Surface Of Retinal Pigment Epithelial Cells Research Articles

Membrane Attack Complex Mediates Retinal Pigment Epithelium Cell Death in Stargardt Macular Degeneration.

Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE cells derived via induced pluripotent stem-cell from a molecularly and clinically diagnosed STGD1 patient exhibited reduced ABCA4 protein and diminished activity compared to a normal subject. Consequently, STGD1 RPE cells accumulated intracellular autofluorescence-lipofuscin and displayed increased complement C3 activity. The level of C3 inversely correlated with the level of CD46, an early negative regulator of the complement cascade. Persistent complement dysregulation led to deposition of the membrane attack complex on the surface of RPE cells, decrease in transepithelial resistance, and subsequent cell death. These findings are strong evidence of complement-mediated RPE cell damage in STGD1, in the absence of photoreceptors, caused by reduced CD46 regulatory protein.

Pleiotropic Roles of Scavenger Receptors in Circadian Retinal Phagocytosis: A New Function for Lysosomal SR-B2/LIMP-2 at the RPE Cell Surface.

The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process.

Proteome-wide Identification of Glycosylation-dependent Interactors of Galectin-1 and Galectin-3 on Mesenchymal Retinal Pigment Epithelial (RPE) Cells

Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the β-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor β PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.

Integrin-α5 Mediates Epidermal Growth Factor-Induced Retinal Pigment Epithelial Cell Proliferation and Migration

Proliferation and migration of retinal pigment epithelial (RPE) cells play a crucial role in proliferative vitreoretinopathy (PVR)-related pathology. Cytokines, including EGF, can result in RPE cell activation and cause PVR. In this study, integrin-α₅ expression was first studied in PVR membranes by immunofluorescence. Then the effect of EGF on integrin-α₅ expression was determined by flow cytometry, Western blot analysis and the reverse-transcription polymerase chain reaction (RT-PCR) in the ARPE-19 cell line. Proliferation and migration of ARPE-19 cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide and Boyden chamber assays. We found that a higher level integrin-α₅ was present at the RPE cell surface in PVR compared with normal retina. EGF could dose dependently increase integrin-α₅ mRNA and protein levels in vitro. EGF promoted ARPE-19 cell proliferation and migration. Neutralizing integrin-α₅ by specific anti-integrin-α₅ antibody abolished most of the effects of EGF. The study provided evidence that EGF might influence PVR by promoting integrin-α₅ expression and subsequent proliferation and migration of RPE.

Role of Nectin-1, HVEM, and PILR-α in HSV-2 Entry into Human Retinal Pigment Epithelial Cells

Herpes simplex virus-type 2 (HSV-2) can cause acute retinal necrosis (ARN), which can lead to exudative and rhegmatogenous retinal detachment, yet little is known about the cellular and molecular mechanisms of HSV-2 entry into retinal pigment epithelial (RPE) cells. The goal of this study was to establish the identity of the critical receptors used by the virus for infection. A reporter HSV-2 virus, which expresses beta-galactosidase, was used to quantify entry into RPE cells, and viral replication was ascertained using a plaque assay. Flow cytometry and immunocytochemistry were used to determine cellular expression of entry receptors. Localization of these receptors to the apical or basal surface of RPE cells was determined with immunocytochemistry. The necessity of these receptors, individually and in combination, for viral entry was established using receptor-specific antibodies and siRNAs. RPE cells are highly susceptible to HSV-2 entry and replication. Several assays demonstrated the expression of the entry receptors nectin-1, HVEM, and PILR-alpha and their localization primarily to the apical surfaces of RPE cells. Receptor-specific antibodies and siRNA knockdown of receptors significantly reduced viral entry and implicated nectin-1 as an important receptor, with HVEM and PILR-alpha potentially also contributing to entry. HSV-2 is capable of developing a productive infection in RPE cells by using nectin-1 as an important entry receptor. To lesser degrees, HVEM and PILR-alpha may also contribute to HSV-2 entry into RPE cells.

Retinal Pigment Epithelium-Retinal G Protein Receptor-Opsin Mediates Light-dependent Translocation of All-trans-retinyl Esters for Synthesis of Visual Chromophore in Retinal Pigment Epithelial Cells

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.

