title: Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.
abstract: OBJECTIVE
  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.
authors.affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. argueso@vision.eri.harvard.edu
authors.forename: Pablo
authors.initials: P
authors.lastname: Argüeso
authors.forename: Ann
authors.initials: A
authors.lastname: Tisdale
authors.forename: Sandra
authors.initials: S
authors.lastname: Spurr-Michaud
authors.forename: Mika
authors.initials: M
authors.lastname: Sumiyoshi
authors.forename: Ilene K
authors.initials: IK
authors.lastname: Gipson
mesh_terms: D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000166:cytology; D005293:Ferritins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D015636:Magnesium Chloride / Q000494:pharmacology; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D018396:Mucin-1 / Q000378:metabolism*; D055264:Mucin-4; D009077:Mucins / Q000378:metabolism*; D018384:Oxidative Stress; D012395:Rose Bengal / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen; D005456:Fluorescent Dyes; C451759:MUC16 protein, human; C524658:MUC4 protein, human; D008565:Membrane Proteins; D018396:Mucin-1; D055264:Mucin-4; D009077:Mucins; C010297:polycationic ferritin; D015636:Magnesium Chloride; D012395:Rose Bengal; D005293:Ferritins; D002122:Calcium Chloride