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P. Argueso, F. Mantelli, Z. Cao, I. K. Gipson, N. Panjwani; An Apical Epithelial Barrier at the Ocular Surface Is Formed by the Association of Cell Surface Mucins With Galectin-3. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2350.
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We have previously shown that cell surface mucins on apical ocular surface cells act as counter-receptors for galectin-3, a ß-galactoside-binding protein that may participate in the stabilization of mucosal barriers by interacting with carbohydrates on the cell surface glycocalyx. The purpose of this study was to determine whether inhibition of galectin-3 association with mucins results in uptake of rose bengal, a diagnostic dye clinically associated with ocular surface pathology.
Human corneal-limbal epithelial (HCLE) cells were grown in conditions that induce stratification and mucin biosynthesis. Cells were then incubated with 0.1M ß-lactose (Galβ1-4Glc), a competitive inhibitor of galectin-3 binding. Controls were disaccharides (sucrose [Glcα1-2Fruc] and maltose [Glcα1-4Glc]) with no affinity for galectin-3. Barrier function was assayed by incubation of cultures with 0.1% rose bengal, and dye uptake quantified using Spot Advanced software. Biotinytated galectin-3 at the cell surface and galectin-3 in culture media were detected by Western blot using a rat anti-human galectin-3 antibody and quantified using densitometry. Rose bengal uptake was also evaluated on C57BL/6 mouse epithelial cell surfaces (cornea, oral mucosa, esophagus, colon, vagina).
Induction of mucin biosynthesis in HCLE cells resulted in protection against rose bengal uptake. Competitive inhibition of galectin-3 binding with ß-lactose resulted in increased areas of rose bengal uptake as compared to sucrose and maltose (p<0.05). By immunoblot, cell surface galectin-3 significantly decreased after addition of ß-lactose, concomitant to an increase in culture media, indicating that galectin-3 was released from the glycocalyx. By comparison, no changes in galectin-3 levels were detected in the cell surface or culture media in cells treated with control sugars. In mice, no rose bengal staining was observed on the mucosal epithelial cell surfaces tested. When mouse corneas were treated with ß-lactose, diffuse rose bengal staining was observed throughout the epithelium, as compared to sucrose and maltose.
These results indicate that association of galectin-3 with cell surface mucins contributes to the formation of a protective barrier that prevents rose bengal uptake. Alteration of this association in patients with ocular surface disease may result in disruption of the epithelial barrier and deterioration of tear film stability.
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