Phagocytosis Of Rod Outer Segments Research Articles

Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

Natural, high-mannose glycoproteins inhibit ROS binding and ingestion by RPE cell cultures

Previous studies have suggested that a mannose receptor mediates the phagocytic uptake of effete rod outer segments by retinal pigment epithelial cells. In the present study, the effect of adding a soluble ligand for the mannose receptor, horseradish peroxidase, was examined. Cultured retinal pigment epithelial cells from Long Evans rats were preincubated with various concentrations of horseradish peroxidase for 20 min followed by a challenge of FITC-labeled bovine rod outer segments for 3 h. Both counts of total rod outer segments (bound and ingested) and ingested rod outer segments were determined. Rod outer segment uptake was reduced, in a concentration-dependent fashion, by an average of 60% of control values when horseradish peroxidase was added to retinal pigment epithelial cultures. Similarly, total rod outer segment values were reduced to 50% of controls in the presence of at least a 10 μg ml −1 horseradish peroxidase concentration. Horseradish peroxidase inhibition of retinal pigment epithelial phagocytic capacity was reversible. Other high mannose glycoproteins, such as invertase, β-glucoronidase, and ovalbumin, were equally effective in preventing rod outer segment ingestion by retinal pigment epithelial cells. These data further support the hypothesis that a mannose receptor on the retinal pigment epithelial apical surface facilitates phagocytosis of rod outer segments.

Characterization of rod outer segment plasma membrane proteins which bind to the mannose receptor.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Transforming growth factor-beta regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway.

Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of many ocular diseases, including proliferative vitreoretinopathy. We examined the effect of TGF-beta on the phagocytosis of rod outer segments by retinal pigment epithelium (RPE), which is a major function of RPE, and investigated the dependence of this effect on the protein kinase C (PKC) pathway. Phagocytotic uptake of fluoresceinated bovine rod outer segments was determined by flow cytometry. RPE cells were treated with TGF-beta 1 or TGF-beta 2 and their effects on phagocytosis were examined. The effects of various PKC inhibitors (calphostin C, staurosporine, and extended exposure to phorbol 12-myristate 13-acetate, PMA) and a stimulator (brief exposure to PMA) on RPE phagocytosis was evaluated. Both TGF-beta 1 and TGF-beta 2 up-regulated RPE phagocytosis and PMA abolished the up-regulating effect of TGF-beta. In contrast, PKC inhibition by staurosporine and calphostin C resulted in increased phagocytosis. A combination of TGF-beta and PKC inhibitor treatment did not produced any additive effect on phagocytosis. We concluded that TGF-beta up-regulates human RPE phagocytosis, but that this effect is counteracted by PKC activation. It is possible that this TGF-beta-induced effect is due, in part, to a negative modulation of the PKC-dependent pathway.

Nitric oxide decreases in vitro phagocytosis of photoreceptor outer segments by bovine retinal pigmented epithelial cells.

The presence of nitric oxide synthase (NOS) in the retina, the constitutive isoform in photoreceptor outer segments and the inducible form in retinal pigmented epithelial (RPE) cells, has been demonstrated, but the role of the free radical NO produced, remains unknown. We have investigated the effect of NO on the process of rod outer segment (ROS) phagocytosis. Using an in vitro assay for phagocytosis in primary cultures of bovine RPE cells, we demonstrate that NO released by SIN-1 (3-morpholinosydnonimine) in the culture medium inhibits the phagocytosis of ROS. Furthermore, endogenous NO, produced by RPE cells cotreated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), is also able to decrease RPE cell phagocytic activity. This effect depends upon the continuous presence of NO during the assay and is abolished by the scavenging of NO by hemoglobin or by the inhibition of NO synthase activity by L-arginine analog, NG-monomethyl-L-arginine. Pretreatment of ROS with SIN-1 failed to impair subsequent phagocytosis, demonstrating that NO directly affects the RPE cells ability to phagocytose ROS. The inhibitory effect of NO is cGMP independent, since 8-bromo-cGMP does not modify this process. This decrease of ROS phagocytosis by RPE cells caused by NO may occur as a result of retinal inflammation, and could lead to photoreceptor degeneration.

