How good is the evidence to suggest that phagocytosis of ROS by RPE is receptor mediated?

CHAPTER 6

How Good is the Evidence to Suggest that Phagocytosis of ROS by RPE is Receptor Mediated? *BARBARA J. MCLAUGHLIN, *NIGEL G. F. COOPER and +VIRGINIA L. SHEPHERD *Department of Ophthalmology and Visual Sciences, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40292, USA +Department of Veterans Affairs and the Departments of Medicine and Biochemistry Vanderbilt University, Nashville, TN 3 7212, USA CONTENTS ....................................................................... 1. Introduction 1.l. Non-Specific Versus Specific Phagocytosis ......................................... ................................................. 1.2. Receptor-Mediated Phagocytosis ........................................................ 2. Phagocytosis by Macrophages 2.1. Non-Specific Phagocytosis ....................................................... ................................................. 2.2. Receptor-Mediated Phagocytosis 2.2.1. Fcreceptor .............................................................. 2.2.2. C3 receptor .............................................................. ................................................. 2.2.3. Mannosylfucosylreceptor ........................................................ 2.2.4. /.I-Glucanreceptor 3. Phagocytosis by Retinal Pigment Epithelium ........................................... 3.1. Non-Specific Phagocytosis ....................................................... ................................................. 3.2. Receptor-Mediated Phagocytosis 3.2.1. Fcreceptor .............................................................. 3.2.2. Mannosylfucosyl receptor ................................................. 3.2.3. Other receptors .......................................................... 4. Potential Ligands for Receptor-Mediated Phagocytosis by RPE .......................... ........................................................... 4.1. MannoseasaLigand ......................................................... 4.2. Rhodopsinas aLigand 4.3. Other ROS Ligands for the Mannose Receptor ..................................... 4. Conclusions/Future Directions ........................................................ Acknowledgements......................................................................l6 References ............................................................................ 1. INTRODUCTION

0 1994 Pergamon Press Ltd Printed in Great Britain.

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specialized phagocytes, the retinal pigment epithelial cells (RPE), which continually phagocytize the underlying photoreceptor outer segments (ROS) as part of the normal visual cycle. In addition to being phagocytic, macrophages and retinal pigment epithelial cells also share other features, including the presence of receptors for Fc and C3bi (Elner et al., 1981), mannosedphosphate (Tarnowski et al., 1988) and mannose

Phagocytosis is a form of endocytosis in which large particles are ingested in endosomal compartments called phagosomes. Macrophages, which are professional phagocytes, phagocytize senescent and damaged cells and micro-organisms every day as part of the body’s defense mechanism. Likewise, in the retina there are Progress in Retinal and Eye Research Vol. 1, No. 1

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B. J. MCLAU(;IHIN el al.

(McLaughlin et al., 1987; Tarnowski et al., 1988; Shepherd et al., 1991) as well as a number of other immunophenotypic characteristics (Einer et al., 1992). As the evidence for receptor-mediated phagocytosis in macrophages has been well established, this chapter will attempt to use those data for comparison as well as criteria for establishing that a similar receptor-mediated process occurs during ROS phagocytosis.

1.1. Non-Specific Versus Specific Phagocytosis

Macrophages are capable of ingesting an enormous quantity of material as well as a variety of particles, including senescent erythrocytes, circulating tumor cells, autologous tissue debris, denatured proteins, micro-organisms, hormones, lipoprotein, inert colloids, and immune complexes. Phagocytosis of most of these particles is facilitated by coatings on the particle surface called opsonins, for which macrophages have specific receptors (Coleman, 1986; Chakravarti and Chakravarti, 1987). The macrophage receptors are clustered at clathrin-coated regions of the plasma membrane and interact with specific opsonic ligands on the surface of the particle. This process results in the engulfment of the foreign particle into a phagosome as part of the more general mechanism of receptor-mediated endocytosis or specific phagocytosis (Montesano et al., 1983). While the majority of phagocytic events mediated by macrophages are known to be carried out by specific receptors on the macrophage surface, phagocytic uptake of latex beads is generally referred to as a non-specific interaction, possibly involving charge or hydrophobic interactions (Karnovsky et al., 1975). This socalled non-specific phagocytic interaction, nevertheless, is similar to receptor-mediated phagocytosis in that it involves clathrin-coated regions of membrane in the area of the forming phagosomes (Aggeler and Werb, 1982). Despite these structural similarities, the biochemical nature of this type of phagocytic interaction remains unknown (Karnovsky et al., 1975) and for that reason the process is referred to as non-specific.

Non-specific versus specific phagocytosis is besl illustrated when the retinal pigment epithelium is used as the phagocytic model system. The retinal pigment epithelium, like its macrophage counterpart, is capable of non-specific phagocytosis of latex beads and carbon particles (Hollyfield and Ward, 1974; Custer and Bok, 1975; Funahashi et al., 1976; Feeney and Mixon, 1976; Hollyfield, 1976). The RPE is also capable of specific phagocytosis of ROS. However, when there is an inherited defect in the RPE, specific phagocytic uptake of ROS becomes inhibited (Goldman and O'Brien, 1978), but non-specific uptake of carbon or latex particles continues to occur (Custer and Bok, 1975; Edwards and Szamier, 1977; Essner et al., 1978; Seyfried and McLaughlin, 1983). This suggests that different mechanisms exist for the uptake of ROS versus latex beads or carbon particles and that there is some specific interaction which underlies the phagocytosis of ROS by the RPE.

1.2. Receptor-Mediated Phagocytosis

Earlier studies by Griffin and co-workers (Griffin et al., 1975, 1976) have elegantly demonstrated that receptor-mediated phagocytosis by macrophages involves a 'zippering' interaction between specific receptors on the cell surface and the ligand coating on the particle, which requires that either receptor or ligand be distributed uniformly over the two interacting surfaces in order for engulfment and phagocytosis to occur. If either ligand or receptor is not distributed evenly over the two surfaces, binding occurs but engulfment is prevented. While this zipper interaction has never been tested in RPE phagocytosis, it nevertheless remains the model for receptor-mediated uptake of particulate material by macrophages. One of the strongest indicators that the RPE is capable of specific or receptor-mediated phagocytosis of ROS comes from the work of Laird and Molday (1988) and Philp et al. (1988) who have shown that iodinated ROS binding and phagocytosis can be competitively inhibited with unlabeled ROS. This suggests that specific interactions are occurring during the binding and

