Abstract

SO.04.07

Membrane-associated mucins are regulated by inflammatory mediators on ocular surface epithelial cells

Kakkassery V.^1,2, Spurr-Michaud S.², Perez B.², Blalock T.², Gipson I. K.^2
1Zentrum für Augenheilkunde, Universitätsklinikum Essen, ²Schepens Eye Research Institute, Harvard Medical School, Boston

Objective: Membrane associated mucins are altered on the ocular surface of non-Sjögren’s dry eye patients. Objective of this study was to evaluate the effect of inflammatory mediators present in tears of dry eye patients on ocular membrane-associated mucins (MAM) at the level of gene expression, protein biosynthesis or shedding using an immortalized corneal epithelial cell line (HCLE).
Methods: Human corneal limbal epithelial cell line (HCLE) cells were grown to confluence. Receptor mRNA of these mediators were verified in HCLE cells by reverse transcriptional polymerase chain reaction. To induce differentiation and stratification and mucin expression, cells were cultured for seven days in serum containing media. After serum starvation for 24h, cell were treated with interleukin (IL)-1a, 6 and 8, and tumor necrosis factor-a (TNF-a) for one, six as 24 hours. Effect of the mediators on membrane associated mucins (MUC1 and MUC16) gene transcription was evaluated by real-time polymerase chain reaction using TaqMan primers. Effects on protein translation and shedding of the mucins were detected by the immunoblot amount analysis of MUC1 and MUC16 in cell lysates and culture media.
Results: Expression of Interleukin 1 receptor I, Interleukin 6 receptor, Glycoprotein 130, Tumor necrosis factor receptor I and II in HCLE was demonstrated by conventional RT-PCR. No significant alterations in MUC1 and MUC16 gene transcription or protein translation were seen after treatment cells with IL-1a, IL-6 IL-8, or TNF-a in HCLE. After 24 hours of treatment, MUC1 protein shedding was significantly increased after treatment with IL-6, and MUC1 and MUC16 protein shedding after treatment with TNF-a.
Conclusions: The shedding rate of MUC1 and MUC16 is regulated by IL-6 and TNF-a in HCLE cells. These results suggest a mechanism for the decreased H185-epitope (MUC16) amount in non-Sjögren’s dry eye patients.

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