Temporal Change in Pro-Inflammatory Cytokine Expression from Immortalized Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress

Abstract

Purpose To determine the metabolic activity, and cytokine expression over time from immortalized human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. Methods HCECs were cultured and expanded in DMEM/F-12 with 10% FBS. The cells were exposed to either normal media (295 mmol/kg) or hyperosmolar media (500 mmol/kg) for 0.25, 3, 6, and 12 hours. After each exposure duration, metabolic activity was quantified using alamarBlue, and a panel of pro-inflammatory cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α, IFN-γ, and IL-17A) was quantified using multiplexed electrochemiluminescence (Meso Scale Diagnostics, Rockville, MD). Results Metabolic activity of the HCEC exposed to hyperosmolar conditions was significantly reduced at the 3-, 6-, and 12-hour mark compared to the control (all p < 0.01). There was no significant difference in cytokine expression between the hyperosmolar media and control at the 0.25- and 3-hour mark for all cytokines (all p ≥ 0.28). The difference in cytokine expression between the hyperosmolar media and the control was significant for IL-1β, IL-4, IL-6, IL-8, IL-12p70, IL-13, and TNF-α at the 6-hour mark (all p ≤ 0.02). No significant change in cytokine expression between the hyperosmolar media and control was noted for IL-2, IL-10, IL-17A, and IFN-γ (all p ≥ 0.74) at the 6-hour mark. Conclusion Hyperosmolar stress reduced cell metabolic activity and increased expression of IL-1β, IL-4, IL6, IL8, IL-12p70, IL-13, and TNF-α over a 6-hour period in an immortalized HCEC line.