Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro

Abstract

Tear hyperosmolarity is known to cause ocular surface inflammation in dry eye syndrome. Benzalkonium chloride (BAK), an eyedrop preservative, is known to induce dry eye in long-term-treated patients. Analyzing the modulation of the proinflammatory potential of hyperosmolarity in the presence of BAK on the conjunctiva could give new insights into the effect of this preservative on the disease. In a hyperosmolar model on a conjunctiva-derived cell line, and in the presence of BAK, we evaluated key inflammatory markers [CCL2, IL-8, IL-6, macrophage migration inhibitory factor (MIF) and intercellular adhesion molecule (ICAM)-1] as well as the osmoprotectant element nuclear factor of activated T cells (NFAT)5 using ELISA, RT-qPCR or immunofluorescence staining. Hyperosmolarity highly stimulated CCL2 and NFAT5 in these cells. BAK alone only increased IL-6 expression. The stress-combined condition stimulated CCL2, NFAT5, MIF and IL-8 secretion. ICAM-1 was not modulated by any of the conditions tested. In this model, hyperosmolarity and BAK induced the release of different proinflammatory mediators, and, when combined, they lead to the release of additional inflammatory cytokines. This in vitro study highlights the importance of avoiding long-term ophthalmic treatments containing BAK, as tear film hyperosmolarity can be a result of its detergent action.