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Christophe Roubeix, Chloé Clouzeau, David Godefroy, William Rostène, Christophe Baudouin, Francoise Brignole-Baudouin; Apoptotic And Proinflammatory Effects Of Desiccative Stress On Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2328. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the effect of a desiccative stress (DS) on conjunctival epithelial cells in order to develop a new in vitro model of dry eye and to determine the impact of benzalkonium chloride (BAK), a commun preservative in eye drop, in such condition.
Desiccative stress was induced on Wong-Kilbourne-derived of Chang Conjunctiva (WKD) cell line by removing cell culture medium and cells were further incubated in a drying chamber (relative humidity: 17±3%). We compared the cytotoxic effects of a subtoxic BAK concentration (3.10-3%) for 30 min and a desiccative stress for 1 or 3h and of the combinations the two stresses using spectrofluorimetry techniques for cell viability and apoptosis (Neutral red, YO-PRO-1, Hoechst33342). An assessment of cytokine production was also performed after a recovery time of 1h in the supernatants and in the cell lysates (Proteome Profiler array, R&D systems). Expressions of proinflammatory mediators such as IL-6, MCP-1 or MIF, as well as membrane proteins as ICAM-1 and chemokines receptors, CCR-2 or CXCR-4 were confirmed using immunofluorescence, western-blot and quantitative PCR.
: DS induced a slight decrease of cell viability and a moderate increase of apoptosis in a time dependant manner and sensitized the WKD to the proapoptotic effects of BAK. During desiccation, the WKD acquired a proinflammatory phenotype expressing high levels of inflammatory factors IL-6, MCP-1, MIF as well as ICAM-1, CCR-2 or CXCR-4.
This model of DS reproduces apoptotic and proinflammatory cell response, which are known to be induced in conjunctival epithelial cells during dry eye disease. These results also highlight the increased cytotoxic effects of BAK combined with DS. Furthermore, this cellular model offers the possibility to better understand the inflammatory mechanisms occurring during dry eye, to evaluate anti-inflammatory strategies and to investigate new therapeutic targets.
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