Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

Immunoreactivity of rhodopsin and opsin.

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.

A radioimmunoassay specific for opsin.

A radioimmunoassay is developed for bovine opsin using a rabbit antiserum against bovine rod outer segment membranes. The assay is specific for opsin. Rhodopsin, bacteriorhodopsin and hemoglobin do not show cross-reaction. It can be carried out rapidly, has a sensitivity of 0.01 pmol bovine opsin and gives accurate results, even in the presence of a large excess of rhodopsin. Under the conditions described, the assay can be used to measure bovine opsin and rhodopsin in each other's presence by running a sample before and after illumination, with a sensitivity 2000-times higher than with spectrophotometric methods. The opsin content of rather crude preparations such as bovine retina homogenates can be accurately determined. Rabbit and mouse opsin can also be assayed with a reasonable degree of accuracy using the same rabbit antiserum.

Detergent-induced specificity of an antirhodopsin serum for opsin. Micro-complement fixation studies.

An antiserum elicited in rabbit against dark-adapted rod outer segment membranes has been characterized by means of the micro-complement fixation technique. Both particulate rhodopsin and opsin, either biochemically intact or denatured and either membrane-bound or in the absence of lipids, are able to interact with the antiserum. Solubilization of the antigens in increasing concentrations of Emulphogene BC-720 leads to complete loss of complement fixation with both rhodopsin and opsin, but in the case of opsin this requires almost 10-times more detergent. In the case of opsin this masking phenomenon is preceded by a drastic exposure of antigenic sites which in the membrane vesicles are not accessible to the antibodies. Absorption experiments show that the antigenic sites on membrane-bound rhodopsin and opsin, as well as on Emulphogene BC-720-solubilized opsin, are of the same nature. Competition experiments show that the masking effect of the detergent is due to an inhibition of the primary antigen-antibody interaction and not to the inhibition of lattice formation. The use of detergents other than Emulphogene BC-720 further demonstrates that detergents more efficiently mask the antigenicity of conformationally intact than of denatured rhodopsinoids. The balance between the masking and the denaturing efficiency of a particular detergent determines whether a detergent-induced immunological discrimination can be observed between rhodopsin and opsin. The detergent-induced masking effects described are typical for the present antiserum and are probably dependent on methodological details of the immunization procedure.

Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity.

Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.

Carbohydrate components recognized by the cone-specific monoclonal antibody CSA-1 and by peanut agglutinin are associated with red and green-sensitive cone photoreceptors.

Previous investigations have demonstrated that the monoclonal antibody CSA-1 and peanut agglutinin label specifically cone photoreceptor cells. In the present study, we compared the binding of CSA-1 and peanut agglutinin to that of the monoclonal antibodies COS-1 and OS-2, which have been shown to recognize the red/green- and blue-sensitive cone visual pigments, respectively. Using lectin and immunocytochemistry on serial semithin sections of the pig retina, we have demonstrated in the present study that both CSA-1 and peanut agglutinin label specifically the red-, and green-sensitive, but not the blue-sensitive cone cell outer segments. Peanut agglutinin does bind, however, to the cone matrix sheaths associated with all three types of cones. These observations support the idea that red-, and green-sensitive cone cells share some common molecular epitopes and may represent a differentiation line of cones, considerably different from that of blue-sensitive cones.

Identification of the blue-sensitive cones in the mammalian retina by anti-visual pigment antibody.

Monoclonal antibodies to visual pigments produced in our laboratory were applied to analyze the distribution of color-specific photoreceptor cells in the retina (photoreceptor mosaic). We demonstrated in two ways that the monoclonal antibody OS-2 specifically recognized the blue-sensitive cone cells in the mammalian retina. First, rabbit photoreceptors damaged selectively by intense blue light were recognized by OS-2 antibody. Second, OS-2-positive cones in the ground squirrel were those with thick inner segments, which is known to be characteristic of the blue-sensitive cones. In addition, the OS-2-positive cones in monkeys have a distribution and pattern characteristic of blue-sensitive cones in that species. In several other species (human, rabbit, cow, and pig), the OS-2-positive cones represent an appropriate minority of the population of photoreceptor cells. The visual pigment recognized by the OS-2 antibody had a relative molecular weight of 36,000, as shown by immunoblotting of 3 mammalian species. All other cones were recognized by another monoclonal antibody, COS-1, which is regarded as specific to middle-to-long-wavelength-sensitive photoreceptors.

Four cone types characterized by anti-visual pigment antibodies in the pigeon retina.

Using three antibodies to visual pigments (monoclonal antibodies COS-1 and OS-2, and a polyclonal anti-opsin serum), four different types of cone cells could be distinguished in the red area (dorsoposterior part with the highest density of cones) of the pigeon retina. Both members of the double cone and the single cone with the red oil droplet were labelled with our monoclonal antibody COS-1 (type I cone). The single cone with the orange oil droplet was positive both with anti-opsin and monoclonal antibody OS-2 (type II cone). The single cone exhibiting a yellowish-green oil droplet, fluorescent in ultraviolet light, also reacted with anti-opsin but lacked the antigenic determinant recognized by OS-2 (type III cone). The thin cone with the small colorless oil droplet was negative with both COS-1 and anti-rhodopsin (type IV cone). We propose that the four immunologically distinguishable cone types correspond to cones expressing visual pigments with different (long-, middle-, short-wavelength and ultraviolet) color sensitivities.

