Morphological observation on cell death and phagocytosis induced by ultraviolet irradiation in a cultured human lens epithelial cell line

The purpose of this study is to observe dynamic morphological changes induc ed by ultraviolet (UV) irradiation in a cultured human lens epithelial cell line using electron microscopy, cell viability staining, time-lapsed video graphy and immunohistochemistry. Human lens epithelial cell line SRA 01-04 was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20% feta l bovine serum. Subconfluent cells were irradiated under a bank of UV lamps , which emitted 275-400 nm radiation with a maximum at 310 nm. The UV inten sity was 20 muW cm(-2) at dosages from 0 to 10 mJ cm(-2) Alterations in the morphology of the living cells were monitored and recorded with phase-cont rast microscopy and time-lapsed videography. At different times, the cells were fixed and examined by transmission electron microscopy (TEM), diamidin ophenolindole (DAPI) staining, and in situ immunohistochemistry using TdT-m ediated dUTP-biotin nick end labeling (TUNEL). Cell viability was also asse ssed with crystal violet staining. At low doses of UV exposure (2-5 mJ cm(- 2)), time-lapsed videography revealed definitive cell death that appeared t o be primarily apoptotic. The dead cell debris was engulfed and phagocytose d by neighboring living cells. Phase-contrast microscopy and TEM demonstrat ed that, at UV 10 mJ cm(-2), the cells not only showed typical apoptosis su ch as nuclear membrane shrinkage, chromatin condensation, and fragmentation into apoptotic bodies, but also necrosis such as swelling of the nucleus a nd cell body and disruption of the plasma membrane. In support. DNA stainin g and in situ immunohistochemical reactions in the UV irradiated cells were both positive. The phagocytotic process was also seen with TEM. UV irradia tion thus appears to cause both apoptosis and necrosis in the cultured huma n lens epithelial cell line. Active migration and phagocytosis of the cells appear to be stimulated by UV-induced damage. These findings may also aid in the understanding of UV injury and repair mechanisms of lens epithelial cells in vivo. (C) 2000 Academic Press.