January 2002
Volume 43, Issue 1
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Retinal Cell Biology  |   January 2002
Retinal Pigment Epithelium of the Rat Express CD81, the Target of the Anti-proliferative Antibody (TAPA)
Author Affiliations
  • Eldon E. Geisert, Jr
    From the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee.
  • Haley J. Abel
    From the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee.
  • Liying Fan
    From the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee.
  • Grace R. Geisert
    From the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee.
Investigative Ophthalmology & Visual Science January 2002, Vol.43, 274-280. doi:
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      Eldon E. Geisert, Haley J. Abel, Liying Fan, Grace R. Geisert; Retinal Pigment Epithelium of the Rat Express CD81, the Target of the Anti-proliferative Antibody (TAPA). Invest. Ophthalmol. Vis. Sci. 2002;43(1):274-280.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The present study focuses on the role of CD81, the target of the anti-proliferative antibody (TAPA), in the regulation of the growth of retinal pigment epithelium (RPE).

methods. RPE of 8-day-old rat pups was cultured. The level of CD81 in the cultures was defined by immunoblot methods, and the distribution of the protein was examined using indirect immunohistochemical methods. In addition, the effects of the antibody binding were tested in culture.

results. CD81 was found in all layers of the normal retina with a distinct absence of labeling in the inner and outer segments of the photoreceptors. Based on the authors’ original immunohistochemical analysis, it was difficult to determine whether CD81 was expressed by RPE. By examining cultures of RPE it was demonstrated that CD81 was expressed on the surface of these cells and that it was concentrated at regions of cell–cell contact. Indirect immunohistochemical methods using a peroxidase-labeled secondary antibody in albino mice revealed heavy labeling of the RPE in the intact eye. When the AMP1 antibody (directed against the large extracellular loop of CD81) was added to cultured RPE, the mitotic activity of the cells was depressed.

conclusions. CD81 was found in the normal rat retina. Previous studies demonstrated that CD81 was expressed in retinal glia, the Müller cells that span the thickness of the retina, and astrocytes found in the ganglion cell layer. The present study demonstrated that CD81 was also expressed by RPE. The dramatic effects of the AMP1 antibody and the location of CD81 at regions of cell–cell contact support the hypothesis that this molecule is part of a molecular switch controlling contact inhibition.