PHAGOCYTOSISOF ROS BY RPE ingestion of rod outer segments, involving a ligand on the ROS surface with a receptor on the RPE apical membrane. Retinal pigment epithelial cells in in vitro culture conditions are also capable of phagocytizing red blood cells, bacteria, algae and yeast, albeit in substantially smaller numbers than their preferred target, the rod outer segments (Mayerson and Hall, 1986). Although the possibility has been raised that retinal pigment epithelial cells may use mechanisms similar to macrophages for ingesting red blood cells and rod outer segments by opsonization, coating of these two particle types with serum or antibodies does not enhance phagocytic uptake (Mayerson and Hall, 1986; Reid et al., 1992). On the other hand, competition for ROS ingestion using an equal amount of mannan-coated particles (yeast) yields a significant reduction in ROS phagocytosis (Mayerson and Hall, 1986). The same inhibition occurs when yeast is used to compete for macrophage phagocytic uptake of zymosan (Sung et al., 1983) and indicates not only that these particles can be effective inhibitors during both RPE and macrophage phagocytosis, but that a mannose receptor-mediated process is involved. 2. PHAGOCYTOSIS BY MACROPHAGES 2.1. Non-Specific Phagocytosis As mentioned above, macrophages are capable of mediating uptake of latex beads by non-specific mechanisms that may involve recognition of charge densities on the bead surface or binding to proteins secreted by macrophages that adhere to the bead as a coating (Aggeler and Webb, 1982). It must be noted, however, that uptake of all particulate materials, from the so-called nonspecific phagocytosis of latex beads to the specific uptake of opsonized particles is regarded by workers in this field as cellular events governed by ligand - receptor interactions (Wright and Silverstein, 1983). Latex bead uptake is referred to as non-specific only because the biochemical nature of the interaction has yet to be fully characterized. 2.2. Receptor-Mediated Phagocytosis Macrophages have binding sites for at least forty ligands on their plasma membranes for

149

which only a few receptors have been identified that are known to promote ingestion of particulate substances (Wright and Silverstein, 1983). Of these, receptors for the Fc fragment of IgG, complement and oligosaccharides are the best characterized.

2.2.1. Fc RECEPTOR The Fc fragment of the immunoglobulin G (IgG) molecule is an important opsonin for phagocytosis by the immune system. Several types of Fc receptors are present on different cells of the immune system (monocytes, macrophages, polymorphonuclear leukocytes, lymphocytes, mast cells and several non-lymphoid cells) with a molecular weight range reported to be from 45 to 70 kDa (Unkeless et al., 1981; Chakravarti and Chakravarti, 1987). Binding of the Fc receptors with particle-bound immunoglobulins initiates transmembrane signals that activate an array of metabolic activities beginning with engulfment and phagocytosis to the secretion of peroxide and arachidonate metabolites and histamine release. The 55 kDa Mr form of the receptor is thought to be a ligand-dependent ion channel which is not linked to the cytoskeleton and is freely diffusible in the plane of the membrane (Unkeless et al., 1981; Young et al., 1983).

2.2.2. C3 RECEPTOR The third component of complement (C3) is also an important opsonin for macrophage phagocytosis. Human macrophages express two receptors for the cleavage products of the third component of complement (C3). One receptor recognizes C3b and is called CR1; the other receptor recognizes a cleavage product of C3b (C3bi) and is called CR3. Both receptors can exist in two different states. The inactive state binds ligand but does not signal phagocytosis, whereas, the active receptor binds ligand and initiates phagocytosis. The two receptors function independently and are mobile in the plane of the macrophage plasma membrane. C3 receptors can be activated by lymphokines (Griffin and Griffin, 1979), fibronection or phorbol esters (Wright et

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al., 1984). Activation can be reversed after these substances are removed and can also be stopped by microtubular depolymerizing agents (Griffin and Griffin, 1979; Wright and Silverstein, 1982). In some cases, the complement receptors can function synergistically with Fc receptors to promote phagocytosis. However, unlike the Fc receptors, C3 receptors do not initiate the release of hydrogen peroxide or arachidonic acid even when they are phagocytically active. In addition, laminin has been observed to enhance both Fc and complement-mediated phagocytosis by cultured human macrophages (Bohnsack et al., 1985). As described above, IgG and the third component of complement are the best known opsonins which stimulate phagocytosis and for which the Fc and Ca receptors have been widely studied. There are, however, other phagocytic interactions which occur in the absence of serum opsonins which play an important role in nonimmune host defense mechanisms. These are the carbohydrate-lectin interactions involving the mannose receptor and /3-glucan receptor pathways.

2.2.3. MANNOSYLFUCOSYL RECEPTOR

Zymosan is a commonly used test particle for studying opsonin-independent phagocytosis by monocytes, macrophages and polymorphonuclear leukocytes. Phagocytic uptake of zymosan, whose major constituents are a-mannans and/3-glucans, occurs via the mannosylfucosyl receptor (Warr, 1980; Ezekowitz et al., 1983; Stahl et aL, 1978; Sung et al., 1983). This receptor has specificity for glycoconjugates terminating in mannose or fucose (Ezekowitz and Stahl, 1988) and functions in receptor-mediated endocytosis of soluble mannose-containing ligands such as lysosomal enzymes (Stahl et al., 1978) and peroxidases (Shepherd and Hoidal, 1990; Rabinovitch et al., 1985). In addition, this receptor-mediates phagocytosis of particulate mannose-containing substances such as zymosan, Pneumocystis carinii, Candida albicans, P s e u d o m o n a s acruginosa and Mycobacterium arium (Sung et al., 1983; Zimmerman et al., 1990; Ezekowitz et al., 1991; Marodi et al., 1991; Speert et al., 1989; Bermudez et al., 1991). It

is not known, however, whether these two types of uptake (soluble versus particulate) are mediated by different populations of receptor molecules. The mannosylfucosyl receptor is a 175 kDa membrane glycoprotein (Wileman et al., 1986) that recognizes glycoconjugates bearing more than one terminal mannose residue (Maynard and Baenziger, t981). This macrophage-specific surface receptor recognizes oligosaccharides terminating in fucose as well as mannose (Stahl et al., 1976, 1978; Schlesinger et al., 1978; Shepherd, et al., 1981) and this has given rise to the term mannose/fucose receptor. The monosaccharide, mannose, is a poor inhibitor of mannose receptor binding of soluble ligands but is more effective at high concentrations (Sung et al., 1983). In addition, both mannan and mannose-BSA block binding of soluble ligands to the mannose receptor (Wileman et al., 1986). The receptor requires Ca 2~ for its binding activity and binds ligands avidly at neutral pH but poorly at pH 5 (Lennartz et al., 1987a,b). One of the most effective inhibitors of mannose receptor activity in phagocytic uptake of particulate substances is horseradish peroxidase (Sung et al., 1983), with micromolar concentrations of mannan and somewhat higher concentrations of other mannose-containing oligosaccharides also being capable of inhibiting zymosan binding and phagocytosis. The mannose receptor has now been cloned and sequenced and is an integral membrane protein containing eight carbohydrate recognition domains, a transmembrane region and a cytoplasmic tail (Ezekowitz et al., 1990; Taylor et al., 1990). The involvement of this receptor in phagocytosis has been clearly demonstrated by the finding that when non-phagocytic cells (COS-I) are transfected with mannose receptor cDNA, they become capable of phagocytosis (Ezekowitz et al., 1990, 1991). 2.2.4. fl-GLUCANRECEPTOR The role of /3-glucan receptor in particulate phagocytosis has been studied primarily in monocytes but is present also on macrophages. This receptor recognizes the/3-glucan constituent of zymosan (Czop and Austen, 1985; Czop, 1986). Like the mannose receptor, the/3-glucan receptor functions independently of the Fc receptor for IgG