Kinetic Studies on Phagocytosis and Lysosomal Digestion of Rod Outer Segments by Human Retinal Pigment Epithelial Cells in Vitro

Using novel methodology, this study describes the kinetics of rod outer segment (ROS) phagocytosis and digestion by human retinal pigment epithelial (RPE) cells in vitro and examines the effect of certain lysosomal enzyme inhibitors on ROS digestion in these cells. Human RPE cells displayed saturation of phagocytosis with respect to both ROS concentration and time. While surface-binding and ingestion phases of ROS phagocytosis saturated after 24-36 h, the rate of ROS digestion reached a maximal level within 24 h. Increasing the concentration of zinc in the culture medium from 1.9 to 100 μM had no significant effect on ROS digestion. The effects of swainsonine (an α-mannosidase inhibitor), pepstatin (an aspartic protease inhibitor), and leupeptin (a cysteine protease inhibitor) were also examined. At 6 h, ROS digestion was reduced 27.3 ± 15.3% by swainsonine, 69.4 ± 20.9% by pepstatin, and 77.0 ± 14.4% by leupeptin. The effect of these inhibitors declined with increasing time. This study is the first to demonstrate the functional importance of cysteine and aspartic proteases in the digestion of ROS by RPE cells in vitro.

A Comparison of Phagocytosis by the Retinal Pigment Epithelium in Normal and Delayed Amelanotic Chickens

The retinal pigment epithelium (RPE) performs a critical role in maintaining the integrity and renewal of the retinal system by clearing the shed photoreceptor outer segment debris through the process of phagocytosis. If phagocytosis is impaired, accumulation of the debris occurs leading to the degeneration of the retina. Phagocytosis, measured by polyvinyl toluene bead uptake is a normal cell activity exhibited by the embryonic retinal pigment epithelium even before complete differentiation of the neuronal layers. It is significant that embryonic pigment epithelium from either normal or delayed amelanotic (DAM) strain is competent in the process of non-specific particle uptake. In normal chickens non-specific phagocytic activity is observable as early as embryonic day 9 and continues through the rest of the developmental period and throughout adult life. The retinal pigment epithelium in DAM chickens exhibits early phagocytic activity at embryonic day 9 and then through day 16, but by day 19 this function is lost. This loss of phagocytic capacity in RPE of the DAM strain precedes and may be instrumental in the gradual retinal degeneration exhibited by these animals. Specific phagocytosis of rod outer segments (ROS) was examined in 4-week-old post-hatch normal and DAM RPE. Phagocytosis of ROS was greatly reduced in DAM RPE implying that the defect in the DAM is expressed in non-specific and specific phagocytosis. This study indicates that the DAM retinal system can now serve as an alternative and comparative model for studying different aspects of retinal degeneration.

Phagocytosis of outer segments by retinal pigment epithelium: phagosome-lysosome interaction.

We studied phagocytosis of rod outer segments (ROS) by the retinal pigmented epithelium (RPE) using rapid freezing, freeze-drying, and electron microscopic immunocytochemistry. Phagocytosis of photoreceptor outer segment tips by the RPE occurs daily, and in rats the shedding of these tips is light-entrained to a circadian rhythm. We studied the phagocytic process 5, 30, 90, and 150 min after light onset or after subjective light onset in rats entrained to a 12-hr dark-12-hr light cycle. Lysosomes were labeled with antibodies to cathepsin D, a major lysosomal enzyme responsible for opsin degradation. Phagosomes and phagolysosomes were recognized because of the lamellar structure of their photoreceptor-derived contents. We found a population of lysosomes that fuse with one another before they interact with phagosomes. This fusion can be triggered either by light or by endogenous circadian mechanisms. We also found that lysosome-phagosome interaction occurs after the ingestion stage is completed and that this interaction occurs in two steps. First, smaller lysosomes fuse with phagosomes. Subsequently, larger lysosomes appear to interact with phagosomes via pore-like or bridge-like structures. It is proposed that interchange of contents takes place through these structures.

