Exp. Eye Res. (1991) 53 , 765-771


Antibodies which Recognize Endoplasmic in the Retinal Pigment Epithelium HIROSHI







of Fine Morphology, The University of Tokyo, Institute of Medical Science, 4-6-l Shirokanedai, Minato-ku, Tokyo 108, Japan

(Received 28 September

7990 and accepted in revised form 31 January


Balb/c mice were immunized with dissociated chick retinal pigment epithelial cells and monoclonal antibodies prepared. Retinas of chick, bovine, rabbit, rat and mouse were examined with the monoclonal antibodies by immunofluorescence microscopy. Among the antibodies obtained, two (S5D8 and S5H8) stained the cytoplasm of epithelial cells of the animals examined in finely granular pattern and recognized a 63-kDa protein in retinal pigment epithelial cells. These antibodies, however, did not react with the rest of the chick eye (cornea, iris, ciliary body, lens and sclera) nor with extraocular tissues (liver, kidney, intestine, brain, testis, adrenal gland, heart, gizzard and skeletal muscle). Immunoelectron microscopy revealed that the antigen was localized in the endoplasmic reticulum of retinal pigment epithelial cells. Key words: retinal pigment epithelium ; smooth endoplasmic reticulum ; monoclonal antibody ; chick retina ; immunohistochemistry : immunoelectron microscopy.

epitopes restricted to membranes of the endoplasmic reticulum (ER) of RPE cells.

1. Introduction Retinal pigment epithelial (RPE) cells have a wide

variety of functions such as absorption of excesslight, exchange of metabolites between the neural retina and choriocapillaris, vitamin A metabolism and phagocytosis of shed outer segmentsof photoreceptors (Bok and Young, 1979; Young and Bok, 1979). Adapted to these functions, the RPEcells have several unique plasmalemmal and intracellular structures such as apical projections, basal infoldings and abundant smooth endoplasmic reticulum (Porter and Yamada, 1960; Hudspeth and Yee, 1973; Kuwabara, 1979). Correlation of the structure of these cells with their polarized function is an area which has been actively investigated. For example, retinol bindingprotein receptors have been localized autoradiographically to the basolateral plasmalemma (Bok and Heller, 1976), Na+-, K+-ATPase to the apical plasma membrane (Jaffe,Burke and Geroski, 1989) and transferrin receptors to cell surfaces (Hunt, Dewey and Dowis, 1989). The recent development of the monoclonal antibody (MAb) techniques (KGhler and Milstein, 1975) has allowed several investigators to begin localizing specific protein antigens of the pigment epithelium. For example, generation of MAbs against RPE cells from several species has been reported (Donoso et al., 1988; Hooks et al., 1989; Chu and Grunwald, 1990 : Klein, Macleish and Wiesel, 1990). With the accumulation of MAbs specific to RPE cells and knowledge of the morphological differentiation of these cells, a better understanding of the interrelationship between structure and function may be gained. In the present study, we describe the generation, characterization and immunohistochemical localization of two MAbs, SSD8 and SSH8, which recognize 00144835/91/120765+07


2. Materials and Methods

Preparation of Monoclonal Antibodies White Leghorn chickens (from a local supplier) were killed by an overdose of sodium pentobarbital and, following enucleation, eyes were dissected into anterior and posterior halves, Sheets of RPE cells were peeled from the retina in phosphate-buffered saline (PBS) containing 2 mM EDTA and dissociated into single cells. Balb/c mice were immunized by intraperitoneal injection of RPE cells (2 x 10“ cells ml-l), after being emulsified in complete Freund’s adjuvant. Three weekslater, mice were boosted with an intraperitoneal injection of RPE cells. Three days after the booster, mice were killed and the spleensremoved. Dissociated spleen cells were then fused with myeloma cells (Ag 8-653) using polyethylene glycol 4000 (Merck, Germany). The supernatants of the resulting hybridomas were screenedfor antibody reactivity with RPE by immunofluorescence microscopy of chick eye frozen sections. Two cultures (S5D8 and SSH8) reactive with the cytoplasm of RPEcells were cloned by the method of limiting dilution. Ascites fluids were obtained in pristane-primed Balb/c mice by the intraperitoneal injection of antibody-secreting cloned hybridoma cells. These ascites were used as the source of antibodies for immunofluorescence microscopy and immunoelectron microscopy. Affinity-purified IgG of the MAbs was used for Western blot analysis. Immunojluorescence Microscopy

