LIPOFUSCIN ACCUMULATION IN CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS REDUCES THEIR PHAGOCYTIC CAPACITY

Purpose. Retinal pigment epithelial (RPE) cells slowly accumulate lipo fuscin pigment within their acidic vacuolar apparatus as a result of e xtra- and/or intralysosomal oxidative alterations of phagocytosed phot oreceptor outer segments (POS) with consequent imperfect degradation o f these structures. In old age, lipofuscin accumulation may become qui te substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age- related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosome s would affect the further phagocytic ability of the cells. Methods. I n the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin ) for 4 weeks, resulting in a pronounced lipofuscin accumulation of th e cells. Fluorescent latex beads (labelled with a red fluorophore) wer e added to unloaded control cultures at 0 and 4 weeks after their esta blishment, and to lipofuscin loaded cells after 4 weeks of feeding wit h artificial lipofuscin. Cellular amounts of lipofuscin, and their pha gocytotic activity, were quantified by static fluorometry measuring li pofuscin-specific and red bead-specific fluorescence, respectively. Th e intracellular location of the beads was verified by confocal laser s canning microscopy. Results. Unloaded, and thus almost lipofuscin-free , control cells exposed to latex beads showed numerous cytoplasmic par ticles emitting reddish fluorescence, while few particles were taken u p by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significant ly (p < 0.001) higher levels in unloaded control cells than in lipofus cin-loaded ones. In the second part of the investigation, unloaded con trol cultures and lipofuscin-loaded cultures were exposed to native bo vine Texas Red-X-labelled POS 4 weeks after the establishment of the c ultures. Unloaded control cells showed a large number of cytoplasmic P OS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red -X-specific fluorescence, thus quantifying the phagocytic ability of t he cells, showed significantly (p < 0.001) higher levels in control ce lls than in lipofuscin-loaded ones. Conclusions. Severe lipofuscin acc umulation of RPE cells appears to result in a greatly decreased phagoc ytic capacity. The resulting reduction in ability to cope with the nee ds of the overlying photoreceptor cells, in order to eliminate the obs olete tips of their POS, may well be of significance in the developmen t of age-related macular degeneration.