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

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  < 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  ≥ 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  ≤ 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  ≥ 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.