For immunocytochemical analysis, the eyes of 0 1991 Academic Press Limited


chicks, cows, rabbits, rats and mice were used, as well as several tissues and organs of chick, including liver, kidney, intestine, brain, testis, adrenal gland, heart, gizzard and skeletal muscle. Tissues were fixed in PLP fixative (2 % formaldehyde, 0.01 M sodium m-periodate, 0.075 M lysine, 0.0375 M phosphate buffer : McLean and Nakane, 19 74), or in 0.1% glutaraldehyde, 2 % formaldehyde in 0.1 M phosphate buffer, pH 7.4, for 4-6 hr at room temperature. They were then rinsed in PBSand cryoprotected by infusion with 30% sucrose in PBS. Frozen sections (about 10 ,um thick) were cut and mounted on poly+lysine coated glass slides (Mazia, Schatten and Sale, 1975). Slides were washed in PBSand successivelytreated with 1% glycine for 5 min and with 1% bovine serum albumin (BSA) for 10 min to reduce non-specific antibody binding. Sections were then incubated with primary antibodies (SSD8,SSH8) (1:200-l : 1600 dilution) for 30 min and, after three washes in PBS they were labelled with FITC-conjugated goat anti-mouse IgG (1: 100, MBL, Japan) for 2 hr at room temperature. Slides were washed four times in PBS, mounted and viewed with a fluorescence microscope (Olympus BH2, Japan). Sections stained without the primary antibodies served as negative controls. ImmunoelectronMicroscopy Indirect immunoperoxidase methods were used for ultrastructural localization of antigen in RPE cells. Chick retinas fixed in the same way as in the immunofluorescence studies were washed and embedded in agar before being cut into SO-pm thick sections with a Vibratome. Sections were washed with three changes of PBSand treated with 0.05 % saponin in PBScontaining 1% BSA. They were then incubated overnight with MAb S5D8 (1: 200, diluted with PBS containing 0.05 % saponin and 1% BSA). After several washes in PBS,the sections were incubated with HRPconjugated goat anti-mouse IgG (Fab’) (MBL) for 6-8 hr. After washing, they were processed for diaminobenzidine (DAB) reaction. The sections were post-tied in phosphate-buffered 1% osmium for 1 hr, dehydrated and embedded in Epon. Unstained thin sections were examined in an electron microscope (JEOL 1200EX). Some sections were lightly stained with uranyl acetate (0.1% in 70% ethanol for 1 min) to distinguish rER from SER. BiochemicalCharacterization Dissociated chick RPE cells were homogenized with a teflon-glass homogenizer in buffer solution (20 mM Tris, 5 mM MgCl,, 1 mM EDTA and 1 mM dithiothreitol) and centrifuged at 100 g for 5 min to remove nuclei and unlysed cells. The supernatant was removed and further centrifuged at 10000 g for 10 min and the resulting pellet washed twice. After being mixed with an equal volume of sample buffer




