A monoclonal antibody that binds to the surface of photoreceptors

DevelopmentalBrain Research, 39 (1988) 117-123 Elsevier

117

BRD 50697

A monoclonal antibody that binds to the surface of photoreceptors Vance Lemmon Departmentof Neurobiology, Anatomy and CellScienceand the Centerfor Neuroscience, Universityof Pittsburgh, Pittsburgh, PA 15261 (U.S.A.) (Accepted 8 September 1987) Key words: Monoclonal antibody; Photoreceptor; Chick retina; Development

A monoclonal antibody (MAB), 7Hll, that binds to the surface of photoreceptors has been produced. Using immunoaffinity procedures, proteins with molecular weights of 53, 48 and 42 kDa were found to bind a MAB 7Hll column, although the 48- and 42-kDa proteins may be proteolytic breakdown products of the 53 kDa protein. Electron microscopic-immunohistochemical studies show that the antigen is concentrated on the external surface of inner and outer segments of photoreceptors. It is likely that the 7Hll antigen is a component of the interphotoreceptor matrix and, therefore, may contribute to some aspect of photoreceptor-pigment epithelial interactions. However, the expression of the 7Hll antigen in cell culture suggests that it is expressed independently of direct cellcell interactions with either pigment epithelial cells, Mtiller cells or other photoreceptors.

INTRODUCTION Studies on the cell-cell interactions that contribute to the formation of a complex tissue, such as the retina, have been greatly facilitated over the past several years by the production of monoclonal antibodies that recognize cell surface molecules on specific classes of cells. These types of antibodies permit several different types of experimental approaches that would otherwise be impossible. These include identification of the cell type of live cells, identification of cell types in aggregates in vitro or in tissue transplanted to ectopic locations in the central nervous system (CNS), and the purification of specific cell types using affinity procedures or fluorescence activated cell sorters. The chick retina has been a popular choice of developmental biologists studying cell-cell interactions. As a result, a number of putative cell recognition molecules have been identified in the chick retina. These include cognin 14, neural cell adhesion mol-

ecule (N-CAM) l°, n e u r o n - g l i a cell adhesion molecule (Ng-CAM) 11 and the related 8D9 antigen 19, T O P 22, and N-cadherin 13. Despite these numerous studies on cell recognition molecules in the chick retina, there are few markers for the different cell types in the chick retina. Well characterized markers presently include monoclonal antibodies (MABs) against ganglion cell axons 19, Miiller cells 16'17, cones is'2°, and rods 12. However, most of these are against intracellular antigens. Cell surface markers for amacrine cells, bipolar cells, horizontal cells, photoreceptors and Miiller cells in the chick retina are not available. This report describes observations on M A B 7 H l l which binds to the surface of photoreceptors in the chick retina. In addition to characterizing the biochemical properties of the 7 H l l antigen and describing its ultrastructural location, we have found that the antigen is expressed on individual photoreceptors that are not in contact with other cells in vitro. This suggests that the 7 H l l antigen is expressed as part of a differentiation program in photoreceptors that is in-

Correspondence: V. Lemmon, Department of Neurobiology, Anatomy and Cell Science, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.

118 dependent of signals requiring direct cell-cell contact with other cells including other photoreceptors, pigment epithelial cells or Mfiller cells. MATERIALS AND METHODS BALB/c mice were obtained from Charles River Labs. White leghorn chicken eggs were obtained from S. Sacks and Son, Evans City, PA. Hybridoma production. Mice were immunized with material solubilized by NP-40 from embryonic day 19 (El9) retinal plasma membranes as described below and elsewhere ts. Hybridomas were prepared following standard fusion procedures 9 using NS-1 cells. Supernatants were first screened using a dotblot immunoassay with the same material that was used to immunize the mice el. Light microscopic immunohistochemistry. Wells containing supernatants that were positive on the dot-blot were subsequently tested using immunohistoiogical procedures with cryostat sections of El9 chick retina previously fixed with 4% paraformaldehyde and 0.01% glutaraldehyde. After incubating the sections with hybridoma supernatant, the sections were washed with Dulbecco's phosphate-buffered saline (PBS) and then incubated with a l:500 dilution of fluorescein-labeled goat anti-mouse IgG (Cappel Labs). Wells containing supernatants that bound to restricted classes of retinal cells were cloned by limiting dilution.