Tetraspanin CD81 is required for the αvβ5-integrin-dependent particle-binding step of RPE phagocytosis

Retinal pigment epithelial (RPE) cells are among the most active phagocytes in the body. Every morning, circadian shedding of outer segment fragments by photoreceptor cells activates a synchronized phagocytic response by RPE cells that is critical for vision. RPE cells require alpha v beta5 integrin receptors for particle binding that triggers engulfment. Here, we show that tetraspanins CD81 and CD9 reside in a complex specifically with alpha v beta5 integrin but not the engulfment receptors Mer tyrosine kinase and CD36 at the apical, phagocytic surface of RPE cells. Function blocking and RNA silencing of CD81 but not of CD9 specifically diminish particle binding. CD81 but not CD9 overexpression is sufficient to increase particle binding and surface levels of alpha v beta5 integrin. Wild-type and mutant RPE cells defective in particle engulfment equally reduce and increase particle binding in response to CD81 inhibition and CD81 overexpression, respectively. By striking contrast, neither CD81 inhibition nor CD81 overexpression has any effect on particle binding by RPE lacking alpha v beta5 integrin. These results identify a novel and important role for CD81 in phagocytosis. CD81 does not function as a binding receptor by itself but promotes outer segment particle binding through functional interaction specifically with alpha v beta5 integrin.

Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling.

Integrity of retinal pigment epithelial cells is necessary for photoreceptor survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors and is assumed to be a target for lipid peroxidation during cell damage. We have previously shown, in contrast, that docosahexaenoic acid is also the precursor of neuroprotectin D1 (NPD1), which now we demonstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Furthermore, we show that neurotrophins, particularly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion. Moreover, docosahexaenoic acid (DHA) elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by pigment epithelium-derived factor and DHA uncovered synergistic cytoprotection with concomitant NPD1 synthesis when cells were challenged with oxidative stress. Also, DHA and pigment epithelium-derived factor synergistically modify the expression of Bcl-2 family members, activating antiapoptotic proteins and decreasing proapoptotic proteins, and by attenuating caspase 3 activation during oxidative stress. Thus, our findings demonstrate that DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, our results identify neurotrophins as regulators of NPD1 and of its polarized apical efflux from RPE cells. Taken together, these findings imply NPD1 may elicit autocrine actions on RPE cells and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix.

15-Deoxy-12,14-prostaglandin J2 inhibits interferon gamma induced MHC class II but not class I expression on ARPE cells through a PPAR gamma independent mechanism

Retinal pigment epithelial (RPE) cells constitute the external part of the blood–retinal-barrier and play a pivotal role in the regulation of retinal immunity. In the present work, we investigated the effects of 15-deoxy-12,14-prostaglandin J2 (15 PGJ2), an endogenous ligand of PPARγ, on the IFNγ-induced expression of MHC class II on RPE cells. Indeed, pathological expression of MHC class II molecules at the surface of RPE cells is a common feature of many blinding conditions. We demonstrated that 15 PGJ2 inhibited the IFNγ-mediated induction of MHC class II on RPE cells without affecting the level of MHC class I and CD54 expression. The other PPARγ agonist rosiglitazone or troglitazone had no similar effects. Moreover, the inhibitory effect of 15 PGJ2 was not abrogated by co-incubation with PPARγ antagonists and did not involve the modulation of STAT-1, AKT or ERK1/2 phosphorylation, nor CIITA, IRF1 or IRF2 transcription. In conclusion, 15 PGJ2 inhibits strongly and specifically the IFNγ-induced MHC class II expression on RPE cells by a PPARγ independent mechanism. Given the differential role of MHC classes I and II in the development of autoimmune uveitis and the potential toxicity of 15 PGJ2, our data's suggest that the development of novel small molecules targeting similar PPARγ independent pathways would be useful for the future management of uveitis.