The phagocytosis of ROS by RPE cells is inhibited by an antiserum to rat RPE cell plasma membranes

A polyclonal antiserum to a rat retinal pigment epithelium (RPE) plasma membrane-enriched fraction has been utilized to identify candidate receptor proteins which may be involved in the phagocytosis of rod outer segments (ROS) by the RPE. Immunoblots of RPE cell extracts show that the antiserum recognizes a number of glycoproteins, including two with M rs of 174 and 75 kDa. The antiserum also recognizes their non-glycosylated counterparts, with M rs of 169 and 65 kDa, respectively, which are synthesized after treatment of the cells with tunicamycin B 2. Immuno-precipitation of [ 35S]-methionine-labeled RPE cell extracts also demonstrates the presence of antibodies to these same glycoproteins as well as to other proteins. The antiserum inhibits the binding of ROS to the RPE, which subsequently results in a decrease in the ingestion of ROS. ROS phagocytosis by the RPE is inhibited by 97% in the presence of a 1 : 10 dilution of the IgG fraction of the antiserum. Phagocytosis recovers to normal levels after 4–6 hr of chase in the absence of antibodies. After sequential adsorption of the IgG fraction to monolayers of fixed RPE cells, which removes RPE surface-specific IgGs, the extent of inhibition of ROS phagocytosis produced by the IgG fraction is reduced. Using immunoblotting we have identified a number of surface-specific immunoreactive bands which are adsorbed out of the antiserum, including the 174 and 75 kDa bands. These data give further support to the hypothesis that ROS phagocytosis is a receptor-mediated process, which occurs via specific cell surface glycoprotein receptors.

Characterization and application of an in vitro detection system for studying the binding and phagocytosis of rod outer segments by retinal pigment epithelial cells

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 125I or fluorescein isothiocyanate (FITC) 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 [ 125I]Rho 4D2 antirhodopsin antibody or [ 125I]streptavidin for radioactivity measurements or with the Rho 4D2 antibody and FITC-goat anti-mouse Ig or FITC-streptavidin 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-permeabilized 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, 907–922], 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 × 10 7 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 × 10 7 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 1–2 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.

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro

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. Autoradiography 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.

Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of diabetic complications

Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of diabetic complications

Sorbitol, myo-Inositol, and Rod Outer Segment Phagocytosis in Cultured hRPE Cells Exposed to Glucose: In Vitro Model of myo-Inositol Depletion Hypothesis of Diabetic Complications

The "myo-inositol depletion hypothesis" remains a leading but still controversial contender among proposed pathogenetic mechanisms for the chronic complications of diabetes. The multifaceted interrelationships among altered tissue myo-inositol content and metabolism and tissue function have been difficult to elucidate in diabetic animal models due in part to the complex, heterogeneous nature of tissues prone to diabetic complications. The retinal pigment epithelium consists of a homogenous cell monolayer that exhibits related alterations in myo-inositol metabolism and function in diabetic animals. Nontransformed human retinal pigment epithelial (hRPE) cells, which retain their general phenotypic and morphological characteristics during monolayer culture in vitro, were examined for parallel alterations in myoinositol metabolism and cell function when grown under carefully controlled conditions in medium containing hyperglycemic concentrations of glucose. Exposure of hRPE cells to 20-40 mM glucose produced time- and dose-dependent increases in sorbitol content and decreases in myo-inositol content that were partially blocked by the aldose reductase inhibitor sorbinil. myo-Inositol was taken up by two Na-dependent transport systems, at least one of which was competitively inhibited by glucose. Exposure to 20 mM glucose impaired the ability of hRPE cells to take up human retinal rod outer segments, an important physiological function of these cells. The impairment of rod outer segment uptake by high glucose levels was prevented by an aldose reductase inhibitor or elevated medium myo-inositol that corrected the fall in myo-inositol content. Thus, hRPE cells provide a new in vitro model in which to examine the biochemical-functional interrelationships of the myo-inositol depletion hypothesis.