(124 mM Tris-HCI, pH 6.8, 4.6% SDS, 50 mM dithiothreitol, 60% glycerol) and dissolved by boiling, samples were run on an SDS-PAGE ( 12 % polyacrylamide gel). The separated proteins were transferred to Hybond-C extra membrane (Amersham, U.K.). Transblots were incubated with the MAbs (1:lOOOO for SSD8, 1:200 for S5H8) for 1 hr, followed by alkaline phosphatase-conjugated antimouse IgG (1: 200, BioRad, U.S.A.) for 1 hr. The antibody-reactive bands were visualized using nitroblue tetrazolium (NBT) as the chromogen. Ouchterlony double-diffusion test against typespecificrabbit anti-mouse antisera showed that both of the MAbs (S5D8 and SSH8) belonged to the IgG, subclass. 3. Results Staining Pattern by Light Microscopy When viewed by immunofluorescence microscopy, the cytoplasm of RPE cells was intensely stained with SSD8 in a finely granular pattern. Diffuse staining was also observed in the apical processes: however, the nuclei were not labelled and looked like holes against the fluorescent cytoplasm. The rest of the retina was not stained with S5D8 [Fig. 1 (B)]. A similar pattern of RPE immunoreactivity was observed with another MAb (SSH8). However, S5H8 also stained the inner segments of some photoreceptors [Fig. 1 (C)l. The stained region of the inner segment was proximal to the oil droplets and, judging from the phase contrast views of frozen sections [Fig. 1 (A)] and Toluidine bluestained plastic sections, seemed to coincide with the paraboloid of cone photoreceptors (manuscript in preparation). There was no labelling of RPEcells when the primary antibody was omitted [Fig. 1 (D)]. Staining Pattern by Electron Microscopy

Because immunofluorescent staining with S5D8 was more intense than with SSH8, ultrastructural immunocytochemistry of RPE cells was carried out with S5D8 as the primary antibody. DAB reaction product was generally observed on the membranes of cytoplasmic tubular structures [Fig. 2 (A)]. The immunoreactive structures appeared to correspond mostly to the smooth endoplasmic reticulum (sER), which is abundant in these cells. Reaction product was also seen in the area of the cytoplasm near immunolabelled SER.The latter may be due to some redistribution of DAB reaction products during tissue processing. In these cells, the rough endoplasmic reticulum (rER) is sparsely distributed among the sER in the apical cytoplasm. Though it was difficult to distinguish rER from sER in unstained sections, it becamepossible in sections lightly stained with uranyl acetate (0.1 y0 in 70% ethanol for 1 min). In these sections. rER also appearedto be immunoreactive [Fig. 2(B)]. Nuclear membrane, rarely studded with ribo-





FIG. 1 . Immunofluorescence micrographs of frozen section of chick retina stained with MAbs SSD8 and S5H8. OLM, Outer lir niting membrane ; BM, Bruch’s membrane (the position is indicated with a horizontal line in each figure) ; P. the area occ:upied b r pigment granules. A, Phase contrast micrograph of chick retina (light-adapted). RPE cells rest on Bruch’s membrane @Ml. Tlieir aI)ical processesare characterized by abundant pigment granules (P). Cone photoreceptors contain oil droplets (axx-ows) in their iInner segments.B, Staining of chick retina with S5D8 (1: 1600 dilution). The cytoplasm of RPEcells is intensely latjelled, wrhile n.uclei are unreactive (arrows). No other retinal cells show positive staining. C, Chick retina stained with MAb SSH8 (I1:200 dilution). Both the cytoplasm of RPE cells and the paraboloids (arrowheads) of photoreceptors are labelled. Nut lei are n ot stai ned (arrows). D, Primary antibody is replaced with PBS. No fluorescence is observable. Bar represents 100 ,~rn

somes in these cells, was not reactive. Neither apical

nor basolateral plasma membranes, including basal infoldings, were stained. Nuclei, Golgi complexes and mitochondria were generally negative for immunoreactivity as well. However, some mitochondrial outer membranes exhibited DAB reaction product, but this staining was confined to regions immediately adjacent to the immunopositive SER.The sER was sometimes observed in apical processes, in which case these membranes were also immunopositive

