Lyar is a New Ligand for Retinal Pigment Epithelial Phagocytosis (original) (raw)
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The Journal of biological chemistry, 2017
One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: human-derived RPE-like cell line, ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterize...
Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture
Methods in Molecular Biology, 2019
Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we provide experimental protocols to synchronize the POS binding and engulfment steps of phagocytosis. Finally, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.
Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture
Experimental Eye Research, 2014
retinal pigment epithelium phagocytosis signaling photoreceptor outer segment renewal engulfment a b s t r a c t RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.
Molecular …, 2009
PurposeTo examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.MethodsHESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel™ matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT–PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology.ResultsHESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures.ConclusionsThe ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.
Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation
The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye. Keywords: Alpha-Melanocyte Stimulating Hormone; Immune Privilege; Macrophages; Neuroimmunomodulation; Neuropeptide Y; Phagocytosis.
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.
Proceedings of the National Academy of Sciences, 1997
Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires ␣v5 integrin, rather than ␣v3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both ␣v3 and ␣v5 integrins, only ␣v3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of ␣v5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, ␣v5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas ␣v3 was expressed basolaterally. Using quantitative f luorimaging to assess in vitro uptake of f luorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, ␣v5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for ␣v5 in phagocytosis, immunof luorescence experiments demonstrated codistribution of ␣v5 integrin with internalized ROS. Control experiments showed that blocking ␣v3 function with antibodies did not inhibit ROS phagocytosis and that ␣v3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor ␣v5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-␣v5 complex. ␣v5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.
Proceedings of the National Academy of Sciences
Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires ␣v5 integrin, rather than ␣v3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both ␣v3 and ␣v5 integrins, only ␣v3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of ␣v5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, ␣v5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas ␣v3 was expressed basolaterally. Using quantitative f luorimaging to assess in vitro uptake of f luorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, ␣v5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for ␣v5 in phagocytosis, immunof luorescence experiments demonstrated codistribution of ␣v5 integrin with internalized ROS. Control experiments showed that blocking ␣v3 function with antibodies did not inhibit ROS phagocytosis and that ␣v3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor ␣v5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-␣v5 complex. ␣v5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.