Structural and Functional Alteration of Corneal Epithelial Barrier Under Inflammatory Conditions

Abstract

Purpose: 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.

Materials and 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.

Conclusion: 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.

KEYWORDS::

• Corneal epithelium
• Corneal barrier
• Inflammation
• Cytokines
• Tight junctions

ACKNOWLEDGMENTS

We thank Itziar Fernández, M.Sc. for statistical analysis advice, Susan Möschter for technical assistance, and the confocal microscopy service from the University of Valladolid, especially Sagrario Callejo for her technical support.

Declaration of interest: This study was supported by grants from the Ministry of Education and Science, Spain (FEDER-CICYT MAT2007–64626-C02-01), also by FPU-MEC, JCyL-Regional, and FPI-University of Valladolid Scholarships Programs, Spain, by DFG grant PA738/9-2 and BMBF Roux program grants FKZ 9/18,12/08,13/08, Germany, and by Bilateral Research Grant Spain/Germany DE2009-0085 and DAAD ID6234017.