PURPOSE: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of human chymase on conjunctival epithelial cells in vitro. METHODS: Human conjunctival epithelial cells were incubated with human chymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. RESULTS: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. CONCLUSIONS: Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.
PURPOSE: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of humanchymase on conjunctival epithelial cells in vitro. METHODS:Human conjunctival epithelial cells were incubated with humanchymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. RESULTS: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. CONCLUSIONS:Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.