The response of the mammalian retina to injury is similar to that occurring in other parts of the central nervous system. Like the brain and spinal cord, the retina contains glial cells (astrocytes in the ganglion cell layer, and Müller cells that span the cellular layers) which display a reactive response after injury. 1 2 The astrocytes and Müller cells can hypertrophy and increase the expression of the intermediate filament protein, glial fibrillary acidic protein (GFAP). 3 4 5 6 The retinal glia and retinal pigment epithelium (RPE) can proliferate after injury. For example, when the retina becomes detached, two distinct sets of processes can occur. First, the Müller cells can send their processes into the subretinal spaces between the photoreceptors and RPE causing a glial scar. 7 This scar appears to contribute to the absence of retinal reattachment and the death of photoreceptors. 8 Second, the retinal glial cells and pigment epithelium can also migrate into the vitreal space, proliferate, and form cellular membranes. 9 This response is known as proliferative vitreoretinopathy, and when these cellular membranes contract, the retina can detach. 10 Proliferation of non-neuronal cells in the retina is a common and deleterious feature of both disease and injury, including diabetes, retinal detachment, 7 photocoagulation, 5 proliferative vitreoretinopathy, 1 10 and mechanical injury. 6 11 Thus, reactive glial responses can have serious consequences, potentially resulting in the loss of sight. 
Understanding molecular mechanisms associated with these changes may provide insights into interventions, which may stop or reverse the detrimental effects of reactive gliosis and cellular proliferation in the retina. The present proposal focuses on the role of CD81, previously known as the target of the anti-proliferative antibody (TAPA). CD81 is a member of the tetraspanin superfamily that consists of an increasing number of members (CD9, CD37, CD53, CD63, TAPA-1[ CD81], CO-029, R2, CD82 [KAI1], CD151, Tspan 1–6, NAG-2, late bloomer, and NET1–7). 12 13 14 15 16 17 18 19 20 21 As a family, the tetraspanins appear to be part of a molecular complex 13 22 23 24 linking cell–cell contact to changes in cellular behavior. In the present study we examine the distribution of CD81 in RPE and define its role in the proliferation of these retinal cells. 
Materials and Methods
Culturing RPE and Antiproliferative Assays
The RPE cells were cultured according to the methods described by Edwards. 25 For these experiments, Long-Evans rats (two litters of rat pups) were used, allowing us to easily identify pigment epithelium due to the presence of brown pigment. Rat pups (6–9 days after birth) were deeply anesthetized with hypothermia and the eyes removed. The globes were placed in the dark at room temperature overnight. The next day the anterior segment of the eye was removed, and the eyes were digested with a combination of trypsin and collagenase. Sheets of RPE were peeled off of Bruch’s membrane and placed in Hanks’ balanced salt solution. At this point, a few of the dissected sheets of RPE were fixed in 4% paraformaldehyde and placed aside for light or electron microscopic examination. The remaining sheets of RPE were treated with 0.1% trypsin in 5 mM EDTA for 10 minutes to create a suspension of single cells. The RPE cells were plated at a density of 5 × 103 cells/cm2 into T-75 culture flasks. The cells were allowed to grow to confluence in Basal Medium Eagle (BME) with 10% fetal calf serum. 
To begin the cell growth experiments, confluent cultures of RPE were treated with 0.1% trypsin in 5 mM EDTA for 10 minutes to create a suspension of single cells. The cells were then plated at a density of 3 × 103 cells/cm2 onto 18-mm diameter poly-l-lysine (PLL)–coated coverslips in 12-well culture dishes. All the cultures were maintained in BME with 2% heat-inactivated fetal calf serum. One day after the initial plating, the cells were transferred into one of the experimental media containing 2% fetal calf serum plus the treatments: no antibodies added, 250 μg nonimmune mouse IgG1 (ICN Immunobiologicals, Lisle, IL), 250 μg/mL of the monoclonal antibody 13-38 (directed against rat neural cell adhesion molecule [N-CAM] 26 ); or 2 μg/mL, 50 μg/mL, or 250 μg/mL anti-microbial protein (AMP)-1 (directed against CD81 27 ). The cells remained in this medium for the next 24 hours. Then BrdU was added to a final concentration of 10 μM and the cells remained in this solution for an additional 24 hours. Four coverslips were used for each treatment condition. After the treatment period was over, the cultures were fixed in 4% paraformaldehyde and processed for BrdU immunohistochemistry. The cultures were rinsed in PBS and incubated in formamide/2× SSC for 2 hours at 65°C. After several rinses in 2× SSC the cells were incubated in 1 N HCl for 30 minutes at 37°C and rinsed again in borate buffer. The cells were incubated in a monoclonal antibody directed against BrdU (G3G4; Developmental Studies Hybridoma Bank, Iowa State University, Iowa City, IA) in PBS with Triton X-100 and 10% fetal calf serum at 4°C overnight. The cultures were rinsed, placed in peroxidase-labeled secondary antibody, and reacted (described later). Then the cultures were counterstained using toluidine blue. 
Immunohistochemistry
Indirect immunohistochemical methods were used to define the cellular localization of CD81 in cultured cells, sections of retina and dissected sheets of RPE. Cultured RPE cells were produced as described. For sections of retina, both Long-Evans (two adult female rats) and Sprague-Dawley (two male) rats were used. Adult rats were anesthetized with a mixture of xylazine (13 mg/kg) and ketamine (87 mg/kg) which was administered by intraperitoneal injection. The rats were perfused through the heart with a solution of 0.1 M PBS (pH 7.5) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.5). All the protocols used in this study were approved by the Animal Care and Use Committee of the University of Tennessee, Memphis, and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. After the eyes were removed from the skull, the cornea was dissected from the globe, and the lens was removed. The eyes were placed in a 30% sucrose solution for at least 2 days. Cryostat sections were taken at 30 μm (Reichert Histostat, Buffalo, NY). 
In general the immunohistochemical methods used were similar, with only minor variations depending on the specific application. The sections were blocked, for 2 hours at room temperature, with 4% BSA (Sigma, St. Louis, MO) in 0.2 M borate-buffered saline (BBS, pH 8.4). The sections were then incubated in the monoclonal antibody AMP1 27 at a dilution of approximately 20 μg/mL of 0.2 M BBS with 1% BSA overnight at 4°C. Sections were washed three times for 10 minutes each in 0.2 M BBS and incubated with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at a dilution of 1:250 in BBS for 2 hours at room temperature. Sections were washed in BBS followed by 0.1 M PBS, three times for 10 minutes each, and incubated in a solution containing 25 mg of 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) per 50 mL phosphate buffer (pH 7.2) and 200 μL 3% hydrogen peroxide for 15 to 30 minutes at room temperature. When staining cultures of RPE or dissected sheets of RPE a fluorescence-labeled secondary antibody was used (Jackson ImmunoResearch Laboratories). 
For electron microscopic examination, the dissected RPE was rinsed in PBS and stained with the AMP1 antibody, followed by a conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.). After the cells were reacted with DAB, they were postfixed in 1% osmium tetroxide for 1 hour at room temperature. The cells were rinsed in PBS, dehydrated, and infiltrated with Spurr embedding media. Silver to gold sections were obtained on a microtome (Ultracut E; Reichert Histostat) and the sections were examined using an electron microscope (2000EX; JEOL, Tokyo Japan). 
SDS-PAGE and Immunoblot Analysis
To analyze the levels of CD81 in the retina and RPE, an immunoblot method was used. 27 Protein samples were taken from four normal eyes (two Sprague-Dawley rats) or cultured RPE and placed in nonreducing sample buffer (2% SDS, 10% glycerol in 0.05 M Tris-HCl buffer [pH 6.8]). The protein samples were balanced and approximately 70 μg protein was run on 4% to 16% acrylamide gels, using a protein gel apparatus (Mini Protein II; Bio-Rad, Richmond, CA). The proteins were transferred to nitrocellulose, and the blots were blocked in borate buffer (pH 8.4), containing 5% nonfat dry milk and probed with the primary antibody. After a rinse in borate buffer, the blots were incubated in peroxidase-labeled secondary antibody, rinsed extensively, and reacted with 0.05% DAB and 0.01% hydrogen peroxide. 
Results
The overall goal of the present study was to determine whether RPE cells express CD81. Our previous studies 28 revealed that CD81 is expressed in the normal rat retina. This initial study used immunofluorescence to study the distribution of CD81 in the retina and the intense autofluorescence from the outer segments made it extremely difficult to determine whether the RPE expressed CD81. As a first approach to defining the expression of CD81 by RPE, we stained sections of the albino retina for CD81 using immunoperoxidase methods. This method eliminated the problem of autofluorescence associated with immunofluorescence methods. In general, there was a dense reticular pattern of immunolabeling throughout the retina that outlines the cell bodies of the neuronal elements within the retina (Fig. 1) . As observed earlier, there was a clear labeling of the outer limiting membrane, indicating a labeling of Müller cells. In an unexpected finding, the most immunopositive region within the albino retina was the pigment epithelium. These cells were densely labeled, and this labeling extended into the most distal part of the outer segments. All surfaces of the RPE were labeled with the anti-CD81 antibody. The ventral surface next to Bruch’s membrane was heavily labeled. In addition, the inner surface next to the outer segments was labeled. We believe that the labeling observed at the distal end of the outer segments is associated with the projections of the RPE into that layer. Thus, in sections of albino rat, high levels of CD81 immunoreactivity are found to be associated with the RPE. 
To further define the distribution of CD81 on rat RPE cells, the RPE of pigmented rats (Long-Evans) was dissected from the remainder of the retina. This sheet of cells was then immunostained for CD81 and examined face on (Figs. 1E 1F) . This allowed us to see the cells directly and confirm they were CD81 positive. In addition, there appeared to be an increased CD81 immunoreactivity at the regions of cell–cell contact (Fig. 1E) . This analysis was extended to the electron microscopic level (Fig. 2) . The distribution of immunoreaction product revealed labeling on both the dorsal and ventral surfaces of the dissected RPE. The enzymatic digestion had removed Bruch’s membrane from the outer surface of the cells (Fig. 2B) . The membrane folds on the basal surface remained relatively intact with prominent immunolabeling on their surfaces. Virtually all the rod outer segments were removed from the inner surface of the cells. Many fine processes were present that appeared to be the membranous projections of the RPE. Although these processes once surrounded the outer segments, they now appear to lay isolated on the inner surface of the RPE (Fig. 2B) . There was a patchy labeling of the membrane along the inner surface of the dissected RPE. Labeling was also observed on the lateral surfaces of the cells (Fig. 2C) . Immunolabeling of the dissected RPE was not observed in cells treated with the secondary antibody only (data not shown). These data confirm the presence of CD81 on all surfaces of the rat RPE: where the cells interact with the outer segments, where the cells bind to Bruch’s membrane, and between RPE cells. 
