HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (11) Citing Articles via Google Scholar Google Scholar Articles by Argüeso, P. Articles by Gipson, I. K. Search for Related Content PubMed PubMed Citation Articles by Argüeso, P. Articles by Gipson, I. K.

Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.

PURPOSE. Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins.

METHODS. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca⁺² and Mg⁺²) in the rose bengal uptake assay.

RESULTS. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake.

CONCLUSIONS. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

This article has been cited by other articles:

				T. D. Blalock, S. J. Spurr-Michaud, A. S. Tisdale, and I. K. Gipson Release of Membrane-Associated Mucins from Ocular Surface Epithelia Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1864 - 1871. [Abstract] [Full Text] [PDF]

				M. Sumiyoshi, J. Ricciuto, A. Tisdale, I. K. Gipson, F. Mantelli, and P. Argueso Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 197 - 203. [Abstract] [Full Text] [PDF]

				T. D. Blalock, S. J. Spurr-Michaud, A. S. Tisdale, S. R. Heimer, M. S. Gilmore, V. Ramesh, and I. K. Gipson Functions of MUC16 in Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4509 - 4518. [Abstract] [Full Text] [PDF]