Electron microscopic (EM) irnrnunohistochemistry. Chickens were deeply anesthetized with 2,2,2 tribromo ethanol (Fluka) and then perfused with 4% paraformaldehyde/0.01% glutaraldehyde in 0.1 M PIPES buffer, pH 7.3, The retinas were removed and blotted onto nitrocellulose filters and then cut into 300-urn wide strips with a tissue chopper. Retinal strips were incubated overnight in primary antibodies at 4 °C. The strips were washed with PBS extensively over 1 h, then incubated with a biotin-labeled secondary antibody (Vectastain) for 90 min. The strips were washed again over 1 h and then incubated with an a v i d i n - b i o t i n - H R P complex for 90 rain. After a final washing for 1 h, the strips were processed with an intensified DAB procedure ~. Strips were postfixed in 2% osmium and stained with lead citrate prior to being embedded in Spurr's plastic. Thin sections were examined in a J O E L 100-CX electron mi-

croscope. For both light microscopic studies and electron microscopic studies controls were always performed that included incubating sections without the primary antibody but with the secondary antibody. This allowed us to determine if the secondary antibody bound to the tissue. We found no binding of either fluorescein or HRP-labeled antibodies in sections incubated only with secondary antibodies (data not shown). Biochemical procedures. MAB 7H 11 was purified from ascities fluid using Affi-gel Blue D E A E chromatography s. Purified antibody was coupled to CNBr-activated Sepharose 4B affinity heads. For cell fractionation studies >, retinas from E20 chicks were homogenized in 0.32 M sucrose/Tris-buffered saline (TBS)/0.2 mM PMSF/0.5 mglml turkey trypsin inhibitor (Sigma), and layered onto sucrose density gradients with 0.8 M and 1.2 M sucrose steps. The samples were centrifuged in a SW28 rotor at 25,000 rpm for 90 min in a Beckman L7. The plasma membrane fraction at the 0.8/1.2 M sucrose interface was harvested, diluted with an equal volume of TBS and centrifuged at 35,000 rpm for 60 min in a 70.1 Ti rotor. The pellet was homogenized with 1% sodium cholate in 50 mM Tris, pH 8.0 and then centrifuged at 35,000 rpm for 60 min. The various fractions, including soluble fraction (0.32 M sucrose fraction), the sodium cholate soluble and insoluble fractions and the nuclear pellet, were tested for 7 H l l antigen activity using a dot-blot immunoassay 21. The fraction that contained the majority of the 7 H l l antigen was run over the 7 H l l affinity column. The column was washed extensively with TBS and then eluted with 0.1 M diethylamine at pH 10.5, followed by the same buffer at pH 11.5, followed by 4 M guanidine-HCl. The pH of the diethylamine-containing samples was neutralized with solid Tris-HCl and the guanidinecontaining samples were dialysed against TBS to remove the salt. Samples were analyzed using SDSP A G E and a silver stain. Tissue culture. In vitro experiments were conducted on embryonic day 6 (E6) chick retinas s'ls. Retinas were first dissected free of the pigment epithelia prior to cell dissociation so the resulting cultures were made entirely of neural retinal cells. The retinas were incubated in 5 ml of Hanks" calcium, magnesium-free saline solution (CMF) with 0. 125% trypsin

119 for 30 min at 37 °C. Two ml of horse serum and 0.5 ml of DNase I (5 mg/ml) in CMF were added and the retinas were triturated with a Pasteur pipette. The cells were centrifuged and resuspended in Duibecco's modified Eagle's medium with 10% horse serum and antibiotics. Low and high density cultures of 100,000 and 500,000 cells, respectively~ were added to polyL-lysine-coated, 35-ram dishes in 1.5 ml of medium. Cells that had been cultured for 6 days were stained with the MAB 7 H l l , the cultures were washed with PBS and then fixed with 4% paraformaldehyde/ 0.01% glutaraldehyde in 0.1 M phosphate buffer (PB), prior to incubation with a fluorescent secondary antibody.

RESULTS Using the plasma membrane fraction from retina as an immunogen for hybridoma production was an effective way of obtaining cell class-specific antibodies. From this particular fusion several different hybridomas were successfully cloned. These included one which produces an antibody specific for cones 18, two specific for M~iller cells and 4 specific for photoreceptors. One of these photoreceptor-specific antibodies, 7 H l l , was chosen for further characterization because, on preliminary screening, it showed wide species cross-reactivity. Light microscopic immunoh&tochemistry. Chicken

Fig. 1. Light microscopic localization of the 7 H l l antigen. A: E l 4 chick retina has 7H11 antigen on the developing photoreceptors (PR). The location of the outer plexiform layer (OPL) is also indicated. B: E20 chick photoreceptors are stained intensely by MAB 7H11 external to the outer limiting membrane. C,D: MAB 7H11 stains isolated live photoreceptors in culture in a polarized manner. A phase bright inclusion in the cell is indicated by the arrow. E,F: numerous cells in aggregates of retinal cells in culture express 7H11 antigen. A,B: x312; C,D: x625; E,F: x312.