The next step in our analysis was to define the role of CD81 in cultured RPE cells. Primary cultures of the rat RPE were produced according to the methods of Edwards. 25 The cultured cells were plated in T25 flasks, and the cultures were allowed to become confluent. The cells were trypsinized and plated onto PLL-coated coverslips in 12-well plates. These cultures were then stained with the AMP1 antibody while alive, or they were fixed and then stained. In both cases there was prominent labeling of the surface of the RPE (Fig. 3) . When the live cells were stained, the immunofluorescence had a punctatelike appearance, as if the antibody caused small clustering of the antigen to occur (Fig. 3) . As previously observed in cultured astrocytes, the greatest AMP1 immunoreactivity was seen around the periphery of the cells at regions of contact with other cells. 27 Also, it was apparent from comparing two identical photographs, one fluorescent and one in white light, that the cells showing immunoreactivity were the same cells that contained the melanin granules (data not shown). A secondary-only control was run, demonstrating that the labeling was due to the binding of the primary antibody (data not shown). Thus, CD81 is expressed by cultured RPE, and the AMP1 epitope is found on the external surface of the cells. 
To confirm the immunohistochemical findings, the retinal tissue and cultured RPE were analyzed using immunoblot methods (Fig. 4) . Protein samples of cultured RPE, retina from the adult rat and cultured astrocytes (a positive control) were dissolved in nonreducing or reducing sample buffer and separated by SDS-PAGE on 4% to 15% gels. The proteins were transferred to nitrocellulose. As expected, a band representing CD81 was observed at approximately 27 kDa in the nonreduced samples of RPE, retina, and astrocytes. As previously observed, this 27-kDa band was not recognized in reduction protein samples (data not shown). The destruction of the AMP1 epitope in the reduced samples confirms that the 27-kDa band is CD81. 27 An additional (very faint) band at 107 kDa had been observed previously and on sequencing was found to be α-actinin. 28 The 107-kDa band is very prominent in the protein samples from cultured astrocytes and is not destroyed under reducing conditions. A relatively strong band at approximately 54 kDa appeared in the protein samples of cultured RPE. As with the 27-kDa CD81 band, the epitope was destroyed in the reduced samples. We currently believe that this band represents a dimerization of CD81 either with itself or another member of the tetraspanin family; however, further experiments are needed to investigate this speculation. In addition, the 27-kDa band in the protein sample from the RPE was a doublet. This doublet was not observed in the protein samples from cultured astrocytes. The most likely explanation for the doublet is a differential posttranslational processing of CD81 in the cultured RPE cells. CD81 has a putative N-myristoylation site, and biosynthetic labeling has confirmed that the protein can be myristoylated. 22 Control immunoblots were run in which the primary antibody was omitted. None of the described bands was observed in the secondary antibody–only blots, either in reducing or nonreducing conditions. 
As its original name implies (the target of the anti-proliferative antibody, TAPA), CD81 may be associated with cell-cycle regulation. We investigated the role of CD81 in the proliferation of cultured rat RPE. Primary cultures of rat RPE cells were treated with the AMP1 antibody and showed a dramatic decrease in cell proliferation compared with untreated control cultures. To provide an estimate of the number of cells in the S phase, we examined the effects of antibody treatment on BrdU incorporation. RPE were cultured for 24 hours in the antibody treatments: anti-CD81 antibody (AMP1) anti-N-CAM antibody (13-38, another monoclonal antibody of the same isotype as AMP1) or no antibody treatment (see Fig. 3 ). The RPE cells were cultured from juvenile Long-Evans rats (pigmented rats) and could be identified by the melanin granules within the cells. Twenty-four hours after treatment began, BrdU was placed in the culture medium, and the cells were allowed to incorporate the label for an additional 24 hours. The results are shown in graphic form in Figure 3B . In the control cultures, approximately 56% of the RPE were labeled with BrdU. When a control nonimmune IgG1 (250 μg/mL) was added to the cultures, there was a decrease in the mitotic activity with only 42% of the cells being labeled (P < 0.05, Student t-test). In cultures treated with a monoclonal antibody directed against N-CAM (13-38, a mouse monoclonal antibody with the same isotype as AMP1, IgG1) there was a significant decrease in BrdU incorporation (Student t-test, P < 0.05). To aid in defining the relationship between antibody treatment and proliferation, the cells were treated with different concentrations of antibody. Treating the cultured RPE with differing concentrations of antibody (2 μg/mL, 50μ g/mL, or 250 μg/mL) caused a progressive decrease in the number of cells incorporating BrdU (36%, 28%, and 18% respectively). The depression in mitotic activity represents significant decreases in labeling (Student t-test, P < 0.05) with each increase in antibody treatment. Thus, an antibody directed against CD81 on cultured rat RPE is capable of blocking cell cycle progression, and this effect can be regulated by the concentration of the antibody. 
Discussion
The overall goal of the present study was to determine whether CD81 is expressed by RPE. Previously, we demonstrated that CD81 is expressed in the retina and that the overall levels of the protein are upregulated after injury. 28 However, because of the methods used in this study, it was difficult to unequivocally demonstrate the expression of CD81 in RPE. Several different approaches were used to demonstrate its presence in rat RPE. The first was standard immunohistochemistry on frozen sections of the albino rat retina. The pattern of immunoreactivity was consistent with the expression of CD81 by the RPE, and this protein was expressed on apical as well as the basal surface of the cells. A second approach was to stain dissected layers of RPE from the pigmented rat, and these also showed a strong immunoreactivity to antibodies directed against CD81. The expression of CD81 is maintained in culture, and antibodies directed against this protein can depress the mitotic activity of the cultured RPE. Finally, the presence of CD81 in these cells and tissues was confirmed using immunoblot methods. Thus, RPE express CD81. 
Proliferation of RPE in development and after injury is a critical issue in normal vision and preventing the loss of sight. This study demonstrates that CD81 is expressed by RPE and that it is involved in the control of RPE proliferation. For the past several years our laboratory has focused on the role of CD81 in the regulation of glial cell proliferation. CD81 is a member of the recently defined tetraspanin family of proteins. 12 13 14 15 16 17 18 19 20 21 The tetraspanins are part of a molecular complex 23 29 30 31 that are associated with adhesion molecules, 23 32 33 cell migratory behavior, 12 14 34 the maintenance of stable cellular contacts, 24 cell growth and morphology, 27 35 and the regulation of mitotic activity, and they affect signal transduction through second-messenger cascades. 22 36 38 Studies of the burgeoning tetraspanin family demonstrate that these molecules are directly involved in a molecular network controlling cell growth and migration. 
Previously, a considerable amount of work has defined a series of small soluble proteins—growth factors that control the growth of RPE 37 by regulating cell cycle progression. 38 39 40 41 Growth factors also alter the expression of cell-adhesion molecules, 42 the regulation of migratory behavior, and cell survival. 42 43 The present study reveals a role for CD81 in these processes, suggesting that tetraspanins may work in concert with growth factors. Several studies have shown that the effects of growth factors can interact synergistically with extracellular matrix components, 39 42 43 pointing to the possibility that tetraspanins, specifically CD81, may act in concert with growth factors in determining the mitotic activity and migratory behavior of RPE. Future experiments will be directed at defining the interactions between growth factors and CD81 and any convergent signaling pathways. 
In addition to CD81, N-CAM also appears to play a role in the regulation of RPE mitotic activity. RPE are known to express N-CAM 44 and as with other cell–cell interactions adhesive interactions are know to affect cell behavior. For example in the developing brain, as neurons contact astrocytes, the glia exit the cell cycle. 45 This downregulation of glial proliferation is associated with the neuronal adhesion and the neural protein astrotactin. 46 Others 47 48 have shown that N-CAM plays a role in regulating glial proliferation, and N-CAM on neurons can interact with N-CAM on glial cells. Glia–glia interactions are also known to regulate the glial cell cycle. As cultures of glial cells become confluent, they downregulate their own mitotic activity. The evidence presented in this study demonstrates that CD81 is involved in the regulation of the RPE cell cycle. Future studies will define the molecular interactions directly linking CD81 with the adhesive interactions of RPE. 
The control of RPE proliferation is important not only in the normal development of the eye, but it plays a prominent role after injury to the retina. In the retina, proliferation of non-neuronal cells and glial scarring is a common, deleterious response in disease and injury. 6 11 The data presented in this study demonstrate that at least one member of the tetraspanin family, CD81, is directly involved in the regulation of RPE growth. Previously, we have shown that this protein is upregulated after retinal injury. 28 We now know that RPE also expresses CD81, and it may play an important role in the response to injury. Defining the role of CD81 and other tetraspanins in the response of the retina to injury may lead to additional approaches to modulate the role of RPE in retinal injury. By controlling the response of these cells through CD81, it may be possible to modify the proliferative response of retinal glia and RPE, minimizing the deleterious effects of retinal injury and thereby preserving vision. 
 
