Abstract
Purpose: :
There is a constant rebuilding of photoreceptor cells, mainly through the shedding of the photoreceptor outer segment tips, followed by their phagocytosis in the retinal pigment epithelial (RPE) cells. During this process, there is a continuous protein turn over and replacement by RPE cells for maintaining homeostatic regulation. This study was undertaken to determine how the rate of phagocytosis of oxidized-rod outer segments (ox-POS) behave under daily sub-lethal oxidative stress and/or in combination with a lysosomal inhibitor in retinal pigment epithelial cells in culture.
Methods: :
Duplicate ARPE-19 cell cultures were seeded into 96 well plate and were left untreated or treated daily with a sub-lethal dose of 40uM H2O2 and/or in combination with 40uM leupeptin for 3 and 7 days following standard incubation protocols. Purified bovine POS were oxidized under UV irradiation (302 nm light source) for 16 hours and labeled using Rhodamine conjugation kit (Invitrogen). Rhodamine-labeled ox-POS (1x106/ml) were fed to the cultures (both treated and untreated) for a period of 5 hours and continuous phagocytosis was assessed every 20 min using live cell confocal imaging system (BD pathway 855). Also, fluorescence was measured at time 0 and time 5 hrs in a fluorometer plate reader at the excitation filter of 530/25 nm and emission filter of 590/35 nm after careful washing to read only the phagocytosed ox-POS.
Results: :
We found that there was a decrease in the rate of phagocytosis at the end of 3 days of stress both in H2O2 group and in combination group that received H2O2 and leupeptin when compared to the untreated groups. However, at the end of 7 days of oxidative stress treatment we found that in both the treatment groups there was an increase in the rate of phagocytosis with time following the order of H2O2+ leupeptin > H2O2 > control. The linear slope for the rate of phagocytosis per cell was 12.11 for combination group, 10.43 for H2O2 and 5.48 for the control group (mean average from 3 experiments). Additionally, we noted that there was a delay in the ingestion of ox-POS in the control group where as the process was faster in both the oxidant-induced groups after 7 days of exposure to oxidative stress.
Conclusions: :
Our studies conclude that RPE cells respond differentially to a single stress or a combination of stressors with respect to phagocytosis. This synergistical effect of oxidative stress may contribute to the formation of lipofuscin in the RPE cells which may have direct implications in ageing and age-related macular degeneration.
Keywords: retinal pigment epithelium • phagocytosis and killing • oxidation/oxidative or free radical damage