Transforming Growth Factor Beta Induces Urokinase Receptor Expression in Cultured Retinal Pigment Epithelial Cells

The effect of transforming growth factor-β₁ (TGF-β₁) and interferon-γ (IFN-γ) was studied on urokinase receptor (uPAR) expression of cultured human retinal pigment epithelial (RPE) cells. Human RPE cells were incubated with 1, 5 or 10 ng/ml of TGF-β₁ or with 10, 100 or 1,000 IU/ml of IFN-γ to measure total cellular uPAR protein and released uPAR by enzyme immunoassay. uPAR at cell surface was measured by flow cytometric analysis at 8, 12, 24 and 48 h. uPAR mRNA levels were assayed by Northern blotting at 2, 6, 12 and 24 h. The increase in uPAR gene expression in RPE cells exposed to TGF-β₁ paralleled enhanced uPAR level at the cell surface and in conditioned medium. TGF-β appeared to induce also membrane-bound uPA activity and the release of active plasminogen activator inhibitor-1, indicating that TGF-β has the potential to regulate plasminogen activation at the RPE cell surface. The increase in uPAR gene expression by IFN-γ did not seem to translate into the protein level. We conclude that TGF-β regulates the pericellular proteolysis in RPE cells by increasing uPAR expression.

A Novel Retinal Pigment Epithelial Protein Suppresses Neutrophil Superoxide Generation. I. Characterization of the Suppressive Factor

In acute intraocular inflammation, neutrophils release a variety of agents, that are potentially toxic to the surrounding tissues. The reactive oxygen metabolites, including superoxide are among these injurious agents. In the present study, retinal pigment epithelial (RPE) cells were found to inhibit by 70% to 80% the production of superoxide by neutrophils that were stimulated either by the receptor-coupled activator, N-formyl-methionyl-leucyl-phenylalanine, or by the non-receptor-coupled activator, phorbol myristate acetate. The inhibition is effective with a relatively small number of RPE cells (0.65×10 5) mixed in a large pool of neutrophils (7.6×10 5). The transduction of this effect does not require a neutrophil–RPE cell surface contact since the RPE culture supernatants also effectively inhibit the superoxide production. This protein is secreted specifically by cultured and noncultivated intact RPE cells, but not by fibroblasts, corneal epithelial cells or intact choroidal tissues. The protein is not cytotoxic to the neutrophils, and the inhibitory effect occurs in a dose-dependent manner up to the concentration tested. This factor is unrelated to either transforming growth factor-β, or transferrin. There was no evidence of RPE scavenging of superoxide generated enzymatically by hypoxanthine-xanthine oxidase system, indicating that this factor does not have superoxide dismutase activity. When RPE cells were pre-incubated with 10 μg ml −1cycloheximide, 60% of the activity was lost, suggesting that a de novo protein synthesis is required for the activity and that the protein is a significant steady-state product of RPE cells. Incubation of the released RPP with thermolysin (10 μg ml −115 min, 37°C) also eliminated the activity. The degradation of activity by protease and inhibition of RPP activity by cycloheximide, therefore, confirmed the protein nature of the suppressive factor. This protein from RPE cells appears to act directly on the neutrophils, reducing the release of oxygen metabolites during activation, and thus limiting tissue injury during inflammation.

Müller cell changes precede vascularization of the pigment epithelium in the dystrophic rat retina

In the Royal College of Surgeons rat with inherited retinal dystrophy, photoreceptor cell degeneration is accompanied by retinal pigment epithelial (RPE) cell alterations and Müller cell changes such as increased expression of glial fibrillary acidic protein (GFAP). Vascular changes such as vascularization of the RPE, vascular proliferation, and formation of vitreoretinal membranes (VRMs) are observed later. To study the relationship of Müller cell changes to the vascular alterations in the dystrophic retina, we used immunoperoxidase techniques and antibodies against GFAP and vimentin. Our study showed that during photoreceptor degeneration, Müller cells expressed small amounts of GFAP. As degeneration progressed, GFAP expression increased and morphological alterations occurred in Müller cells. Müller cell apical processes extended and proliferated in the subretinal space and contacted the apical surface of duplicated RPE cells. Later, GFAP reactive fibers surrounded retinal vessels apposed to the RPE. As the vessels became enmeshed within the RPE, the GFAP-positive perivascular processes disappeared. Eventually, the RPE-associated vessels became displaced into the inner retina where VRMs were sometimes observed. Immunoblots showed increased GFAP in dystrophic as compared with control retinas. Studies of vimentin distribution in the dystrophic retina showed results similar to the GFAP study. Moreover, the vimentin study suggested increased number of Müller cell processes in the dystrophic as compared with control retinas. The close temporal and anatomical relationships among Müller cell, RPE, and vascular changes in the dystrophic rat suggest a role for Müller cells in retinal neovascularization and proliferative retinopathy.

Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.

Phagocytic interactions of sialated glycoprotein, sugar, and lectin coated beads with rat retinal pigment epithelium.

Latex beads coated with mucin (a sialic acid containing glycoconjugate), N-acetylglucosamine (GlcNAc), or with the lectins, succinylated wheat germ agglutinin (sWGA) (binds to GlcNAc) or Limax flavus agglutinin (LFA) (binds to sialic acid residues) were used as phagocytic particles. Phagocytosis of these coated beads by retinal pigment epithelial (RPE) explants was determined by bead uptake in normal (Long Evans) and dystrophic (Royal College of Surgeons, RCS/p+) rat retinas. Electron microscopy showed that beads coated with mucin or LFA lectin were not phagocytized by either dystrophic or normal RPE. sWGA-coated beads were phagocytized by both dystrophic and normal RPE, while GlcNAc-coated beads were taken up by dystrophic RPE only. Specificity of uptake for sWGA and GlcNAc bead coatings was shown by the reduction in the number of beads phagocytized in the presence of the appropriate competing sugar or lectin. The lack of phagocytic uptake of beads coated with a sialated glycoprotein or a sialic acid binding lectin suggests that sialic acid residues are not recognized as particulate ligands in this phagocytic assay. The data which show the uptake of beads coated with sWGA (binds only GlcNAc) together with results which showed WGA (binds both GlcNAc and sialic acid)-coated beads were not taken up, further suggest that GlcNAc residues may be involved in bead phagocytosis. The most striking difference between normal and dystrophic RPE engulfment of coated beads is the uptake of GlcNAc-coated beads by dystrophic RPE only. These results suggest that the receptor molecules on dystrophic RPE cell surface membranes may be different from those on normal RPE membranes.

Expression of Ia antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis.

Recently, human retinal pigment epithelial (RPE) cells have been demonstrated to express class II, HLA-DR, antigens both in vivo and in vitro. HLA-DR antigens were detected on RPE cells from patients with uveitis and retinitis pigmentosa. In addition, in vitro studies revealed that not only does this cell express HLA-DR antigen but also that this antigen can be modulated by the lymphokine, interferon (IFN)-gamma. In this study we evaluated the development of the murine class II, Ia, antigens on RPE cells in experimental autoimmune uveoretinitis (EAU). Ia antigen was evaluated with the avidin-biotin-peroxidase technique. Ia antigen was not detected on RPE cells from normal rats. However, Ia antigen was detected on the surface of RPE cells from EAU rats four days prior to the development of clinical and histopathological EAU. Moreover, the expression of Ia antigen on RPE cells from EAU rats continued to persist until one and one-half months after immunization. This study demonstrates that during the course of EAU the RPE cell is activated to express Ia antigens. This antigen expression may be important in the initiation and/or perpetuation of immune reactivity in the eye.

Distribution of anionic sites on the surface of retinal pigment epithelial and rod photoreceptor cells.

The distribution of anionic (negatively charged) groups on the surface of retinal pigment epithelial cells (RPE), rod photoreceptor cells (RP) and discarded rod outer segments (R0S) was studied by labeling these groups with cationic ferritin (CF) at different pH values. In normal neonatal rats, characteristic CF binding patterns were observed on the RPE cell surface at pH values ranging from 1.8-7.2. Similar distributions were seen on the surface of RPE cells from RCS-p+ rats with inherited retinal degeneration. Treatment of RPE cells from normal neonates with several different degradative enzyme preparations prior to CF labeling, did not alter the density or distribution of CF molecules at any of the pH values tested. In normal adult rats, CF binding to RPE cells was essentially similar to that observed in neonates. In RP cells, heavy CF labeling at pH 1.8 was seen on the surface of the inner and outer segments and connecting cilium. CF labeling patterns on discarded R0S undergoing phagocytosis were similar to those on RPE cells at all pH values except pH 5.5. The findings indicate that anionic sites are present on the surface of RPE and RP cells and on discarded R0S undergoing phagocytosis. There is no evidence that such sites are involved in the phagocytic defect characteristic of RCS-p+ rats. Adult-like patterns of anionic sites on RPE cells appear early in postnatal development; their resistance to a wide range of degradative enzymes suggests that the carbohydrates or proteins containing these anionic moieties are organized in a complex fashion in the RPE cell surface.