Figure 1.
 
Sections of albino rat retina stained for CD81, demonstrated higher levels of immunoreactivity throughout the retina (A). A very distinct line of immunoreactivity was present at the outer limiting membrane (arrowheads). This labeling is diagnostic of Müller cell labeling. High levels of immunoreactivity were apparent throughout the layers of the retina. In addition, there was a very pronounced staining of the RPE. Arrows: basal surface of the RPE. Note the CD81 immunoreactivity extending up into the outer segments from the RPE. This pattern of labeling was not observed in control sections stained with the secondary antibody only (B). (C) High-magnification photomicrograph illustrates the labeling of the RPE in the retina. An adjacent section, counterstained by the Nissl method (D), revealed the nuclei of the RPE (arrowheads) immediately adjacent to the outer segments. (E, F) Dissected RPE immunostained for CD81. The sheets of cells were free-hand dissected from enzymatic digests of retina, fixed, and stained with AMP1 antibody followed by a fluorescein-labeled anti-mouse secondary antibody. Note the heavy labeling of the cell surface (E). The pigment granules can be seen in the light microscopic photomicrograph (F). These data demonstrate that CD81 is expressed by RPE in vivo. ONL, outer nuclear layer; INL, inner nuclear layer; IPL inner plexiform layer; and GCL, ganglion cell layer. Scale bar, (A, B) 20 μm; (C, D) 40 μm; (E, F) 25 μm.
Figure 1.
 