Characterization of monoclonal antibodies recognizing retinal pigment epithelial antigens.

PURPOSE: To purify and characterize retinal pigment epithelial proteins exhibiting uveitogenic characteristics after immunization of Lewis rats, a broad panel of monoclonal antibodies directed against retinal pigment epithelial (RPE) protein antigens, was isolated. METHODS: Bovine RPE detergent extracts were used to isolate monoclonal antibodies against RPE antigens. Species and tissue specificity within the eye was tested through immunocytochemical analysis. Western blot analysis was used to determine the molecular weight of the RPE antigens. RESULTS: Several RPE-reactive antibodies were obtained. At least four monoclonal antibodies were isolated that reacted with different RPE antigen types. Against most of the antigens more than one hybridoma cell line was isolated. Two hybridoma lines were isolated producing antibodies, which on immunocytochemical analysis showed strong reactivity with the RPE and eye muscle tissue. The latter monoclonal antibody recognizes a 32 kD protein in RPE cells. Five monoclonal antibodies recognize a protein with an apparent molecular weight of 65 kD. One cell line was isolated that produced antibodies showing an irregular reaction pattern with both iris and ciliary body. CONCLUSIONS: RPE cells, striated eye muscle and smooth muscle cells share a 32 kD antigenic protein. This antigen is present in almost all ocular epithelial cells. Based on reaction patterns on Western blot and immunocytochemical analysis, there are at least three different 65 kD RPE antigens, two of which are RPE-specific and one of which is also present in the kidney epithelium.

Rod cell-specific antigens in retinoblastoma.

We investigated rhodopsin immunoreactivity in five well-differentiated retinoblastomas using a panel of monoclonal antibodies directed against specific antigenic sites in the amino- and carboxyl-terminal portions of rhodopsin. All five monoclonal antibodies bound to the rod cell outer segment of nontumorous retina in all 10% formaldehyde solution-fixed, paraffin-embedded tissue sections. A characteristic "halo" cell surface staining pattern was observed in four (80%) of five tumors treated with two monoclonal antibodies, B6-30 (rhodopsin amino-terminal specific) and K16-107 (rhodopsin carboxyl-terminal specific). In each case, the staining pattern was limited to well-differentiated areas of the tumor containing Flexner-Wintersteiner rosettes or fleurettes. One tumor was not stained by any monoclonal antibody, whereas all monoclonal antibodies stained the rod cell outer segments of nontumorous retina. Our studies indicate that selected retinoblastomas may be differentiated along a rod photoreceptorlike cell lineage.

The pigment dispersion syndrome.

The pigment dispersion syndrome was noted in 10% of white and black subjects with and without glaucoma. This suggested pigment dispersion was not a high risk factor in the development of glaucoma. The HLA-B7 antigen was less prevalent and HLA-B13 and Bw17 antigens were significantly more prevalent in individuals with the pigment dispersion syndrome than in subjects without the syndrome. Either HLA-B13 or Bw17 antigen was found in 23 (77%) of 30 patients with the pigment dispersion syndrome and in only 13 (6%) of the 203 subjects without pigment dispersion. Pigmentary glaucoma was characterized by a significantly higher prevalence of combinations of HLA-B12 and B13 or HLA-B12 and Bw17 antigens as compared to the pigment dispersion syndrome without glaucoma, primary open-angle glaucoma, or the general population. This suggested that pigmentary glaucoma differed genetically from primary open-angle glaucoma and was a separate entity.

Two types of lamprey retina photoreceptors immunoreactive to rod- or cone-specific antibodies.

The localization of structures immunoreactive to antisera against opsin (OPS; a specific marker for rods) and against visinin (VIS; a specific marker for cones in the vertebrate retina) was investigated in the retina of the river lamprey (Lampetra japonica) by means of fluorescence microscopy. Different immunoreactive structures were found with the two antisera. A vitreadly located structure in the outer nuclear layer displayed OPS-like immunoreactivity, while a scleradly located one exhibited VIS-like reactivity. Thus, the two antisera applied to the retina of the river lamprey permit the distinguishing of rod and cone immunoreactive photoreceptors such as occurs typically in all vertebrate classes. The former appears in the outer segments of a type of rod cells, and the latter in the cell bodies and axons of cones.

Monoclonal antibodies specific to chicken iodopsin.

