Cytoprotective Effect of Lacritin on Human Corneal Epithelial Cells Exposed to Benzalkonium ChlorideIn Vitro

Abstract

Purpose Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the presence of lacritin confers protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK. Materials and Methods Recombinant human lacritin was cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established using the MTT assay after exposure of subconfluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK. Results BAK reduced CRL-11515 cellular metabolic activity in a time and dose dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (P < 0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 hours prior to BAK exposure significantly dampened levels of LC3-II (P < 0.05) and promoted a significant increase in cellular metabolic activity (P < 0.01) compared to BAK alone. Conclusions These results suggest lacritin protects cultured HCE cells stressed with BAK. Lacritin may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…LACRT (lacritin): A prosecretory mitogen primarily in tears and saliva that transiently accelerates autophagic flux in stressed cells. 1834 Lacritin targets heparanase-deglycanated SDC1 (syndecan 1) on the cell surface, 1835 and accelerates flux by stimulating the acetylation of FOXO3 as a novel ligand for ATG101 and by promoting the coupling of stress acetylated FOXO1 with ATG7. 1836 Laforin: See EPM2A.…”

Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles

“…3A). Downstream of Ca 2+ mediated signaling, 40 HCE-Ts are known to initiate more rapid motility and proliferation, 14, 41 which can be visualized during the closure of a scratch made on a confluent sheet of cells. To visualize the in vitro effect of LSI, we applied a scratch to a sheet of cells and captured the time-lapse healing process (Fig.…”

“…12 Lacritin is the most severely downregulated protein in contact lens related dry eye and is similarly deficient in blepharitis (a common inflammation of the eyelid). 13 Previous in vitro tests have shown that, on the ocular surface, lacritin triggers Ca 2+ wave propagation, 13 in addition to promoting the survival of primary and cultured human corneal epithelial cells stressed with interferon-γ, tumor necrosis factor 14 , and Benzalkonium chloride (BAK), 15 indicative of both mitogenic and cytoprotective activity. This cell targeting specificity is triggered by a unique ‘off-on’ switch controlled by heparanase deglycanation of the cell surface protein, syndecan-1, which exposes a lacritin binding site as a prerequisite for its downstream mitogenic signaling.…”

A Comparison of the Effects of Benzalkonium Chloride on Ocular Surfaces between C57BL/6 and BALB/c Mice

“…Furthermore, the prevalence of discomfort or corneal/conjunctival disruptions in patients was dose dependent, increasing with the amount of preserved eye drops administered [32]. For in vitro cell studies, the BAC levels in culture media were much less than those in the in vivo animal experiments and as low as 0.0003% in some studies [33,34]. In studies involving animal models in which dry eye and toxicity tests were frequently conducted, the concentration of BAC that was topically applied varied case by case from 0.01% to 0.5%, with lower concentrations corresponding to longer durations of observation [35,36].…”