[Fig. 2 (C)l. At

the base of the apical processes,small myeloid bodies (lenticular accumulation of sER)were stained more or less uniformly [Fig. 2 (C)l. However, large myeloid bodies in the cytoplasm appeared immunoreactive only at their periphery [Fig. 2(A)]. This centrenegative profile may be due to insufficient infiltration of the primary or HRP-conjugated antibodies into the

interspace between the cisternal stacks of the myeloid bodies. Specificity of MAbs SSD8 and SSH8

The reactivity of S5D8 and S5H8 for tissues other than retinas was assayed by FITC-immunofluorescence microscopy. Retinas of other animals examined (cow, rabbit, rat and mouse) showed positive staining with S5D8 (Fig. 3, and Table I). MAb S5H8 also labelled bovine, rabbit and mouse RPE cells, but failed to stain those of rat. In all cases,retinal labelling abruptly disappeared at the position of the ora serrata (data not shown). Anterior portions of the chick eye (which included cornea, iris, ciliary body, lens, and sclera) were not stained. Other tissues and organs of chick (liver, kidney, intestine. brain, testis, adrenal





FIG 2. Immunoelectron micrographs of RPE cells stained with MAb S5D8. A, DAB reaction product is mainly localized around tubular elements in the cytoplasm. Myeloid body (MB) is reactive only at their surface. Nuclear membrane (NM) is unlabelled. N. Nucleus of RPE cell; M, mitochondria; OS, outer segment of photoreceptor. Bar represents 1 jlrn. B, Immunoreactive rER (arrows) in the apical cytoplasm of a RPE cell. This section is stained with uranyl acetate solution. Bar represents 0.5 /Lrn. C, Cross section of apical processes containing small myeloid bodies (double arrows) and sER (arrows). Small myeloid bodies and sER are positively stained with MAb SSD8. Bar represents 1 LLrn.

FIG. 3. Frozen sections of bovine retina stained with MAb SSDS. Phase contrast micrograph (A) and FITCimmunofluorescence micrograph (B) of the same field are shown. The cytoplasm of RPE cells are stained intensely, while nuclei remain unstained (arrows), as was the case with chick retina. Connective tissue of the choriocapillaris (ch) is weakly stained. Spot-like fluorescence in the neural retina (double arrows) are retinal vessels, which are fluorescent in specimens without the primary antibody (data not shown). ILM, Inner limiting membrane. Bar represents 100 ym.




TO RPE Western




Reactivity of chick tissues to antibodies S5D8 and S5H8 Reactivity of monoclonal antibodies Tissue




SSD8 46

RPE Chick Bovine Rabbit Rat Mouse


+ + + +

+ f + + +









Other parts of chick eye

Cornea Iris Ciliary body Lens Sclera

Chick other organs Liver

Kidney Intestine Brain Testis Adrenal gland Heart Gizzard Skeletal muscle

gland, heart, gizzard and skeletal muscle) were not labelled with either S5D8 or S5H8 (Table I). Biochemical Characterization of Antigens SDS gel electrophoresis and immunoblot analysis were carried out to determine the molecular size of the antigens recognized by SSD8 and S5H8 using cellular membranes of chick RPE cells. Both of the MAbs (S5D8 and S5H8) reacted with a single polypeptide band which had an apparent molecular weight of 63 kDa (Fig. 4). 4. Discussion To date, five studies have appeared reporting the isolation of MAbs raised against the RPE cells. Donoso et al. (1988) produced two MAbs which recognized a 72-kDa antigen protein and which reacted with plasma membranes of bovine RPE. Antibodies obtained against bovine RPE cells (McKechnie et al.. 1988) have been used in immunocytochemical experiments which indicate that the RPE shares certain characteristics with glandular epithelium and myoepithelium during cell migration and epiretinal membrane formation. MAbs against human RPE cells have also been reported (Hooks et al., 1989). These MAbs, one of which recognized a 42-kDa antigen protein in Western blots, stained the cytoplasm of bovine RPE cells. Chu and Grunwald (1990) reported four MAbs