PHAGOCYTOSIS OF

and does not require IgG or complement fragments for the phagocytic response. Similar to the Fc and complement receptors, it may rely on interactions with other opsonic proteins present in the extracellular matrix, such as fibronection (Chakravarti and Chakravarti, 1987). 3. PHAGOCYTOSIS BY RETINAL PIGMENT EPITHELIUM 3.1. Non-Specific Phagocytosis Although retinal pigment epithelial cells are capable of phagocytizing non-specific particles such as carbon particles or latex beads (Hollyfield and Ward, 1974; Custer and Bok, 1975; Funahashi et al., 1976; Feeney and Mixon, 1976; Hollyfield, 1976; Seyfried and McLaughlin, 1983), particles with one micron-sized diameters are preferred and are phagocytized before smallersized particles (Funahashi et al., 1976). Studies on the developing rat retina have demonstrated that this non-specific uptake of latex beads by the pigment epithelium of 10-15 day old neonatal animals precedes the more specific phagocytic uptake of ROS and suggests not only that different mechanisms exist for the two types of uptake systems, but that the specific surface constituents are either not present or not functional prior to differentiation and shedding of ROS (Philp and Bernstein, 1981). A more recent study, however, has shown that rat pigment epithelium is capable of phagocytosing ROS in vitro (Ershov and Stroeva, 1989) at all the neonatal stages, including the newborn. This study, however, shows that even though newborn pigment epithelium is capable of ROS phagocytosis, only low numbers of ROS are actually phagocytized before postnatal day 15. At that time, there is a dramatic increase (5-fold) in ROS phagocytosis, which suggests that there are some developmentally-dependent differences in the specific mechanisms required for ROS uptake. 3.2. Receptor-Mediated Phagocytosis In order to examine the evidence for whether ROS phagocytosis is a receptor-mediated interaction, it is important to first look at the criteria which are used to make this determination. The first requirement is that there must be a

ROS BY RPE

151

competitive inhibition of the ligand-receptor interaction by the putative ligand. The second requirement is that the ligand-receptor binding should demonstrate saturability within a concentration range. The third requirement is that the binding interaction should demonstrate high affinity. In the past several years, evidence has been growing in support of these criteria which demonstrates that ROS phagocytosis is receptormediated. Mayerson and Hall (1986) have demonstrated that the target particle, ROS, is selectively preferred over other particles such as red blood cells, yeast, algae and bacteria. More importantly, Laird and Molday (1988) have demonstrated a 40o7o inhibition of binding of iodinated ROS to cultured RPE cells when a 2fold excess of unlabeled ROS is used as the competitive ligand. Kinetic studies by Hall and Abrams (1987) have shown that the binding of ROS to cultured rat RPE cells can reach saturation with respect to time and concentration and that binding and ingestion are temperaturedependent. Evidence to support the glycoprotein nature of the RPE phagocytic receptor using proteolytic modification of the RPE surface membrane has shown that inhibition of ROS phagocytosis can be correlated with the removal of several RPE glycoproteins (Colley et al., 1987). Further support has come from experiments which interfere with the glycosylation of putative receptor proteins on the RPE cells and show that ROS phagocytosis can be inhibited as a result of these changes (Hall et al., 1987, 1990; Boyle and McLaughlin, 1990). Other evidence into the glycoprotein nature of phagocytic receptors continues to accumulate from studies that use antibodies which recognize specific RPE membrane glycoproteins to block ROS phagocytosis (Guo et al., 1991; Gregory and Hall, 1992). More recently, ROS ingestion by RPE cells has been shown to be linked to transmembrane signaling mechanisms common to other ligand-receptor systems (Hall et al., 1991; Heth and Schmidt, 1991). Of interest is the finding that this mechanism may be malfunctional in dystrophic RPE cells that manifest a phagocytic defect (Heth and Schmidt, 1992).

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B. J. McLAu¢itn 1N et al.

3.2.1. Fc RECEPTOR While these various studies have served to accumulate evidence in support of the idea that ROS phagocytosis by the RPE is receptormediated, they nevertheless have not been able as yet to identify a specific phagocytic receptor. One logical place to begin would be to determine if RPE cells share any receptor similarities with macrophages, such as the presence of Fc or C3 receptors. Elner et al. (1981), have reported that IgG and complement coated erythrocytes can selectively bind and be phagocytized by RPE from monkey eyes, which suggests the presence of receptors for both Fc and C3bi on the RPE. It is generally thought, however, that while these receptors are present on RPE cells, they do not function as receptors for ROS phagocytosis. This is based on data showing that rat ROS coated with polyclonal antibodies (Mayerson and Hall, 1986) and bovine ROS coated with monoclonal antibodies to antibovine rhodopsin (Laird and Molday, 1988) are both bound and ingested by RPE cells to the same degree as when unopsonized ROS are used as the phagocytic particles. On the other hand, it must be added that serum factors other than IgG do appear to play an important role in enhancing ROS phagocytosis (Mayerson and Hall, 1986) specifically when present at the RPE apical surface (Edwards, 1991).

3.2.2. MANNOSYLFUCOSYLRECEPTOR

For the past decade, considerable evidence has accumulated to show that specific recognition between a phagocyte and its target is mediated by the interaction of carbohydrate-binding proteins or lectins which leads to phagocytosis. This process has been termed lectinophagocytosis (Ofek and Sharon, 1988). Sugar-specific recognition is now well established as an important determinant in a number of c e l l - c e l l interactions and so it is not surprising to find that a similar recognition process has received considerable attention in the RPE phagocytic system. In particular, a mannose recognition system has been proposed for receptor-mediated phagocytosis by the RPE which would involve

