Structural and functional alteration of corneal epithelial barrier under inflammatory conditions.
OBJECTIVE
The aim of the study was to determine the effect of inflammatory conditions on the expression of tight junction (TJ) and adherens junction (AJ) proteins between human corneal epithelial cells and, consequently, on corneal epithelial barrier integrity.


METHODS
Zonula occludens proteins ZO-1 and ZO-2, claudin-1 and -2 (CLDN-1 and CLDN-2), occludin (OCLN) as well as E-cadherin (E-cad) expression were analyzed in a human corneal epithelial cell line (HCE) at basal conditions and after stimulation with inflammatory cytokines (TNFα, TGFβ, IL-10, IL-13, IL-17, IL-6), using real time RT-PCR, Western blotting and immunofluorescence. Actin cytoskeleton staining was performed after all stimulations. Transepithelial electrical resistance (TER) and fluorescein transepithelial permeability (TEP) were measured as barrier integrity functional assays.


RESULTS
ZO-1, ZO-2, CLDN-1, CLDN-2, OCLN and E-cad were detected in HCE cell membranes at basal conditions. Cytokine stimulation resulted in significant changes in the expression of TJ and AJ proteins, both at mRNA and protein level, a remarkable change in their localization pattern, as well as a reorganization of actin cytoskeleton. Pro-inflammatory cytokines TNFα, TGFβ, IL-13, IL-17 and IL-6 induced a structural and functional disruption of the epithelial barrier, while IL-10 showed a barrier protective effect.


CONCLUSIONS
Simulated inflammatory conditions lead to an alteration of corneal barrier integrity by modulating TJ, and to a lesser extent also AJ, protein composition, at least In Vitro. The observed barrier protective effects of IL-10 support its well-known anti-inflammatory functions and highlight a potential therapeutic perspective.
Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain.
Laura
L
Contreras-Ruiz
Ute
U
Schulze
Laura
L
García-Posadas
Isabel
I
Arranz-Valsero
Antonio
A
López-García
Friedrich
F
Paulsen
Yolanda
Y
Diebold
D022005:Adherens Junctions / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002463:Cell Membrane Permeability; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007634:Keratitis / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000235:genetics* / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000096:biosynthesis / Q000235:genetics; D062827:Zonula Occludens-2 Protein / Q000096:biosynthesis / Q000235:genetics
D016207:Cytokines; C569042:TJP1 protein, human; C569046:TJP2 protein, human; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D012313:RNA