk
authors.forename: Sarah
authors.initials: S
authors.lastname: Runswick
authors.forename: Thomas
authors.initials: T
authors.lastname: Mitchell
authors.forename: Paul
authors.initials: P
authors.lastname: Davies
authors.forename: Clive
authors.initials: C
authors.lastname: Robinson
authors.forename: David R
authors.initials: DR
authors.lastname: Garrod
mesh_terms: D000818:Animals; D001249:Asthma / Q000276:immunology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004285:Dogs; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000276:immunology; D062793:Occludin; D010447:Peptide Hydrolases / Q000502:physiology*; D011058:Pollen / Q000201:enzymology*; D012220:Rhinitis / Q000276:immunology*; D019108:Tight Junctions / Q000276:immunology*
chemical_list: D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D062793:Occludin; D010447:Peptide Hydrolases