mannosylated side chains of ROS plasma membrane glycoproteins with a RPE receptor which specifically recognizes mannose residues (Philp and Bernstein, 1980; Heth and Bernstein, 1984; Mayerson and Hall, 1986; McLaughlin el al., 1987; Tarnowski el al., 1988; Boyle et al., 1991; Shepherd el al., 1991). This proposal has evolved over the years from an original suggestion by O'Brien (1976) that changes in rhodopsin oligosaccharides could trigger ROS phagocytosis as well as the demonstration of mannosylated side chains on ROS and RPE cell plasma membranes (Hall and Nit, 1976; McLaughlin and Wood, 1980; Bridges, 1981; Molday and Molday, 1987). Continued support for this idea has come from the finding that, next to ROS, yeast particles that are coated with mannan are a preferred target for phagocytic uptake by the RPE (Mayerson and Hall, 1986). As yeast particles are also phagocytized by macrophages using a mannose recognition system, a similar mannosylfucosyl receptor may be operative during ROS phagocytosis. A mannose-dependent recognition system similar to that identified in macrophages has been demonstrated in RPE for both pinocytic uptake of soluble mannose ligands (mannan-BSA) as well as for the phagocytic uptake of particulate mannosecoated beads (McLaughlin el al., 1987; Tarnowski el al., 1987; Shepherd et al., 1991). Further, immunoblots of rat and human apical RPE membranes stained with antibodies to macrophage mannose receptor have demonstrated a single immunoreactive band at 175 kDa (Boyle et al., 1991). Immuno-electron microscopy of RPE cells using antibodies to macrophage mannose receptors have demonstrated the presence of mannose receptors on apical membranes and microvilli (Boyle et al., 1991; Shepherd el al., 1991). Despite these findings and the obvious existence of a mannose recognition system in RPE cells, concerns have been raised by others (Lentrichia et al., 1987; Shirakawa el al., 1987; Philp et al., 1988; Laird and Molday, 1988; Hall and Abrams, 1991) that mannose is not involved in the l i g a n d - r e c e p t o r interaction. A suggestion has been made that some of the conflicting results may be due to the use of synthetic particles (latex beads) instead of ROS as target particles in phagocytic assays (Hall and Abrams, 1991).

PHAGOCYTOSIS OF ROS BY R P E 2500

2000

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500

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1

2

3

4

5

6

7

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Experimental =

Total

s

Ingested

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Conditions

ROS ROS

FIG. 1. Bar graph of bound and internalized ROS after the following conditions: (1, 2) RPE incubated with ROS alone; (3, 4) RPE incubated in the presence of antiserum against macrophage receptor; (5, 6) preincubated with anti-mouse receptor preabsorbed with purified mannose receptor protein; (7, 8) RPE incubated with ROS preabsorbed with purified mannose receptor protein; (9, 10) RPE preincubated with antiserum against mannose-6phosphate receptor.

In response to these concerns, ROS have been used as the target phagocytic particle in a more recent study of mannose-receptor activity (Boyle et al., 1991). The data from this study show that phagocytosis of ROS can be inhibited by 80% in the presence of an antibody to the mannose receptor as compared with the control RPE incubated with ROS alone or with ROS in the presence of preimmune serum (Fig. 1). As shown in Fig. 1, even when the RPE cells are incubated with an antiserum to another RPE cell-surface receptor (mannose-6-phosphate) in the presence of ROS, there is no inhibitory effect. This control is important because it rules out the possibility of any steric interactions of the primary (mannose receptor) antibody with other RPE membrane proteins which could potentially interfere with phagocytosis. More importantly, when the ROS themselves are preabsorbed with the purified mannose-receptor protein, there is a dramatic

153

inhibition (930/o) of ROS phagocytosis (see Fig. 1). In the face of these compelling data, which strongly suggest that the mannose receptor plays a role in the ligand-receptor interaction of ROS phagocytosis, are the equally compelling counter arguments. These are as follows: in establishing whether a ligand - receptor interaction occurs, it is essential to demonstrate that the receptor action can be competitively inhibited by its putative ligand and that the inhibitory action can reach saturation levels. In the case of the mannose receptor, mannose, mannan or mannose-containing glycoconjugates are the competitive inhibitors of choice. It has been shown, however, that soluble ligands such as monosaccharides of rhodopsin (mannose and N-acetylglucosamine) do not inhibit binding of ROS to RPE cells (Lentrichia et al., 1987). Moreover, other attempts using glycopeptides released from rhodopsin to inhibit ROS phagocytosis have been equally unsuccessful (Laird and Molday, 1988; Philp et al., 1988). To date, the strongest argument against mannose recognition is the data showing that mannan and mannose-BSA do not have an inhibitory effect on ROS phagocytosis (Philp et al., 1988; Hall and Abrams, 1991). On this basis, it has been suggested that if the mannose receptor is the phagocytic receptor on the RPE, it would require stringent binding to an oligosaccharide structure not found in rhodopsin or in the simple sugars tested, and would, in fact, have to be quite different from that of the macrophage mannose receptor (Hall and Abrams, 1991). The following arguments can be made against these points. First, it has been shown in competition studies for mannose receptor-mediated phagocytosis in macrophages that soluble ligands as compared with particulate ligands are ineffective as competitive inhibitors (Benton and Gordon, 1983). Second, it has been demonstrated in macrophage studies that monosaccharides, such as mannose are poor antagonists for mannose receptor-mediated events (Stahl et al., 1978). Third, the mannose receptor from both RPE and macrophages has been affinity purified from mannose and fucose columns (Shepherd et al., 1991; Stephenson et aL, 1987) and receptor from both cell types co-migrate on SDS-PAGE as a

154

B.J. MCLAUGHI.INet al. TABLE 1. Inhibitory Effects of Glycoproteins on ROS Phagocytosis HRP M, Potential N-link site Mannose Fucose Inhibition

Ovalbumin

Invertase

Glucuronidase

40,000

45,000

120,000

290,000

5- 8

1

14

4

+++

++

+

-

-

-

60070

52%

60070

54°70

single 175 kDa protein. Antibodies against the macrophage receptor cross-react with the RPE mannose receptor (Boyle et al., 1991; Shepherd et al., 1991). Fourth, antiserum to the mannose receptor blocks the phagocytosis of ROS (Boyle et al., 1991). Likewise, purified mannose-receptor protein preabsorbed to ROS also inhibits phagocytosis of ROS (Boyle et al., 1991). Fifth, RPE and macrophages both phagocytize yeast which contain mannose-bearing ligands (Benton and Gordon, 1983; Mayerson and Hall, 1986; Sung et al., 1983). This is of interest because yeasts are particulate, mannose-bearing particles and they are also effective inhibitors of ROS binding to RPE cells (Mayerson and Hall, 1986). Finally, it has been demonstrated recently that ROS are avidly phagocytized by macrophages (Shepherd and McLaughlin, unpublished data), and that ROS recognition can be partially inhibited by zymosan (90%), mannan (60%), mannose-BSA (33%) and antimannose receptor antibody (35%). One effective ligand which may be used to help clear up this controversy and which could be used to compete for ROS phagocytosis is horseradish peroxidase (HRP), a mannose-rich glycoprotein. HRP has been used as an effective competitive ligand for mannose receptor-dependent phagocytosis in macrophages (Sung et al., 1983) and recently has been shown to be endocytosed by RPE cells by a mannose-sensitive mechanism (Heth and Bernstein, 1991). HRP and several other mannose-rich glycoproteins have now been used to test their effectiveness as competitive ligands for ROS phagocytosis (Guo et al., 1992;

+++++

+

Lutz et al., 1992). These data show that HRP inhibits both binding and ingestion of ROS by cultured rat RPE cells. The inhibition is both concentration-dependent and reversible with a significant inhibitory effect (or saturation) reached at 100/ag/ml of HRP. The RPE cells recover approximately 70% of their phagocytic capacity in 90 min after removal of HRP from the culture media and approximately 90°70 of their phagocytic capacity after 180 min. Similar inhibitory effects are observed when three other mannose-rich glycoproteins are substituted for HRP as the competitive ligand. When control experiments are done in the presence of albumin, ROS phagocytosis is not inhibited (not shown). Table 1 summarizes these data and provides further support for a mannose receptor-dependent system in ROS phagocytosis.

