Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity.
OBJECTIVE
Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite 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 lysosomes would affect the further phagocytic ability of the cells.


METHODS
In 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 the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at O and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy.


RESULTS
Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones. In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS 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 the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones.


CONCLUSIONS
Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.
Sundelin
S
S
Department of Ophthalmology, Linköping University, Sweden.
Wihlmark
U
U
Nilsson
S E
SE
Brunk
U T
UT
D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D008062:Lipofuscin / Q000378:metabolism*; D018613:Microscopy, Confocal; D008863:Microspheres; D010587:Phagocytosis / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D011817:Rabbits; D012374:Rod Cell Outer Segment / Q000502:physiology; D014966:Xanthenes
D005456:Fluorescent Dyes; D008062:Lipofuscin; D014966:Xanthenes; C034657:Texas red