The phagocytosis of ROS by RPE cells is not inhibited by mannose-containing ligands

We have examined the ability of mannose and the mannose-rich ligands, mannan and mannosylated BSA, to inhibit the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. Mannose, at concentrations up to 0·25 m, had no effect on either the binding or the ingestion of ROS. At concentrations above 0·25 m, the cells were rounded and showed detachment from the substrate, and phagocytosis was markedly inhibited. Neither mannan (2 mg ml −1), nor mannosylated BSA(0·8 mg ml −1), affected the phagocytosis of ROS. These results suggest that the phagocytosis of ROS is probably not mediated by a mannose receptor on the surface of the RPE cells.

Stimulation of rod outer segment phagocytosis by serum occurs only at the RPE apical surface

The phagocytosis of isolated rod outer segments by cultured rat retinal pigment epithelium (RPE) previously has been shown to be stimulated by serum in the culture medium. In vivo, serum is normally in contact with the basolateral surface of the RPE. Components of serum have also been detected in the interphotoreceptor matrix, raising the possibility that these components may be in contact with the apical RPE surface. However, with conventional culture techniques it is not clear whether the stimulation of phagocytosis by serum occurs at the basolateral or apical surface. To resolve this uncertainty, phagocytosis was studied using RPE cultured on microporous filters, permitting control of which RPE surfaces are in contact with serum. Serum was found to have no influence on phagocytosis when present at the RPE basolateral surface, regardless of the serum concentration (2 or 20%). In contrast, phagocytosis was elevated three- to fivefold when 2% serum was present at the apical RPE surface, irrespective of the presence of serum at the basolateral surface. It is concluded that serum stimulates the phagocytosis of ROS only when the serum is present at the RPE apical surface.

ROS ingestion by RPE cells is turned off by increased protein kinase C activity and by increased calcium

The activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) rapidly inhibits the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. PMA, at a concentration between 3·3 and 10 n m, blocks ROS ingestion by 50%, but does not inhibit the binding of ROS. The Ca 2+ ionophore, A 23187, also inhibits ROS phagocytosis, with an IC 50 of about 0·5–1·0 μ m and interferes with the ability of RPE cells to bind ROS. The effects of both of these drugs are reversible after drug washout. When PMA and A 23187 are applied to cells consecutively, the effects are additive. These results suggest either that PMA and A 23187, act upon the same proteins in the pathway which controls ROS ingestion, or that A 23187 affects phagocytosis at the ROS binding level, while PKC affects steps further along the ingestion path. The effect of this process is to shut down the ingestion of ROS, as is seen during the prolonged feeding of ROS to RPE cells in culture.

RPE cells from normal rats do not secrete a factor which enhances the phagocytosis of ROS by dystrophic rat RPE cells

Retinal pigment epithelial (RPE) cells from normal and dystrophic rats were grown separately and in mixed culture for 7 days, without a change of growth medium. Isolated rod outer segments (ROS) were suspended in the conditioned medium from these cells, and were fed to the mixed or pure RPE cell cultures. No increase or decrease in the phagocytosis of ROS by dystrophic or normal RPE cells, respectively, was observed. These results suggest that normal RPE cells do not secrete a diffusible factor(s) which enhances the phagocytosis of ROS by dystrophic RPE cells.

Specific Radioimmunoassay to Investigate Rod Outer Segment Phagocytosis by Retinal Pigment Epithelium in vitro

A sensitive radioimmunoassay has been developed which allows rapid quantitation of rod outer segment (ROS) phagocytosis by retinal pigment epithelial (RPE) explants in vitro. It involves the use of an antiopsin antiserum, in conjunction with 125I-protein A as a second antibody, and utilizes permeabilization with ethanol to distinguish between the binding and ingestion phases of phagocytosis. This procedure will be used in the future to investigate potential regulatory factors of ROS phagocytosis by retinal pigment epithelium and to evaluate animal models of retinal degeneration.

Effect of glycoconjugates on rod outer segment phagocytosis by retinal pigment epithelial explants in vitro assessed by a specific double radioimmunoassay procedure

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.