3.2.3. OTHER RECEPTORS

A novel approach to the study of R P E - R O S interactions has been demonstrated by the two independent laboratories of Hall (Gregory and Hall, 1992) and Cooper (Tien and Cooper, 1989; Guo et al., 1991; Tien et al., 1991). These two groups have used immunological methods to attempt to identify putative receptors for ROS. These studies have been initiated, in part, because extensive molecular analyses of RPE membrane fractions have found nothing noteworthy in the protein compositions of membranes isolated from normal rats and membranes isolated from RCS rats with an inherited defect in phagocytosis

PHAGOCYTOSIS OF ROS BY RPE

(Clark and Hall, 1986; Clark et al., 1986; Cooper et al., 1987; Tien et ai., 1991). These studies have been largely unsuccessful in explaining the absence of phagocytosis in the RCS rats from the gross appearance of protein patterns as observed in oneand two-dimensional polyacrylamide gels. To date, the differences observed between the proteins isolated from the RPE of normal and dystrophic rats have been confined to their oligosaccharide chains (Clark, 1989; Cooper et al., 1987; Tien et al., 1991). Cooper's laboratory has shown differences in the lectin affinities of glycoproteins with molecular weights of 175 and 86 kDa in the RCS rat that appear to be underglycosylated. Clark (Clark and Hall, 1986; Clark, 1989) has identified glycoproteins with molecular weights of 186 and 175 kDa in the RCS rat with reduced levels of fucose content. These studies have indicated that minor alterations in the structure of a RPE membrane glycoprotein might be involved in the inherited retinal dystrophy and have also raised the possibility that minor alterations in epitopes within the polypeptide chains themselves may go undetected with this type of screening. These studies have led to a search for the immunological identity of functional epitopes as a logical next step. While the immunological studies described below have not yet led to a definitive identity of the putative phagocytic receptor, they have already contributed some interesting data concerning the nature of the phagocytic process. The basic assumption of these immunological approaches is that a receptor for some ROS ligand will be present in the plasma membrane of the RPE. Thus, a membrane fraction isolated from the rat RPE cells has been used as an immunogen. The two laboratories have adopted different approaches to the isolation of antigens, and, for the production of polyclonal antiserum, both have had similar success. Hall and his colleagues have used differential centrifugation to isolate a plasma membrane-enriched fraction from cultured RPE (Clark and Hall, 1986) which has been used to immunize New Zealand white rabbits (with the antisera being prepared commercially). Cooper and his colleagues have used a novel, bead isolation technique to obtain an apical membrane enriched fraction from in situ RPE (Cooper et al.,

155

1987; Tien et al., 1991). The proteins of the apical membrane have been fractionated by polyacrylamide gel electrophoresis and then three different molecular weight regions of the gels have been used separately to immunize three different groups of Balb/c mice. The first antiserum (ASPE1) is to RPE proteins in the molecular weight range of 7 0 - 100 kDa of the polyacrylamide gel, the second (ASPE2) to proteins in the 100-200 kDa range, and the third (ASPE3) to proteins in the > 200 kDa range. The resultant antisera from both groups of investigators have been used in a phagocyticinhibition assay in an attempt to perturb the putative receptor- ligand interaction that is assumed to underlie the phagocytic process. In the assay, cultured RPE cells are pretreated for 1 hr with either non-immune or immune antisera. The cells are washed and then fed for 4 hr with ROS that have been previously isolated and labeled with fluorescein isothiocyanate (FITC). FITCROS are counted in the cell cultures using fluorescence microscopy to obtain a total ROS count per unit area. The fluorescence contributed by surface bound ROS is then quenched by washing the cells with Trypan blue. A subsequent count of FITC-labeled ROS provides the number of ROS that have been internalized during the feeding period. When the number of internalized ROS is subtracted from the total ROS count, an estimate of the ROS that are bound to the cell surface is produced. From these three counts of total-ROS, bound-ROS and internalized-ROS, certain aspects of the phagocytic process can be demonstrated. Gregory and Hall (1992) have obtained an antiserum that inhibits the binding of ROS to the RPE and also significantly reduces the ingestion phase. Their demonstration that the antiserum inhibits ROS binding to the RPE, lends support to the possibility that inhibition of ROS binding is causally related to the reduced number of internalized ROS. These data are supportive of a model of phagocytosis in which binding and internalization can be viewed as two phases of a sequential process, with the binding of ROS to a membrane receptor inducing, via some transmembrane signal, the internalization of the receptor - ligand complex.

156

B. J. MCLAUGHLIN et EFFECT OF ANTISERA (1 : 100) ON RPE PHAGOCYTOSIS OF ROS IN THE LE RAT

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PREINCUBATION CONDITIONS r---i TOTAL

~

INGESTED

FIG. 2. Bar graph comparing the effects of three antisera, ASPE1, ASPE2 and ASPE3 on total and internalized ROS using cultured RPE from Long Evans rats. The data are expressed as a percentage of total and internalized ROS observed in control experiments with normal mouse serum (NMS) Note that ASPE2 and ASPE3 affect both total and ingested ROS, whereas, ASPE1 affects only the ingested ROS. The bound ROS (total minus ingested) are reduced by ASPE2 and ASPE3, but increased by ASPE1 (see Table 2). However, the possibility that ROS binding is not the single requisite event causing internalization becomes evident when the phagocytic defect in the RCS rat is considered. In the RCS rat, it has been shown that RPE can bind ROS but that there is little internalization of ROS by the dystrophic R P E (Chaitin and Hall, 1983). This latter state has been mimicked, to some degree, by one of the three antisera (ASPEI) generated in the laboratory of Cooper and his colleagues (Fig. 2). This figure shows that ASPE1 (1:100 dilution) inhibits internalization by 50% but does not inhibit binding. Binding is actually increased by 57~0 in this condition (Table 2). This result is somewhat analogous to the situation inherent in the RCS rat. It remains to be seen if a more concentrated dilution of this antisera can completely inhibit internalization without inhibiting binding. In any case, with A S P E I at this dilution, the data indicate that internalization can be inhibited while binding is unaffected. The antisera, ASPE2 and ASPE3, on the other hand, affect both binding and internalization of ROS by the R P E of Long Evan's (LE) rats (Fig. 2). Striking differences in effects of the three antisera, particularly with respect to binding, are

al.