FIG. 4. SDS-PAGEand western blot analysis of chick RPE cell membranes. The SDS-PAGEgel stained with coomassie blue shows several major bands, one of which has a relative molecular mass of 63 kDa. Both MAb S5D8 and S5H8 stain this 63 kDa protein. Bovine serum albumin is 96 kDa, and 46 kDa is ovalbumin


raised against embryonic chick RPE cells. They showed that these MAbs could serve as indicators of cellular differentiation at distinct stages of development. Most recently, antibodies have been generated which are specific for mature amphibian RPE cells (Klein et al., 1990). In the present study, we report on two MAbs which recognize a 63-kDa protein in Western blots. The antigen(s) recognized by these MAbs are evidently different from those identiiled in the earlier studies. Although S5D8 and SSH8 stain proteins of almost the same size by SDS-PAGE, we have as yet no evidence to indicate that they recognize different epitopes of the same protein, or different proteins of the same size. The only differences we have noted is that, unlike SSD8, S5H8 stains paraboloids of photoreceptors in addition to the RPE. Immunoelectron microscopy with SSD8 revealed the precise localization of the antigenic protein in the cytoplasm of RPE cells. Although DAB reaction product may occasionally have undergone diffusion, precipitating out onto different types of cell organelles, labelling was mainly localized to the SER. Membranes of this organelle. which elsewhere have been ascribed functions as diverse as drug and metabolite detoxification and metabolism of steroids (Remmer and Merker, 1965 ; Jones and Fawcett, 1966 ; Nussdorfer, 1980) are especially prominent in RPE cells (Porter and Yamada, 1960). Although, they often have been assumed to play a role in the metabolism of retinoids (Hirosawa and Yamada, 19 76 ; Young and Bok, 19 79 ; Berman et al., 1980), there is currently no immunohistochemical evidence to show the localization within


these structures of enzymes involved in the vitamin A metabolism. The sER of the liver also participates in the metabolism of vitamin A (Futterman and Andrews. 1964). However, our MAbs did not stain liver membranes. Therefore, we speculate that the MAbs recognize proteins specific for sERin the RPE cells. In the chick RPE, sER regionally piles up to make a peculiar structure named myeloid body. This structure is found only in RPE cells of fish, amphibians, reptiles and birds (Porter and Yamada, 1960 : Yamada, 196 1; Nguyen and Anh, 1971; Braekevelt, 1980). In our study, MAb SSD8 also stained myeloid bodies. We have no functional evidence yet to indicate whether the myeloid body is a type of SER.MAbs obtained here would serve to elucidate functions of myeloid bodies in RPE. DAB reaction product was also observed in rER. In the chick RPE cells, rER is small in number and sometimes continuous to the sER or small myeloid bodies. This labelling is not surprising because the antigenic proteins are synthesized in rER. One of the MAbs presented here (SSD8), like that of a previous study (Gaur et al., 1989), stains the paraboloid region of photoreceptors. The paraboloid is composedof sERpacked with glycogen granules in the inner segment of visual cells and is believed to play a role in glycogen metabolism in photoreceptors (Rodieck, 1973). These MAbs may thus prove to be valuable tools in studying the function of this organelle. Such investigations are currently in progress in our laboratory. The importance of RPE cells in maintaining visual function has long been recognized (Young and Bok, 1979). It is our hope that studies of the molecular constituents of these cells, such as those using MAbs, will help to elucidate the nature of these critical functions. Acknowledgements This study was supported in part by grants from the Ministry of Education, Science and Culture (01304026, 63570006).

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Monoclonal antibodies which recognize endoplasmic reticulum in the retinal pigment epithelium.

Balb/c mice were immunized with dissociated chick retinal pigment epithelial cells and monoclonal antibodies prepared. Retinas of chick, bovine, rabbi...
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