Monoclonal antibodies (mABs) from hybridoma cells were raised and screened with a purified cone pigment, iodopsin, from the chicken retina. Four different methods were used to test these antibodies: (1) dot-immunobinding assay; (2) enzyme-linked immunoabsorbent assay (ELISA); (3) one dimensional immunoblotting and (4) two dimensional immunoblotting. Three classes of antibody producing cell lines were identified. One class produces a mAB specific to iodopsin. The mAB from the second class crossreacts with iodopsin and probably one of the other three cone pigments. The mAB from the third class probably crossreacts with all the four cone pigments in the chicken retina. The mABs from all these classes of hybridoma cell lines were selected so that they do not crossreact with rhodopsin. Two dimensional immunoblotting indicated that iodopsin has a much higher isoelectric point than rhodopsin, as suggested from the known amino acid sequences of human rod and cone pigments.

A monoclonal antibody specific for the phosphorylated epitope of rhodopsin: comparison with other anti-phosphoprotein antibodies.

Monoclonal antibody A11-82P has been prepared and shown to recognize the phosphorylated epitope of bovine rhodopsin. Antibody A11-82P is quite specific for phosphorylated rhodopsin; only the 200K neurofilament protein has been found to cross react, and this requires 10(3) more protein for the same extent of binding as evaluated by competition ELISA. An immunocytochemical study of light-adapted retina showed strong staining of rod outer segments. Survey of a variety of rat tissues showed no specific staining with A11-82P, further demonstrating that this antibody is quite specific for phosphorylated rhodopsin. Two other antibodies were found to bind both phosphorylated rhodopsin and the 200K neurofilament protein: RT-97 (1) and MAP 1B3 (2). Both antibodies also recognized other phosphoproteins and appear to be less specific in their structural requirements for a phosphoprotein epitope than is A11-82P.

Rhodopsin and blindness.

Systemic immunization with purified homologous rhodopsin from retinal outer segments induced blindness in primates (Macaca mulatta). Inflammation and characteristic retinal changes were the earliest clinical signs of the disease. Perivasculitis, subretinal exudations and bullous detachments of the retina were progressive and unrelenting pathological processes leading to rapid and irreversible visual deterioration. Electroretinographic responses (ERG) at this stage of the disorder became abolished. Antibodies and delayed hypersensitivity to rhodopsin were demonstrated only in the experimental diseased animals. Homologous visual purple appears to be organ and immunopathologically specific. Histological confirmation of these findings showed a pathological spectrum of destructive alterations confirmed specifically to the outer segments of the entire retina. The pathologic reaction was supported by a distinct and pronounced granulomatous inflammatory response.

Localization of visual pigment antigens to photoreceptor cells with different oil droplets in the chicken retina.

Frozen semithin sections and unembedded retinal pieces were investigated by immunocytochemistry using two antibodies produced against visual pigments in our laboratory. One was a polyclonal serum (AO) raised against bovine rhodopsin, while the other one was a monoclonal antibody (COS-1) produced against an epitope present in a cone visual pigment. AO stained, as expected, rod outer segments; in addition it also recognized a single cone characterized by a deep yellow oil droplet as well as another single cone with a yellowish green oil droplet. In contrast, COS-1 labelled both members of the double cones; the principal member having a yellowish-green oil droplet and the accessory member. COS-1 also stained a single cone type exhibiting a large red oil droplet.

Monoclonal antibodies to chicken iodopsin.

The protein moiety of chicken iodopsin, R-photopsin, was purified from the chicken retina using a sucrose flotation method followed by two steps of column chromatography. Apparent molecular weights of R-photopsin and scotopsin (the protein moiety of chicken rhodopsin), which was partly purified in the process of purification of R-photopsin, were estimated to be 34,000 and 36,000, respectively, by sodium docecylsulfate-polyacrylamide gel electrophoresis. Using the purified R-photopsin as an antigen, four kinds of hybridoma cells which secreted monoclonal antibodies specific for R-photopsin and iodopsin were prepared. The antibodies thus obtained reacted with neither other chicken cone visual pigments nor rhodopsin as analyzed by immunoblots and immunoprecipitation methods. All the monoclonal antibodies stained the majority of the cone outer segments in chicken retina, while an antiserum raised against cattle rhodopsin stained the rod outer segments as well as some cone outer segments in the retina.

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin.

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.

Anti-lamprey retinal antibodies: immunohistochemistry on the retinas of several species of vertebrates.

Two monoclonal antibodies, H16 and B11, which were raised against lamprey retinal homogenate, were found to react with both short and long photoreceptor outer segments. On Western blotting of the retinal homogenate, both antibodies recognized a 40,000 Da and a 80,000 Da band. H16 antibody stained rod outer segments of all examined vertebrates, all cone outer segments of the turtle and chicken, and certain cone outer segments of the macaque. B11 antibody stained submammalian rod outer segments and some mammalian cone outer segments, leaving all mammalian rod outer segments unstained. The epitope recognized by H16 antibody is considered to be located in a conserved or commonly inherited element of an outer segment-bound molecule, presumably rhodopsin. B11 antibody, on the other hand, seems to recognize a reactive group which has failed to be inherited by mammalian rod cells; why it recognizes all cone outer segments in the turtle and chicken and only a part of them in the cow, cat, and macaque, meanwhile ignoring all of them in the frog and fish, is subject to further study.