demonstrated in a study of ROS binding to RPE from the RCS rat (Fig. 3). Here, in the RCS rat RPE, it can be seen that A S P E I has no effect on binding of ROS, but ASPE2 and ASPE3 reduce ROS binding by 78% and 67%, respectively (Table 2). Together, these studies indicate that (1) when binding is affected, internalization is affected; therefore binding probably influences the efficacy of internalization; (2) when internalization alone is affected, an actual increase in binding is observed. Therefore, binding and internalization are most likely two distinctive parts of the phagocytic process which may not be causally related. This idea is supported by the fact that binding and internalization can also be differentiated by their different temperature sensitivities. Hall and Abrams (1987) have shown that binding of ROS increases rapidly from 10 to 17°C, whereas, little internalization takes place at that temperature range. Raising the temperature above 20°C results in a sharp decrease in binding with a rapid increase in internalization. It is noteworthy that there is a striking parallel between the activities of ASPE1 and antibodies to the mannose receptor with regard to binding and internalization. Antibodies to the rat alveolar macrophage mannose receptor inhibit internalization of ROS by the R P E but do not appear to affect binding (see Fig. 1). In fact, binding seems to be greatly increased relative to the control. A similar finding occurs with ASPE1. These data lead to the suggestion that binding and internalization are subserved by two different molecules and raise the interesting possibility that the mannose receptor may be involved in ROS internalization. Clues to the identity of these two molecules can be obtained from the nature of the antigen pools that are used to produce the three antisera. ASPE 1 is derived from proteins in the molecular weight range of 7 0 - 1 0 0 kDa. The protein subserving internalization may therefore be present in this range. The most effective inhibitor of ROS binding is ASPE2, derived from proteins in the 1 0 0 - 2 0 0 kDa molecular weight range. As proteins >200 kDa range also have some affect on binding, it seems likely that the binding protein should be present in both of the immunogens from

PHAGOCYTOSIS OF R O S BY R P E

157

TABLE 2. LE binding

RCS binding

LE ingestion

ASPE1 (70- 100 kDa)

+52%

0

-57%

ASPE2 (100- 200 kDa)

-57%

-78%

-85%

ASPE3 (>200 kDa)

- 53%

- 67%

- 60%

EFFECT ON ANTISERA (1:100) ON RPE PHAGOCYTOSlS OF ROS IN THE RCS RAT

1500. < z D

1350. 1200. 1050900. 750. 600. 450-

m

300-

z

150-

n

0 ASPE1

ASPE2

ASPE3

IgG

NBSS

PREINCUBATION CONDITIONS F'--I TOTAL

I ~ 1 INGESTED

F]G. 3. Bar graph comparing the effects of three antisera, ASPE1, ASPE2 and ASPE3 on ROS binding to cultured RPE from RCS rats. ASPE2 and ASPE3 reduce the number of ROS bound to the surface of the RPE from RCS rats. ASPE1 does not block the binding of ROS to the RPE. which ASPE2 and ASPE3 are derived. Such a protein would have a molecular weight close to 200 kDa. Studies are underway to test the hypothesis that two proteins, a binding protein and an internalization protein, are involved in the phagocytosis of ROS.

4. P O T E N T I A L MEDIATED

LIGANDS

FOR

PHAGOCYTOSIS

RECEPTORBY RPE

4.1. M a n n o s e as a Ligand

A ligand is required for any receptor-mediated interaction to be fully characterized. In the case of

ROS phagocytosis by the RPE, a ligand on the surface of the ROS plasma membrane will potentially be recognized by a receptor on the RPE, followed by binding and internalization o f the ROS. The first studies to show a relationship between mannose-recognition events and ROS phagocytosis were those of the Bernstein group (Philip and Bernstein, 1981; Heth and Bernstein, 1984) who showed that by adding mannose to a phagocytic assay, ROS phagocytosis could be inhibited. Later, a mannose-recognition system was proposed again by Mayerson and Hall (1986) when they observed a preference of RPE cells to phagocytize yeast which have mannose coats. At that time, they suggested that mannose could be involved in recognition, binding and even the ingestion steps of ROS by RPE cells and that rhodopsin, or some other mannose-containing ROS protein, could be the likely candidate. Although there have been a number of studies using lectins to probe for cell surface sugars on ROS which could serve as potential ligands for receptor-mediated phagocytosis by the RPE (Hall and Nir, 1976; McLaughlin and Wood, 1980; Bridges, 1981; Seyfried and McLaughlin, 1983; McLaughlin and Boykins, 1984; SeyfriedWilliams et al., 1984; Tarnowski and McLaughlin, 1987; Molday and Molday, 1987), an identifiable l i g a n d - receptor interaction has remained largely speculative. Now that a mannose receptor for the phagocytic recognition of ROS has been identified on RPE cells (McLaughlin et al., 1987; Tarnowski et al., 1988; Shepherd et al., 1991; Boyle et al., 1991), there continues to be speculation about

158

B. J. MCLAUGHHNet al.

whether the mannose receptor is the RPE phagocytic receptor and whether mannose is the ROS ligand (Hall and Abrams, 1991). These concerns are based on the inability of mannose and mannan to cause an inhibitory action on ROS phagocytosis. The new data presented here, showing that mannose-rich glycoproteins act as very competent inhibitors for ROS phagocytosis, breathe new life into a role for the mannose receptor in ROS phagocytosis.

4.2. Rhodopsin as a Ligand

It is well established that rhodopsin is the major glycoprotein constituent of ROS membranes and contains primarily N-acetylglucosamine and mannose oligosaccharide side chains. Rhodopsin is also present in the ROS plasma membrane with its oligosaccharide groups oriented externally on the surface (Kamps et al., 1982). It is, therefore, the obvious candidate for participating in a mannose recognition system. In the past, a number of studies have attempted to define (Shirakawa et al., 1987; Lentrichia et al., 1987) the role of carbohydrates in the binding of rhodopsin-containing membrane systems to the RPE. In two of these studies, it has been shown that excess mannose or N-acetylglucosamine do not inhibit binding of ROS discs or rhodopsinliposomes. This has led to the conclusion that the major carbohydrate components of rhodopsin, mannose and N-acetylglucosamine, do not act as recognition signals for the binding of rhodopsincontaining membranes. Further studies by Laird and Molday (1988) and by Philp et al. (1988) have demonstrated that glycopeptides released from rhodopsin do not interfere with the binding and phagocytosis of ROS, even when ROS are prelabeled with an N-terminal-specific, rhodopsin monoclonal antibody (Laird and Molday, 1988). These studies have contributed to the popular opinion that rhodopsin is not the ROS ligand that binds to the RPE cells. In spite of the evidence, there has been one report (Kean et al., 1990) showing that purified rhodopsin can bind specifically to the RPE, which is suggestive that there are rhodopsin receptors on RPE cells which might participate in the binding of rhodopsin-

containing membranes. It may be that more definitive experiments need to be done in which membrane-bound or purified rhodopsin has been de-glycosylated and then presented to the RPE as the phagocytic particle, and its effect tested, before rhodopsin and/or mannose can be ruled out as phagocytic ligands.

