Abstract
Purpose: :
To determine how photodynamic damage to ARPE–19 cells affects their phagocytic activity.
Methods: :
ARPE–19 cells were subjected to sub–lethal doses of photodynamic treatment by irradiating cells with green light in PBS for 30 min. in the presence of various concentrations of merocyanine 540 or Rose Bengal. The survival of cells was determined by standard cellular techniques such as MTT reduction assay, trypan blue exclusion method and the release of LDH. In all experiments, the cell survival was in the range of 90–100%. After exposure to FITC–labeled latex beads or bovine ROS, cells were thoroughly washed with PBS, trypsinized, re–suspended in PBS and examined by flow cytometry. To determine whether the photodynamic treatment resulted in a measurable oxidation of the cell lipids, chloroform–methanol extracts of the control and treated cells were examined by HPLC–EC(Hg) set for detecting characteristic cholesterol hydroperoxides.
Results: :
The photodynamic treatment of ARPE–19 cells induced a dose–dependent inhibition of both their specific and non–specific phagocytic activity. The extent of the inhibition of the specific phagocytic activity was significantly higher than that of the non–specific phagocytic activity. At the cell survival close to 100%, the photodynamic treatment resulted in 40–60% inhibition of the phagocytosis. Our preliminary results indicate that the photodynamic treatment of ARPE–19 cells was accompanied by a measurable formation of singlet oxygen– and free radical–dependent cholesterol hydroperoxides.
Conclusions: :
Photodynamic damage to ARPE–19 cells is accompanied by a distinct inhibition of the cell phagocytic activity. Our data suggest that the degree of phagocytosis inhibition can be correlated with the extent of the cell lipid peroxidation. Therefore, it can not be ruled out that age–related increase in the membrane lipid peroxidation contributes to the reduction of the cell phagocytic activity.
Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium