Phagocytosis by retinal pigment epithelium explants in culture (original) (raw)
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Phagocytosis in the light-damaged albino rat eye: Light and electron microscopic study
American Journal of Anatomy, 1974
Retinal photoreceptor degeneration was induced by exposing albino rats to fluorescent illumination at elevated environmental temperatures. Fine carbon particles were injected intravenously or directly into the vitreous body or anterior chamber of the eye. The resulting pattern of invasion, migration, and egression of carbon-filled phagocytes in eyes with degenerated retinas was reconstructed from a time sequence series of light and electron microscopic tissue sections. Retinal debris, such as damaged photoreceptor outer segments and carbon particles, was most frequently removed by two populations of cells possessing phagocytic properties: mononuclear cells of vascular origin and pigment epithelial cells. After retinal damage, mononuclear cells appeared first in the vitreous body and later, in time sequence, progressively deeper in the inner plexiform layer and out to the bipolar nuclear layer, where they were seen within, or partially within, retinal capillaries. After intravenous carbon injection, however, marked phagocytes were not seen in the retina. Carbon-filled phagocytic cells were observed in the choroidal connective tissue and blood vessels after intravenous injection, but not after intravitreal injections of carbon. Therefore, retinal phagocytes did not appear to leave the eye through the choroidal circulation. Pigment epithelial cells proliferated by mitotic activity, occurred as single cells separated from Bruch's membrane, and were seen among the degenerated outer segments. After direct exposure to carbon particles, pigment cell phagosomes contained both carbon and lamellated discs of degenerated outer segments. Whether these cells exited from the eye through retinal capillaries or returned to Bruch's membrane to reestablish continuity in the pigment epithelium could not be determined. Exposure of rats and other mammals to high fluorescent illuminance for short periods causes photoreceptor damage (Noell et al., '66; Gorn and Kuwabara, '67). Photoreceptor degeneration can also be induced by longer, continuous exposure of albino rats to fluorescent illuminance at levels commonly found i n laboratories and animal rooms (OSteen, '70; OSteen and Anderson, '71). The rate and degree of photoreceptor damage depends on the intensity of the illuminance source, the environmental and body temperature of the animal, and the exposure time (Noell et al., '66; Hansson, '70; OSteen and Anderson, '72). The resulting massive volume of damaged photoreceptor cells disappears from the retina in a short time after exposure, but Kuwabara and Gorn ('68), and Grignolo et al., ('69), observed
Pharmacology and Toxicology, 2001
The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 mM), toremifene (1-20 mM) and chloroquine (1-1000 mM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC 50 7.2 mM for D407 cells and 3.6 mM for pig retinal pigment epithelial cells) and toremifene (EC 50 6.2 mM and 3.1 mM respectively) than to chloroquine (EC 50 77.2 mM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.
Biology of the Cell, 1992
i Summary -The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiog.raphy of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.
Experimental Eye Research, 1992
Direct and indirect radioactivity and fluorescent assays have been developed to study the interaction of rod outer segments (ROS) with retinal pigment epithelial (RPE) cells. In the direct assays ROS labelled with izsI or fluorescein isothiocyanate (FIX) have been used to measure total phagocytosis, i.e. surface binding and ingestion. In the indirect assays RPE cells were first treated with unlabelled ROS or biotinylated ROS and subsequently probed with [lz51]Rho 4D2 antirhodopsin antibody or [1251]streptavidin for radioactivity measurements or with the Rho 4D2 antibody and FIX-goat anti-mouse Ig or FITCstreptavidin for fluorescent counting. In these indirect methods the number of surface bound ROS were distinguished from the number of ingested ROS by comparative labelling of non-permeabillzed and permeabilized ROS-treated RPE cells. Using these assays, we have studied the binding and ingestion of bovine ROS with cultured bovine RPE cells. As in the case of newborn cultured rat RPE cells [Hall and Abrams (1987) Exp. Eye Res. 45.907-221, binding and ingestion of bovine ROS by bovine RPE cells was saturable with respect to ROS concentration and time. At 37 "C ROS binding reached a saturating concentration at 1 x lo7 ROS per well: the number of bovine ROS ingested by bovine RPE cells, however, was less than the number of rat ROS ingested by rat RPE cells. When 1 x lo7 ROS per well was used, maximal surface binding of bovine ROS to bovine RPE cells was obtained after 2-3 hr, whereas after an initial delay, ingestion rapidly increased to a maximum at l-2 hr. In contrast to cultured newborn rat RPE cells, however, little, if any binding of ROS to bovine RPE cells was observed at 23 "C or 17 "C. Using direct binding assays and competitive inhibition assays, we have also shown that bovine RPE cells recognize rat ROS to the same degree as bovine ROS, and rat RPE cells bind bovine ROS in addition to rat ROS. This suggests that the ROS ligand and RPE receptor have conserved binding domains for these mammalian species. The assays described here should prove useful for future studies on the identification and characterization of components involved in ROS-RPE cell interaction during phagocytosis.
British Journal of Ophthalmology, 2002
Aim: To investigate the capability of retinal pigment epithelium (RPE) cells to phagocytose T lymphocytes and to further analyse the immunobiological consequences of this phagocytosis. Methods: Human RPE cells pretreated or not by cytochalasin, a phagocytosis inhibitor, were co-cultured with T lymphocytes for different time points. Phagocytosis was investigated by optic microscopy, electron microscopy, and flow cytometry. T cell proliferation was measured by 3 H thymidine incorporation. RPE interleukin 1β mRNA expression was quantified by real time PCR. Results: RPE cells phagocytose apoptotic and non-apoptotic T lymphocytes, in a time dependent manner. This is an active process mediated through actin polymerisation, blocked by cytochalasin E treatment. Inhibition of RPE cell phagocytosis capabilities within RPE-T cell co-cultures led to an increase of lectin induced T cell proliferation and an upregulation of interleukin 1β mRNA expression in RPE cells. Conclusions: It is postulated that T lymphocyte phagocytosis by RPE cells might, by decreasing the total number of T lymphocytes, removing apoptotic lymphocytes, and downregulating the expression of IL-1β, participate in vivo in the induction and maintenance of the immune privilege of the eye, preventing the development of intraocular inflammation.
Scientific Reports
retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the Rpe of the RcS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. in both dystrophic strains, the Rpe cells were pleomorphic and polymegathic.
Journal of Biological Chemistry, 1996
Expression of early response genes during rod outer segment phagocytosis by normal Long Evans and Royal College of Surgeons-rdy ؉ p ؉ rats and by dystrophic Royal College of Surgeons-p ؉ rat retinal pigment epithelial cells was studied in primary cell culture. Northern analysis revealed that the abundance of zif-268 (egr-1), c-fos, and tis-1 (NGF1-B) mRNA was rapidly and transiently increased in normal retinal pigment epithelial cells during rod outer segment phagocytosis but not during phagocytosis of latex particles. No increase in gene expression was found in Royal College of Surgeons-p ؉ dystrophic retinal pigment epithelial cells challenged with rod outer segments. As shown by electrophoretic mobility shift assay, a prominent short term increase in the intensity of the gel-shifted band was detected using nuclear protein extracts derived from rod outer segment-challenged, control retinal pigment epithelial cells and zif-268, AP-1, AP-2, or tis-1 consensus oligonucleotides. No such increase was detected when using nuclear factor B consensus oligonucleotide or when the early response gene prostaglandin H synthase-2 mRNA was measured over the time course studied. The results suggest that in retinal pigment epithelial cells, rod outer segment-specific phagocytosis is accompanied by the selective expression of early response genes coding for transcription factors. The specific pattern of the induction of these transcription factors is predicted to modulate the expression of gene cascades.
Reactive Changes in the Human Retinal Pigment Epithelium in Vitro
Acta Ophthalmologica, 2009
Explants from the retinal pigment epithelium and the underlying choroid and sclera were dissected from human eyes and transfered to culture wells. The mechanical trauma caused by the dissection and removal of the explants, and the changes in biological milieu caused by transfer of the tissue to an in vitro system causes injury, necrosis and detachment of cells from Bruchs membrane. In the retinal pigment epithelium, cells adjacent to damaged, spherical and detaching cells and smaller cell free zones form rosettes. At the periphery of big defects, the cells spread out to cover the denuded areas of Bruch's membrane. The present work has shown that cell injury in the human retinal pigment epithelium is followed by reactive cellular changes in vitro. The result of these reactive changes are increased variation in cellular form and magnitude and in pigment concentration per unit area.