Finnemann, SC, Bonilha, VL, Marmorstein, AD and Rodriguez-Boulan, E. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization. Proc Natl Acad Sci USA 94: 12932-12937 (original) (raw)
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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.
Photoreceptor Cell Biology and Inherited Retinal Degenerations, 2004
Advances in genomics and molecular biology have led, over the last decade, to the identification of numerous molecules that form the phagocytic machinery of the retinal pigment epithelium (RPE). This research has also shown that the RPE phagocytic mechanism belongs to a group of related clearance mechanisms that share common phagocytic receptors. However, the precise roles of any of these receptors in the daily phagocytic burst characteristic of the RPE remain to be explored. Here, we focus on contributions of the integrin receptor αvβ5 to outer segment phagocytosis by RPE cells. In vitro functional assays and developmental expression studies indicate an important function for αvβ5 receptors in phagocytosis by human, rat, and mouse RPE. We discuss recent evidence that signaling pathways via αvβ5 integrin in both directions across the RPE plasma membrane may promote synchronized, timely, and repeated phagocytosis unique to RPE. αvβ5 integrin engagement initiates a downstream signaling response that may serve to coordinate the activity of multiple components of the RPE phagocytic machinery. Furthermore, regulation of activities of αvβ5 integrin receptors themselves by RPE signaling mechanisms may prevent phagocytosis by RPE cells upon completion of their daily clearance of shed photoreceptor outer segment fragments.
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.
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.
Two distinct cytoplasmic regions of the β2 integrin chain regulate RhoA function during phagocytosis
2006
α M β 2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. α M β 2 , Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during α M β 2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to α M β 2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of β 2 , but not of α M , abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the β 2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for β 2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not suffi cient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was suffi cient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.