4.3. Other ROS Ligands for the Mannose Receptor

Since a mannose receptor-dependent interaction is occurring during ROS phagocytosis, a logical next step would be to determine what other candidates are available on ROS plasma membranes to interact with the mannose receptor. Philp et al. (1988) has shown that glycopeptides enzymatically released from whole ROS do interfere with ROS phagocytosis and could act as potential ligands. In addition, whole (unlabeled) ROS are able to competitively inhibit uptake of iodinated ROS very effectively (Laird and Molday, 1988; Philp et al., 1988) suggesting an abundance of potential ligands. More importantly, several mannose-containing glycoproteins are present in ROS plasma membrane isolates in addition to rhodopsin (Molday and Motday, 1987), which could provide a number of ligands for the RPE mannose receptor other than rhodopsin. In order to probe for some of these ligands, ROS plasma membranes have been isolated using a ricin-agarose bead method (Molday and Molday, 1987; Boesze-Battaglia and Albert, 1989). The procedure is begun by first isolating ROS from fresh bovine eyes according to the method of Chaitin and Hall (1983). The ROS are then treated with neuraminidase (Sigma, St Louis, MO). The suspension is diluted and centrifuged and the pellet resuspended in buffer and incubated with ricin-agarose beads. Ricinagarose labeled ROS are then disrupted by hypotonic lysis and the disc membranes dissociated from ROS plasma membranes with trypsin digestion. The ROS membranes are separated by linear sucrose gradient centrifugation followed by elution with buffer containing /3methyl-galactose (Sigma, St Louis, MO). The resulting membrane suspension is solubilized and

159

PHAGOCYTOSIS OF ROS BY R P E

affinity purified overnight on a mannose-receptor sepharose column containing CaCI2. Unbound proteins are removed from the mannose-receptor column by washing the column with buffer containing CaC12. Bound proteins are eluted from the column with buffer containing methyl-a-Dmannoside (Sigma, St Louis, MO) and EDTA. Four different membrane preparations are then characterized by SDS-polyacrylaminde gel electrophoresis. They are: (1) Intact ROS that have been neuraminidase-treated; (2) ROS disc membranes that have been trypsinized following ricin-agarose labeling; (3) ROS plasma membranes that have been isolated with ricin-agarose beads; and (4) ROS plasma membranes that have been isolated with ricin-agarose beads and affinity-purified on a mannose-receptor sepharose column. All samples are prepared at room temperature for 30 min prior to gel electrophoresis (Molday and Molday, 1979) and electrophoresed on a 6 - 15 % polyacrylamide gradient slab gel followed by silver staining (BioRad, Grand Island, NY). For immuno- and lectin-blot analysis, proteins are separated by SDS-PAGE and then electrophoretically transferred to nitrocellulose paper (Bio-Rod, Grand Island, NY) (Towbin et al., 1979). The primary antibody which has been used for this analysis is antirhodopsin monoclonal antibody (MAbKlb-107) provided by Dr P. Hargrave (Gainesville, FL). Blots have been incubated with MAb K16-107 (1:100) in Tris biffered saline (TBS) containing 0.5% Tween 20 for 2 hr at room temperature. Next the blots are incubated with peroxidase-labeled goat antimouse IgG (1:1000) for 1 hr at room temperature. Antibody binding to ROS plasma membrane ligands is visualized by the addition of TBS containing 0.5/~g/ml 3.3' -diaminobenzidine (DAB) and 0.0010/0 hydrogen peroxide (Nakane and Pierce, 1967). For lectin blots, 10 mM Cacl2 and 10 mM MnC12 are added to TBS in all of the procedures. After blocking the non-specific binding sites with 1% bovine serum albumin, blots are incubated with peroxidase-labeled 0.01 ~g/ml concanavalin A (Con A-specific for a-D mannose and a-D-glucose) in TBS for 2 hr at room temperature and lectin binding is visualized by the addition of 0.05% DAB and 0.001% peroxide in TBS.

208]=,.:i ii!i!ii:iii~

iiiil ¸~ iii~i~ii!~~,ii

...........

i~!j~i

100]=" 71~,--

fff 43~"

29~--

a

b

c

d

e

FIG. 4. Silver-stained, SDS-polyacrylamide gel of bovine ROS membrane fractions. Lane a: standard molecular weight; Lane b: intact ROS; Lane c: ROS disc membranes; Lane d: ROS plasma membrane isolated with ricin-agarose beads; Lane e: ROS plasma membrane protein eluted from a mannose-receptor column.

Figure 4 illustrates the silver-stained bands of these membrane fractions. In intact ROS and ROS disc membranes, there is a major dense band with Mr of 36 kDa in the region of rhodopsin. ROS disc membranes show a similar staining pattern to intact ROS although there are fewer bands. ROS plasma membranes isolated with ricin-agarose beads exhibit a different protein pattern. There is a dense band at 36 kDa in the region of rhodopsin and some minor bands with a Mr of 50, 62, 71 and 110 kDa are also visible. In contrast, ROS plasma membrane proteins eluted from a mannosereceptor column show very few bands with a dense band at 36 kDa and two other faint bands with a Mr of 61 and 72 kDa. Con A stained blots (Fig. 5) indicate which silver stained proteins are glycoproteins with oligosaccharides containing mannose or glucose. In the intact ROS preparation, there are numerous stained bands in the region from 45 to 220 kDa in addition to a major dense band at 36 kDa. In ROS disc membranes, there are numerous stained

160

B.J. MCLAU(iHIANel al.

