971 INTRODUCTION The ocular surface is a mucosal structure directly exposed to a great variety of environmental agents, some of them noxious, such as pathogens, allergens or irritants. In this structure the corneal epithelium forms a barrier that not only regulates the passive movement of molecules through the paracellular pathway, but also prevents foreign material from entering the eye. 1 An altered corneal epithelium result in a vulnerable cornea 2 and it is frequently associated with an increased risk of sterile or infectious corneal ulceration or persistent epithelial defects, for instance. An intact corneal epithelium is therefore essential to maintain ocular surface homeostasis, and intercellular junctions, such as tight junctions (TJs) and adherens junctions (AJs), play a key role in the formation and maintenance of this epithelial barrier. TJs are cell–cell junctions that seal adjacent cells together, preventing the passage of most solute molecules from one side of the epithelial layer to the other. They form a complex structure that mainly consists of transmembrane proteins, such as claudins and occludin, as well as cytoplasmic proteins, such as the zonula occludens protein family. 3–5 Their molecular composition plays a major role in the Current Eye Research, 37(11), 971–981, 2012 © 2012 Informa Healthcare USA, Inc. ISSN: 0271-3683 print/1460-2202 online DOI: 10.3109/02713683.2012.700756 Received 02 April 2012; revised 23 May 2012; accepted 03 June 2012 Correspondence: Yolanda Diebold, Ph.D. IOBA-University of Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, E-47011 Valladolid (Spain). Tel: +34–983-18 47 50. Fax: +34–983-18 47 62. E-mail: yol@ioba.med.uva.es ORIGINAL ARTICLE Structural and Functional Alteration of Corneal Epithelial Barrier Under Inflammatory Conditions Laura Contreras-Ruiz 1,2 , Ute Schulze 3 , Laura García-Posadas 1,2 , Isabel Arranz-Valsero 1,2 , Antonio López-García 1,2 , Friedrich Paulsen 3,4 , and Yolanda Diebold 1,2 1 Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain, 2 Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain, 3 Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany, and 4 Department of Anatomy II, Friedrich Alexander University Erlangen-Nürnberg, Germany 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 Curr Eye Res Downloaded from informahealthcare.com by Universitaetsbibliothek Erlangen-Nuernberg on 10/02/12 For personal use only.