Phagocytosis of Human Retinal Pigment Epithelial Cells: Evidence of a Diurnal Rhythm, Involvement of the Cytoskeleton and Interference of Antiviral Drugs

Abstract

Retinal pigment epithelial (RPE) cells provide crucial functions for the maintenance of the retinal environment. We investigated the phagocytotic mechanisms of RPE cells evaluating the question whether particle uptake underlies a diurnal rhythm. Additionally, a possible connection of volume regulation and the phagocytotic function of RPE cells was studied. As antiviral nucleoside analogues influence cell-volume-regulating mechanisms, we tested several antiviral drugs. Cultured primary RPE cells and a permanent cell line (ARPE-19) were tested for uptake of europium-labeled microspheres quantified by time-resolved fluorometry. Cells were also exposed to cyclic illumination or continuous light and dark culture conditions. Inhibitors of cytoskeleton (microtubuli, actin) and osmotic swelling were also tested. Ingested FITC-labeled microparticles were found in phagosomes strongly associated which the cytoskeleton as they could not be easily moved by laser tweezer microscopy. Phagocytosis was observed predominately during dark intervals and was reduced by continuous light exposure. The diurnal rhythm of unsynchronized RPE cultures was abolished by microtubule inhibitors although no inhibition of overall particle uptake by cytoskeletal blockers was observed. Hypoosmotic swelling of RPE also decreased phagocytosis. Acyclovir was found inhibitory in ARPE-19 cells, whereas azidothymidine showed a protracted inhibiting activity on primary RPE cells and ganciclovir was inactive in both cell types. The presence of a diurnal rhythm also in culture indicates genetic determination of light-regulated particle uptake. This mechanism appears to be influenced by the regulation of cell volume and microtubule function. Inhibition of RPE function by antiviral drugs is a novel finding and in accordance with interferences of the tested drugs with cellular chloride channels described earlier. It may give a hint towards possible ocular side effects in the long-term use of nucleoside-analogous substances.

Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells

“…These include the receptor tyrosine kinase MerTK and its secreted ligands Gas6 and Protein S, as well as the integrin receptor α V β 5 and its secreted ligand MFG-E8 [ 36 ]. ARPE-19 cells are capable of phagocytosing photoreceptor outer segments [ 37 – 39 ], but some differences exist compared to primary cultures. Both require the integrin receptor α v β 5 for the binding and internalization of outer segments [ 21 , 37 ], the main difference being observed at the level of promoter strength, yielding much higher transcriptional activity in ARPE-19 [ 40 ].…”

Distinct Functions between Toll-Like Receptors 3 and 9 in Retinal Pigment Epithelial Cells

“…In addition to monocytes/macrophages, RPE cells are also 'professional' phagocytes [36] . Recently Blander and Medzhitov [37] demonstrated that activation of the TLR 2 and 4 signaling pathway by bacteria regulated phagocytosis at multiple steps including internalization and phagosome maturation.…”

Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion

“…The intracellular location of the beads was verified using scanning laser microscopy [21]. Also the cytoskeletal association of ingested vesicles was tested by optical tweezers [21].…”