Sections of albino rat retina stained for CD81, demonstrated higher levels of immunoreactivity throughout the retina (A). A very distinct line of immunoreactivity was present at the outer limiting membrane (arrowheads). This labeling is diagnostic of Müller cell labeling. High levels of immunoreactivity were apparent throughout the layers of the retina. In addition, there was a very pronounced staining of the RPE. Arrows: basal surface of the RPE. Note the CD81 immunoreactivity extending up into the outer segments from the RPE. This pattern of labeling was not observed in control sections stained with the secondary antibody only (B). (C) High-magnification photomicrograph illustrates the labeling of the RPE in the retina. An adjacent section, counterstained by the Nissl method (D), revealed the nuclei of the RPE (arrowheads) immediately adjacent to the outer segments. (E, F) Dissected RPE immunostained for CD81. The sheets of cells were free-hand dissected from enzymatic digests of retina, fixed, and stained with AMP1 antibody followed by a fluorescein-labeled anti-mouse secondary antibody. Note the heavy labeling of the cell surface (E). The pigment granules can be seen in the light microscopic photomicrograph (F). These data demonstrate that CD81 is expressed by RPE in vivo. ONL, outer nuclear layer; INL, inner nuclear layer; IPL inner plexiform layer; and GCL, ganglion cell layer. Scale bar, (A, B) 20 μm; (C, D) 40 μm; (E, F) 25 μm.
Figure 2.
 