120 retinas were examined for 7HI l-immunorcactivitv at a variety of developmental stages. Very faint staining of developing photoreceptors was first observcd at E l 2 (data not shown) and by E l 4 it was easy to identify (Fig. 1A). The staining was always concentrated external to the outer limiting membrane. At L2(I there was intense staining of photoreceptors between the outer limiting membrane and the pigment epithelia (Fig. 1B). The antigen persists in the photoreceptor layer through adulthood. A variety of species, including rat, quail, monkey, bull frog, and pond slider turtle (Pseudemys scripta elegans), were tested for 7H 11 immunoreactivity. The photoreceptors in all of these species were specifically stained by MAB 7H 11 (data not shown). Cell culture experiments. Cell culture experiments w e r e conducted using chick retina cells in order to determine the cellular location of the 7 H l l antigen.

Adler and associates have demonstralcd that li~e photoreceptors can be stained with peanut Icctm under similar cell culture conditions 5. When live cells, plated at a density of 10(l,0(ll) cells per 35-ram dish, were incubated with MAB 7t]11 there was staining of the surface of a subset of cells in the cultures, lhe staining was intense and it was usually restricted to a particular region of the cell (Fig. 1C, D). This staining w a s opposite the neurite-bearing end of the cell. This pattern of staining is consistent with the pattern of staining seen with MAB 7 H I I of photoreceptors in an intact retina: the receptor region is stained, but no staining is seen on the soma or dendrites in the OPL. Also consistent with the 7HI 1-positive cells being photoreceptors was the presence of small, phase bright inclusions which correspond to the 'lipid droplet' in cones s. These cells were not in contact with other neurons or MOiler cells and there were no pig-

Fig. 2. Electron microscopic immunohistochemistry of 7H11 antigen in adult chicken retina. A: dark D A B reaction product (arrows) is seen between photoreceptors but not inside cells. B: the concentration of D A B reaction product is highest (arrow) immediately adjacent to photoreceptors. Bars = 1 ,urn.

121 ment epithelial cells present in the dish. When cells were plated at higher densities (500,000 cells/35 mm dish), the 7 H l l antibody could be used to identify photoreceptors in large aggregates of cells (Fig. 1E,F). EM immunohistochemistry. In adult chicken retina the regions adjacent to surfaces of inner and outer segments of photoreceptors were heavily stained by MAB 7 H l l while there was no staining inside the photoreceptors (Fig. 2A). The intensity of the staining decreased with distance from photoreceptors (Fig. 2B). It should be noted that not all photoreceptor antigens give this pattern of staining in our hands. For example, 50-1Bll, a cone-specific antibody, stains cone lamellae but not the interphotoreceptor area (see Fig. 3D in ref. 18). In other experiments, the addition of detergent (saponin or Triton X-100) to the retina, prior to incubation with MAB 7 H l l , did not result in intracellular staining of the photoreceptors (data not shown). Biochemical experiments. Attempts to identify the 7 H l l antigen using immunoblots of SDS-polyacrylamide gels 21 failed to give positive results. Therefore, immunoaffinity procedures were used. First, the solubility properties of the antigen were determined by conducting cell fractionation experiments 23. By testing soluble material from the 0.32 M sucrose step, membranes from the 0.8/1.2 M sucrose interface and the nuclear pellet for immunoreactivity on dot-blots, we found that the 7 H l l antigen was concentrated in the soluble fraction (data not shown). A variety of elution buffers were tested for their ability to remove MAB 7 H l l from photoreceptors in cryostat sections of retina, including buffers with pH of 11.5, pH 2.5, 4 M urea, 3 M KI, and 4 M guanidinde-HCl. Only 4 M guanidine-HC1 was effective in removing the antibody from the photoreceptors so this was chosen as the buffer to elute the antigen from the affinity column. The soluble fraction (Fig. 3, lanes A,B) from a retinal homogenate was run over the 7 H l l immunoaffinity column. After extensive washing the column was eluted with two high pH buffers followed by guanidine. The pH 10.5 and 11.5 buffers were used to try to remove any non-specifically bound material from the column. However, when these fractions were examined with S D S - P A G E no protein bands were ob-

