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George Inana, Chris Murat, Weijun An, Ian Harris, Jing Cao; Assessment of Phagocytic Function of Human Retinal Pigment Epithelium in Vitro. Invest. Ophthalmol. Vis. Sci. 2016;57(12):235.
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© ARVO (1962-2015); The Authors (2016-present)
The diurnally regulated phagocytosis of the outer tips of photoreceptors that occur every morning is a key function of the retinal pigment epithelium (RPE) in sustaining the health of the light-sensing photoreceptors. Its importance is illustrated in the well-known animal model of retinal degeneration, the Royal College of Surgeons rat, in which a defect in the RPE phagocytic function leads to retinal degeneration. In fact, the genetic defect (mutation in the Mertk gene) causing this defect has also been found in some humans with retinitis pigmentosa. This brings up the possibility that a defect in the RPE phagocytic function may play a role in other retinal degenerative conditions, including age-related macular degeneration (AMD), especially since AMD is characterized by the accumulation of abnormal intra- and extracellular debris (lipofuscin, drusen) which have been shown to be composed of material from the photoreceptors which ended up in the RPE via phagocytosis. With the goal of studying the status of phagocytic function in normal and AMD human RPE, we first set up an in vitro phagocytosis assay system for the normal human RPE and carried out an initial assessment of the phagocytic function.
Harvesting of human RPE, culture, isolation of rod outer segments (ROS) and labeling with fluorescein, and phagocytosis assay were mostly based on the methods used for rat RPE phagocytosis assay that we have published (McLaren, Inana, Li IOVS 1993). RPE was obtained from 8 post-mortem human eyes. ROS was prepared from human, rat, and pig retinas.
Feeding of fluorescently labeled ROS to human RPE cultured for at least 1 week demonstrated evidence of phagocytosis of the ROS, clearly distinguishable microscopically as ingested phagosomes, after ~8 hours of incubation as seen in our rat RPE phagocytosis assays. The kinetics of phagocytosis by the human RPE was also similar to that of the rat, with binding of the ROS first, then ingestion after 7-8 hours. The human RPE preferred the human ROS over the rat and pig for phagocytosis. A baseline of phagocytic level was established for the normal human RPE, and a moderate decrease in the level was observed with age (28 to 79yo).
Characteristics of ROS phagocytosis by the human RPE were similar to that for the rat, and the initial findings will be useful for the analysis of AMD eyes in the near future.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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