The immunolabeling pattern of dissected rat RPE is illustrated in a series of electron micrographs. (A) Low-magnification micrograph illustrating the entire thickness of a single dissected RPE cell. Boxes: areas that are enlarged in (B) and (C). Note that Bruch’s membrane was missing, presumably removed by enzymatic digestion (A, B ★). Note the patchy labeling of the membrane on the inner surface of the RPE (B; arrows). The presence of apical microvilli was also observed on the inner surface of the cells (arrowhead). (C) Labeling on the lateral surface of the cell (arrows). In addition to this labeling, the basal infoldings on outer surface were labeled (arrowheads). Magnification, (A) ×3000; (B, C)× 10,000.
Figure 2.
 
The immunolabeling pattern of dissected rat RPE is illustrated in a series of electron micrographs. (A) Low-magnification micrograph illustrating the entire thickness of a single dissected RPE cell. Boxes: areas that are enlarged in (B) and (C). Note that Bruch’s membrane was missing, presumably removed by enzymatic digestion (A, B ★). Note the patchy labeling of the membrane on the inner surface of the RPE (B; arrows). The presence of apical microvilli was also observed on the inner surface of the cells (arrowhead). (C) Labeling on the lateral surface of the cell (arrows). In addition to this labeling, the basal infoldings on outer surface were labeled (arrowheads). Magnification, (A) ×3000; (B, C)× 10,000.
Figure 3.
 
(A) Immunostaining of cultured RPE for CD81 antigen. A confluent monolayer of living rat RPE is labeled with the AMP1 antibody. The antibody was added to living cultured cells rinsed with medium and followed by a fluorescein-labeled anti-mouse secondary antibody. The cells were from the first passage of the primary culture. Note the increased labeling at regions of cell–cell contact (arrows). These data demonstrate that CD81 is expressed on the surface of cultured rat RPE. Scale bar, 50 μm. (B) Percentage of culture RPE that incorporated BrdU in control cells and cells treated with monoclonal antibody. The mean for each group is represented by the bar, and the SEM is represented by the error bar. In cultures that did not receive an antibody treatment (Control) approximately 56% of the cells were labeled with BrdU. There was a decrease in RPE proliferation when the cells were cultured in the presence of 250 μg/mL of nonimmune IgG1 (P < 0.05, Student t-test). When the cultures were treated with differing doses of the AMP1 antibody, a clear dose–response relationship was observed, ranging from a modest suppression of mitotic activity at 2 μg/mL to a more than 50% reduction in mitotic activity at 250 μg/mL. Suppression of mitotic activity was also observed when the cells were treated with a monoclonal antibody directed against N-CAM (250 μg/mL).
Figure 3.
 
(A) Immunostaining of cultured RPE for CD81 antigen. A confluent monolayer of living rat RPE is labeled with the AMP1 antibody. The antibody was added to living cultured cells rinsed with medium and followed by a fluorescein-labeled anti-mouse secondary antibody. The cells were from the first passage of the primary culture. Note the increased labeling at regions of cell–cell contact (arrows). These data demonstrate that CD81 is expressed on the surface of cultured rat RPE. Scale bar, 50 μm. (B) Percentage of culture RPE that incorporated BrdU in control cells and cells treated with monoclonal antibody. The mean for each group is represented by the bar, and the SEM is represented by the error bar. In cultures that did not receive an antibody treatment (Control) approximately 56% of the cells were labeled with BrdU. There was a decrease in RPE proliferation when the cells were cultured in the presence of 250 μg/mL of nonimmune IgG1 (P < 0.05, Student t-test). When the cultures were treated with differing doses of the AMP1 antibody, a clear dose–response relationship was observed, ranging from a modest suppression of mitotic activity at 2 μg/mL to a more than 50% reduction in mitotic activity at 250 μg/mL. Suppression of mitotic activity was also observed when the cells were treated with a monoclonal antibody directed against N-CAM (250 μg/mL).
Figure 4.
 