208JP"

208.a=,,~

100~"-

100~

,~

71~="" 71:D,--

k

2 9 J=,,.-

a

b

c

d

e

FIG. 5. Lectin blot analysis of bovine ROS membrane fractions stained with Con A. Lane a: Standard molecular weight; Lane b: intact ROS; Lane c: ROS disc membranes; Lane d: ROS plasma membrane isolated with ricin-agarose beads; Lane e: ROS plasma membrane protein eluted from a mannose-receptor column.

bands but the higher Mr (above 200 kDa) bands are not stained. In the isolated ROS plasma membrane preparation, Con A labels bands with relative Mr of 36, 70, 110 and 130 kDa. Fewer bands are labeled in the ROS plasma membrane preparation eluted from the column, with a major band at 36 kDa and a very faint band at 70 kDa. Western blots with a monoclonal antibody specific for the C-terminus of bovine rhodopsin (MAb k16-107) has been carried out on isolated ROS plasma membrane and ROS protein eluted from mannose-receptor column to determine if the 36 kDa protein is rhodopsin (Fig. 6). A single immunoreactive band at 36 kDa is present in the isolated ROS plasma membrane preparation, as well as in the ROS plasma membrane protein eluted from the mannose-receptor column. In summary, this study shows that rhodopsin and at least two other mannose containing glycoproteins on ROS plasma membranes bind to the

18~=,.-

a

b

c

FIG. 6. lmmuno blot analysis of bovine ROS membrane fractions with monoclonal antibody to C-terminus of bovine rhodopsin (K16-107) Lane a: Standard molecular weight; Lane b: ROS plasma membrane isolated with ricin-agarose beads; Lane c: ROS plasma membrane protein eluted from a mannose-receptor column.

mannose-receptor column and could be potential ligands for the phagocytic interaction. Only functional studies using phagocytic assays will determine if these proteins are actually involved in mediating the binding and internalization of ROS by the RPE mannose receptor.

5. C O N C L U S I O N S / F U T U R E D I R E C T I O N S The evidence presented here clearly demonstrates that ROS phagocytosis by the RPE is receptor-mediated and involves a glycoprotein interaction. The data further suggest that a mannose-receptor system is operating during some phase of the phagocytic interaction, which may facilitate the uptake of rhodopsin-containing

PHAGOCYTOSIS OF ROS BY R P E

membranes but which may not be involved in the initial binding or recognition steps. It must be remembered that macrophages have more than one receptor type, with each receptor targeted for uptake of a specific particle. It is possible that in the case of RPE phagocytosis, several receptor types are targeted for different steps in the phagocytic uptake of one particle, the ROS. This idea is supported by a number of studies which have implicated several RPE membrane glycoproteins in the ROS phagocytic interaction, in addition to those in the 175 kDa Mr range of the mannose receptor (Colley et aL, 1987; Cooper et al., 1987; Clark and Hall, 1986; Clark, 1989; Hall et aL, 1990; Tien et aL, 1991; Gregory and Hall, 1992). New data presented here suggests that binding and internalization may be subserved by different receptor proteins. Circumstantial evidence suggests that the mannose receptor may be functionally recruited during ROS internalization, perhaps to interact with the mannosylated side chains of rhodopsin in the forming phagosome. Future studies will be directed towards identifying which RPE proteins subserve binding and internalization and towards determining how the mannose-receptor protein is related to these two events. Acknowledgements- The authors would like to thank Dr Douglas Lutz for providing the data on horseradish peroxidase inhibition of ROS phagocytosis; Ms Fang Yan for providing the data on ROS ligands for the mannose receptor; and Mr YiHe Guo for the immunological characterization of candidates for other RPE phagocytic receptors. We thank also Mr Jim Musick for his expert assistance in preparing the chapter. This work was supported by NIH grants EY02853 (BJM) and EY02708 (NGFC); by the Kentucky Lions Eye Research Foundation; The Baptist Memorial Hospital, Memphis, TN, and by an unrestricted grant from Research to Prevent Blindness, Inc.

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161

BOHNSACK, J. F., KLEINMAN,H. K., TAKAHASHI,T., O'SHEA, J. J. and BROWN, E. J. (1985) Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Ec-mediated phagocytosis by cultured human macrophages. J. exp. Med. 161: 912-923. BOESZE-BATTAGLIA, K. and ALBERT A. D. (1989) Fatty acid composition of bovine rod outer segment plasma membrane. Expl Eye Res. 49: 699. BOYLE, D. L. and McLAUGHLIN, B. J. (1990) The effect of swainsonine on the phagocytosis of rod outer segments by rat RPE. Curr. Eye Res. 9(5): 407-414. BOYLE, D. L., TIEN, L., COOPER, N. G. F., SHEPHERD,V. and MCLAUGHLIN, B. J. (1991) A mannose receptor is involved in retinal phagocytosis. Invest. Ophthalmol. Vis. Sci. 32(4): 1464-1470. BRIDGES, C. D. B. (1981) Lectin receptors of rods and cones: visualization by fluorescent label. Invest. Ophthalmol. Vis. Sci. 20, 8 - 16. CHAITIN, M. H. and HALL, M. O. (1983) Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 24: 812. CHAKRAVARTI, B. and CHAKRAVARTI D. N. (1987) Phagocytosis: An overview. Path. Immunopath. Res. 6: 316-342. CLARK, V. (1989) 3H-Fucose incorporation into RPE cell surface proteins of normal and dystrophic rats. Invest. Ophthalmol. Vis. Sci. 30: 1542-1547. CLARK, V. and HALL, M. O. (1986) RPE cell surface proteins in normal and dystrophic rats. Invest. Ophthalmol. Vis. Sci. 27: 136-144. COLEMAN, D. L. (1986) Regulation of macrophage phagocytosis. Eur. J. clin. Microbiol. 5(1): 1 - 5 . COLLEY, N. J., CLARK,V. M. and HALL, M. O. (1987) Surface modification of retinal pigment epithelial cells: Effects on phagocytosis and glycoprotein composition. Expl Eye Res. 44:377 - 392. COOPER, N. G. F., TARNOWSKI,B. I. and MCLAUGHL1N,B. J. (1987) Lectin-affinity isolation of microvillous membranes from the pigment epithelium of rat retina. Curt. Eye Res. 6: 969. CUSTER, N. V. and BOK, D. (1975) Pigment epitheliumphotoreceptor interactions in the normal and dystrophic rat retina. Expl Eye Res. 21:153 - 166. CzoP, J. K. (1986) Phagocytosis of particulate activators of the alternative complement pathway: Effects of fibronectin. Adv. Immun. 38: 361- 398. CzoP, J. K. and AUSTEN, K. F. (1985) /3-Glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway. J. Immun. 134: 2588 - 2593. EDWARDS, R. B. (1991) Stimulation of rod outer segement phagocytosis by serum occurs only at the RPE apical surface. Expl Eye Res. 53:229 - 232. EDWARDS, R. B. and SZAMIER, R. B. (1977) Defective phagocytosis of isolated rod outer segments by RCS rat retinal pigment epithelium in culture. Science. 197: 1001 - 1003. ELNER, V. M., SCHAFENER,T., TAYLOR, K. and GLAGOV, S. (I 981) Immunophagocytic properties of retinal pigment epithelial cells. Science 211(2): 74 - 76. ELNER, S. G., ELNER, V. M., NIELSEN, J. C., TORCZYNSKI,E., Yu, R. and FRANKLIN, W. A. (1992) CD68 antigen

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