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R. Kannan, H.J. Gukasyan, V.H. Lee, K.J. Kim; Interferon (IFN/ß/) Isozymes Stimulate GSH Efflux and -Glutamyltranspeptidase Expression in Primary Cultured Rabbit Conjunctival Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3459.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously reported that the conjunctival epithelium secretes GSH and viral (adenovirus type 5) infection significantly decreases intracellular GSH level and its secretion (ARVO 2002). In this study, we investigated the effects of IFNα/ß/γ on GSH efflux and γ-glutamyltranspeptidase (GGT) protein expression in primary cultured pigmented rabbit conjunctival epithelial cells (RCEC). Methods: RCEC were grown on permeable filters (ClearwellsTM) for 7 days. Levels of total cellular and extracellular fluid GSH were measured by an ELISA assay kit (Oxford Biomedical Research, Oxford, MI). GSH efflux was measured in NaCl buffer, after 30 min pretreatment with 1 mM acivicin. GSH levels and efflux were also measured with or without 24 hr exposure of RCEC layers to purified IFNα/ß/γ [(10-1000)/(10-2000)/(10-1000) IU/mL, respectively]. Effect of IFN treatment on the expression level of GGT, the ectoenzyme of GSH degradation, was determined by western blot analysis. Results: Apical GSH efflux after maximal IFNß treatment was more than 2 fold higher than that in untreated RCEC layers (300 vs. 130 pmol/min/106 cells, respectively). At the highest concentrations of IFNα and IFNγ, apical GSH efflux marginally increased from the initial 130 to 150 and 170 pmol/min/106 cells, respectively. Basolateral GSH efflux did not change in any of the treatments. GGT was increased up to an order of magnitude, with maximal levels seen after IFNß treatment. The effect was smaller, in decreasing order, by IFNγ and IFNα treatment. Conclusions: Interferon isozymes appear to regulate the apical efflux of GSH in RCEC layers. At IFN levels that significantly stimulate GSH efflux, the expression of GGT was proportionally higher. These findings suggest that initial cytokine stimulated GSH efflux may be responsible for subsequent depletion of GSH stores and impairment of GSH secretion in ocular Ad5 infection.
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