Immunoblots of protein samples from cultured rat RPE (lane A), cultured rat cortical astrocytes (lane B), or rat retina (lane C) were probed with an antibody directed against CD81 (AC). Lane D: a blot of rat RPE treated in a manner similar to that in lane A with the exception that the primary antibody was omitted. Note the doublet in lane A at approximately 54 kDa, which may represent a dimeric form of CD81. In addition, there is a doublet at approximately 27 kDa in lane A, which may represent a differential posttransitional processing of CD81 in rat RPE (see Ref. 22 ), specifically N-myristoylation. The presence of CD81 in the RPE and retina is confirmed by the presence of a dark band at 27 kDa on the immunoblot. This 27-kDa band was not observed in protein samples run under reducing conditions (data not shown). Molecular weights are indicated to the left in kilodaltons.
Figure 4.
 
Immunoblots of protein samples from cultured rat RPE (lane A), cultured rat cortical astrocytes (lane B), or rat retina (lane C) were probed with an antibody directed against CD81 (AC). Lane D: a blot of rat RPE treated in a manner similar to that in lane A with the exception that the primary antibody was omitted. Note the doublet in lane A at approximately 54 kDa, which may represent a dimeric form of CD81. In addition, there is a doublet at approximately 27 kDa in lane A, which may represent a differential posttransitional processing of CD81 in rat RPE (see Ref. 22 ), specifically N-myristoylation. The presence of CD81 in the RPE and retina is confirmed by the presence of a dark band at 27 kDa on the immunoblot. This 27-kDa band was not observed in protein samples run under reducing conditions (data not shown). Molecular weights are indicated to the left in kilodaltons.
The authors thank Dianna Johnson, Ph.D., for her constructive criticism in the preparation of the manuscript and Bill Orr and Kathy Troughton for technical assistance. 
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Figure 1.
 
Sections of albino rat retina stained for CD81, demonstrated higher levels of immunoreactivity throughout the retina (A). A very distinct line of immunoreactivity was present at the outer limiting membrane (arrowheads). This labeling is diagnostic of Müller cell labeling. High levels of immunoreactivity were apparent throughout the layers of the retina. In addition, there was a very pronounced staining of the RPE. Arrows: basal surface of the RPE. Note the CD81 immunoreactivity extending up into the outer segments from the RPE. This pattern of labeling was not observed in control sections stained with the secondary antibody only (B). (C) High-magnification photomicrograph illustrates the labeling of the RPE in the retina. An adjacent section, counterstained by the Nissl method (D), revealed the nuclei of the RPE (arrowheads) immediately adjacent to the outer segments. (E, F) Dissected RPE immunostained for CD81. The sheets of cells were free-hand dissected from enzymatic digests of retina, fixed, and stained with AMP1 antibody followed by a fluorescein-labeled anti-mouse secondary antibody. Note the heavy labeling of the cell surface (E). The pigment granules can be seen in the light microscopic photomicrograph (F). These data demonstrate that CD81 is expressed by RPE in vivo. ONL, outer nuclear layer; INL, inner nuclear layer; IPL inner plexiform layer; and GCL, ganglion cell layer. Scale bar, (A, B) 20 μm; (C, D) 40 μm; (E, F) 25 μm.
Figure 1.
 
Sections of albino rat retina stained for CD81, demonstrated higher levels of immunoreactivity throughout the retina (A). A very distinct line of immunoreactivity was present at the outer limiting membrane (arrowheads). This labeling is diagnostic of Müller cell labeling. High levels of immunoreactivity were apparent throughout the layers of the retina. In addition, there was a very pronounced staining of the RPE. Arrows: basal surface of the RPE. Note the CD81 immunoreactivity extending up into the outer segments from the RPE. This pattern of labeling was not observed in control sections stained with the secondary antibody only (B). (C) High-magnification photomicrograph illustrates the labeling of the RPE in the retina. An adjacent section, counterstained by the Nissl method (D), revealed the nuclei of the RPE (arrowheads) immediately adjacent to the outer segments. (E, F) Dissected RPE immunostained for CD81. The sheets of cells were free-hand dissected from enzymatic digests of retina, fixed, and stained with AMP1 antibody followed by a fluorescein-labeled anti-mouse secondary antibody. Note the heavy labeling of the cell surface (E). The pigment granules can be seen in the light microscopic photomicrograph (F). These data demonstrate that CD81 is expressed by RPE in vivo. ONL, outer nuclear layer; INL, inner nuclear layer; IPL inner plexiform layer; and GCL, ganglion cell layer. Scale bar, (A, B) 20 μm; (C, D) 40 μm; (E, F) 25 μm.
Figure 2.
 
The immunolabeling pattern of dissected rat RPE is illustrated in a series of electron micrographs. (A) Low-magnification micrograph illustrating the entire thickness of a single dissected RPE cell. Boxes: areas that are enlarged in (B) and (C). Note that Bruch’s membrane was missing, presumably removed by enzymatic digestion (A, B ★). Note the patchy labeling of the membrane on the inner surface of the RPE (B; arrows). The presence of apical microvilli was also observed on the inner surface of the cells (arrowhead). (C) Labeling on the lateral surface of the cell (arrows). In addition to this labeling, the basal infoldings on outer surface were labeled (arrowheads). Magnification, (A) ×3000; (B, C)× 10,000.
Figure 2.
 
