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Decreased Distribution of Lung Epithelial Junction Proteins after Intratracheal Antigen or Lipopolysaccharide Challenge

Correlation with Neutrophil Influx and Levels of BALF sE-Cadherin

GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, United Kingdom

Address correspondence to: Dr. S. M. Evans, GlaxoSmithKline Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. E-mail: sme40185{at}gsk.co.uk

Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.

Abbreviations: adherens junction, AJ • bronchoalveolar lavage, BAL • bronchoalveolar lavage fluid, BALF • ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, EGTA • lipopolysaccharide, LPS • ovalbumin, OVA • optimal cutting temperature, OCT • phosphate-buffered saline, PBS • standard error of the mean, SEM • tris-buffered saline, TBS • tris-buffered saline with 0.1% tween, TBST • tight junction, TJ