served. The guanidine eluate was found to contain 3 principal bands with molecular weights of 53 kDa, 48 kDa, and 42 kDa (Fig. 3C). In addition, a faint band was observed at about 59 kDa. In different preparations the 53-kDa band appeared constantly, while the 48-kDa and 42-kDa bands varied and were sometimes almost absent. A faint band at about 24 kDa was always present, even if a 7 H l l affinity column that had never been exposed to a retinal extract was eluted with guanidine., Therefore, the 24-kDa band was probably an IgG light chain from the column DISCUSSION MAB 7 H l l recognizes an antigen associated with photoreceptors in the chicken retina. Several differ-

Fig. 3. SDS-PAGE of 7Hll antigen. Lanes A and B show 5 and 40 pl of the soluble fraction run over the 7H11 affinity column. Lane C shows the material eluted from the 7H11 affinity column with 4 M guanidine-HCI. The locations of molecular weight standards (in kDa) are indicated at the left.

122 ent lines of evidence indicate that the antigen is on the external surface of photoreceptors and is a component of the interphotoreceptor matrix (IPM). The EM immunohistochemical studies indicate that the 7 H l l antigen is present on the external surface of photoreceptors in the regions between photoreceptors and pigment epithelial cells. Similarly, the ability to stain live photoreceptors in culture demonstrates that the 7 H l l antigen is present on the external surface of photoreceptors. The cell fractionation studies showed that the antigen could be solubilized simply by homogenization in a physiological concentration of salt, with no detergents being required. This raises the question as to how 1 was able to produce an antibody against a soluble component of the IPM using a detergent extract of retinal membranes. While it is possible that the membranes were contaminated with soluble material, a more likely explanation is that the 7H11 antigen is bound, all be it weakly, to the surface of photoreceptors. If this were not the case it would have been difficult, if not impossible, to stain live photoreceptors in culture• Immunoaffinity studies were used to identify the molecular weight of the 7H 11 antigen. A major protein band, with a molecular weight of about 53 kDa, was consistently observed. Less frequently, bands with molecular weights of 48 kDa and 42 kDa were observed. It is possible that these represent proteolyric breakdown products of the higher molecular weight protein, although they may also be distinct proteins that associate with either the 53-kDa protein or MAB 7 H l l . The interphotoreceptor matrix is composed of glycoproteins and glycosaminoglycans 7. An important class of molecules present there are retinol-binding proteins 3. One such molecule is interphotoreceptor retinoid-binding protein which has a molecular weight of 144 kDa in several different spectes -1-.

REFERENCES I Adams, J., Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem., 29 (1981) 775. 2 Adler, A.J. and Evans. C.D., Some functional characteristics of purified bovine interphotoreceptor retinol-binding protein, hn,est. Ophthalmol. Vis. Sci., 26 (1985) 273-282. 3 Adler, A.J. and Martin, K.J., Retinol-binding proteins in bovine interphotoreceptor matrix, Biochem. Biophys. Res. Commun.. 108 (1982) 1601-1608.

Another molecule found exclusively in the IPM is a cGMP phosphodiesterase c' which has subunits with molecular weights of 47 and 45 kDa in bovine eyes. The lack of correspondence of the 7 H l l antigen's molecular weight to these previously identified molecules suggests that the 7H 11 antigen may represent a previously unidentified molecule in the interphotoreceptor matrix. However, Adler and Evans have shown that the IPM contains proteins with molecular weights that correspond closely to that of the 7 H l l antigen (see Fig. 3 in ref. 2). Because M A B 7 H l l stains a variety of species, it should be possible to isolate the antigen in those species, such as monkeys or cows, where the IPM has been more extensively characterized. The cell culture experiments where isolated photoreceptors expressed 7 H l l immunoreactivity reveal two important features of its developmental expression. The first is that direct cell contact with other cells is not required for experession of the antigen. The second is that the 7HI 1 antigen is expressed predominantly on one region of the cell. This is similar to the work of Adler and associates, who have found that isolated chicken photoreceptors in culture express both opsin immunoreactivity and peanut lectin binding in a polarized manner a5. These observations support the concept that developing photoreceptors follow a relatively complex program of differentiation that may require only limited interaction with other cells to produce functional photoreceptors. ACKNOWLEDGEMENTS 1 would like to thank J. Hailey and U. Reuter-Carlson for expert technical assistance and E. Baker for assisting in the antigen purification. Supported by Research Grant RO1-EY05285 from the National Eye Institute.

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