The immunolabeling pattern of dissected rat RPE is illustrated in a series of electron micrographs. (A) Low-magnification micrograph illustrating the entire thickness of a single dissected RPE cell. Boxes: areas that are enlarged in (B) and (C). Note that Bruch’s membrane was missing, presumably removed by enzymatic digestion (A, B ★). Note the patchy labeling of the membrane on the inner surface of the RPE (B; arrows). The presence of apical microvilli was also observed on the inner surface of the cells (arrowhead). (C) Labeling on the lateral surface of the cell (arrows). In addition to this labeling, the basal infoldings on outer surface were labeled (arrowheads). Magnification, (A) ×3000; (B, C)× 10,000.
Figure 3.
 
(A) Immunostaining of cultured RPE for CD81 antigen. A confluent monolayer of living rat RPE is labeled with the AMP1 antibody. The antibody was added to living cultured cells rinsed with medium and followed by a fluorescein-labeled anti-mouse secondary antibody. The cells were from the first passage of the primary culture. Note the increased labeling at regions of cell–cell contact (arrows). These data demonstrate that CD81 is expressed on the surface of cultured rat RPE. Scale bar, 50 μm. (B) Percentage of culture RPE that incorporated BrdU in control cells and cells treated with monoclonal antibody. The mean for each group is represented by the bar, and the SEM is represented by the error bar. In cultures that did not receive an antibody treatment (Control) approximately 56% of the cells were labeled with BrdU. There was a decrease in RPE proliferation when the cells were cultured in the presence of 250 μg/mL of nonimmune IgG1 (P < 0.05, Student t-test). When the cultures were treated with differing doses of the AMP1 antibody, a clear dose–response relationship was observed, ranging from a modest suppression of mitotic activity at 2 μg/mL to a more than 50% reduction in mitotic activity at 250 μg/mL. Suppression of mitotic activity was also observed when the cells were treated with a monoclonal antibody directed against N-CAM (250 μg/mL).
Figure 3.
 
(A) Immunostaining of cultured RPE for CD81 antigen. A confluent monolayer of living rat RPE is labeled with the AMP1 antibody. The antibody was added to living cultured cells rinsed with medium and followed by a fluorescein-labeled anti-mouse secondary antibody. The cells were from the first passage of the primary culture. Note the increased labeling at regions of cell–cell contact (arrows). These data demonstrate that CD81 is expressed on the surface of cultured rat RPE. Scale bar, 50 μm. (B) Percentage of culture RPE that incorporated BrdU in control cells and cells treated with monoclonal antibody. The mean for each group is represented by the bar, and the SEM is represented by the error bar. In cultures that did not receive an antibody treatment (Control) approximately 56% of the cells were labeled with BrdU. There was a decrease in RPE proliferation when the cells were cultured in the presence of 250 μg/mL of nonimmune IgG1 (P < 0.05, Student t-test). When the cultures were treated with differing doses of the AMP1 antibody, a clear dose–response relationship was observed, ranging from a modest suppression of mitotic activity at 2 μg/mL to a more than 50% reduction in mitotic activity at 250 μg/mL. Suppression of mitotic activity was also observed when the cells were treated with a monoclonal antibody directed against N-CAM (250 μg/mL).
Figure 4.
 
Immunoblots of protein samples from cultured rat RPE (lane A), cultured rat cortical astrocytes (lane B), or rat retina (lane C) were probed with an antibody directed against CD81 (AC). Lane D: a blot of rat RPE treated in a manner similar to that in lane A with the exception that the primary antibody was omitted. Note the doublet in lane A at approximately 54 kDa, which may represent a dimeric form of CD81. In addition, there is a doublet at approximately 27 kDa in lane A, which may represent a differential posttransitional processing of CD81 in rat RPE (see Ref. 22 ), specifically N-myristoylation. The presence of CD81 in the RPE and retina is confirmed by the presence of a dark band at 27 kDa on the immunoblot. This 27-kDa band was not observed in protein samples run under reducing conditions (data not shown). Molecular weights are indicated to the left in kilodaltons.
Figure 4.
 
Immunoblots of protein samples from cultured rat RPE (lane A), cultured rat cortical astrocytes (lane B), or rat retina (lane C) were probed with an antibody directed against CD81 (AC). Lane D: a blot of rat RPE treated in a manner similar to that in lane A with the exception that the primary antibody was omitted. Note the doublet in lane A at approximately 54 kDa, which may represent a dimeric form of CD81. In addition, there is a doublet at approximately 27 kDa in lane A, which may represent a differential posttransitional processing of CD81 in rat RPE (see Ref. 22 ), specifically N-myristoylation. The presence of CD81 in the RPE and retina is confirmed by the presence of a dark band at 27 kDa on the immunoblot. This 27-kDa band was not observed in protein samples run under reducing conditions (data not shown). Molecular weights are indicated to the left in kilodaltons.
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