Null mutation in the human 11-cis retinol dehydrogenase gene associated with fundus albipunctatus.

PURPOSE: Recent studies show that mutations in the gene encoding 11-cis retinol dehydrogenase are associated with fundus albipunctatus. The authors wanted to investigate whether additional, more severe, mutations in the 11-cis retinol dehydrogenase gene might be responsible for more severe forms of hereditary retinal diseases. DESIGN: Case-control molecular genetics study. PARTICIPANTS AND CONTROLS: Two index patients, 7 relatives, and 50 control individuals. METHODS: The authors screened two index patients diagnosed with fundus albipunctatus for mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene by direct sequencing. Control individuals were screened for the presence of the mutations using allele-specific oligonucleotide hybridization. MAIN OUTCOME MEASURES: Mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene. RESULTS: In a compound heterozygote, two novel mutations were found: a 4 bp insertion in exon 2 and a missense mutation Cys267Trp in exon 5. In a second pedigree, a homozygous frameshift mutation in codon 43 (Arg42ct[1-bpdel]) was detected. In both families, the mutations segregate with the disease. The mutations were not found in 50 control individuals. CONCLUSIONS: On the basis of our observations, it is unlikely that mutations in the 11-cis retinol dehydrogenase gene are associated with other, possibly more severe, retinal pathologic conditions/dystrophies or syndromic diseases in which the retina is also affected.

Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells.

The effect of retinoic acid on the differentiation of a human retinal pigment epithelium-derived cell line ARPE-19 was studied. Differentiation of ARPE-19 cells is delayed by retinoic acid. The minimum all-trans-retinoic acid concentration needed for delay of ARPE-19 differentiation is 1 microM. A delay of differentiation was also observed using 1 microM 9-cis or 13-cis-retinoic acid. Differentiation at the molecular level was studied by analyzing transcription of two RPE-marker genes, RPE65 and peropsin. In the presence of 1 microM retinoic acid the onset of transcription of both genes was delayed by 2-3 weeks. We conclude that all-trans-, 9-cis-, and 13-cis-retinoic acid delay differentiation of ARPE-19 cells into cells that phenotypically resemble cells from the human retinal pigment epithelium.

Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters.

To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.

Retinoic acid receptors and retinoid X receptors in the mature retina: subtype determination and cellular distribution.

PURPOSE: In the mature retina retinoic acid (RA) biosynthesis was reported to be regulated by light. RA is of vital importance for proper function of the retina. RA activity is mediated by interaction with nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The purpose of this study was to determine if and which RARs and RXRs are present in the mature retina, and to determine their location within the retina. METHODS: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify cDNA fragments encoding RARalpha, RARbeta, RARgamma, RXRalpha, RXRbeta, and RXRgamma from human retinal RNA. RT-PCR products were cloned, sequenced, and used in Northern blot experiments. Antibodies directed against each receptor subtype were used for immunocytochemical analysis. RESULTS: RT-PCR and Northern blot analysis indicated that all RAR and RXR subtypes are present in the mature retina. Western blot analysis, using a cytoplasmic protein fraction isolated from the bovine and human neural retina, showed the presence of RXRalpha. Immunocytochemical analysis of the human, bovine, and rat retina showed that RARalpha is highly expressed in the outer segments of cone photoreceptor cells. RXRalpha expression was observed in the rod inner segment layer. RXRgamma was detected in the nuclei and outer segments of cone photoreceptor cells. CONCLUSIONS: The retinal expression pattern of RARs and RXRs is heterogeneous. The observation that RXRalpha is present in rods whereas RARalpha is present in cones, suggests a mechanism by which RA that is produced upon bleaching, could generate different responses in the two photoreceptor cell subtypes.

The visual cycle retinol dehydrogenase: possible involvement in the 9-cis retinoic acid biosynthetic pathway.

The 11-cis-retinol dehydrogenase (11-cis-RoDH) gene encodes the short-chain alcohol dehydrogenase responsible for 11-cis-retinol oxidation in the visual cycle. The structure of the murine 11-cis-RoDH gene was used to reinvestigate its transcription pattern. An 11-cis-RoDH gene transcript was detected in several non-ocular tissues. The question regarding the substrate specificity of the enzyme was therefore addressed. Recombinant 11-cis-RoDH was found capable of oxidizing and reducing 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinaldehyde, respectively. Dodecyl-beta-1-maltoside used to solubilize the enzyme was found to affect the substrate specificity. This is the first report on a visual cycle enzyme also present in non-retinal ocular and non-ocular tissues. A possible role in addition to its role in the visual cycle is being discussed.

Cloning and structural analysis of the murine GCN5L1 gene.

We recently cloned the murine 11-cis retinol dehydrogenase gene. A second gene, the murine GCN5L1 gene, was found to be situated upstream of the murine 11-cis retinol dehydrogenase gene. We have isolated and sequenced the complete coding sequence of the murine GCN5L1 gene. The distance between the 3'-end of the murine GCN5L1 gene and the 5'-end of the 11-cis retinol dehydrogenase gene is only 776 nt. The murine GCNSL1 gene consists of four exons encompassing approximately 3.5 kb of genomic DNA. Intron/exon splice sites conform to the GT/AG rule. The open reading frame consists of 375 nucleotides encoding a 14 kDa protein. The murine GCN5L1, like the human GCN5L1 protein, displays weak homology (27%) to yeast GCN5. The distance between the murine, human and bovine GCN5L1 and 11-cis retinol dehydrogenase genes appeared to be conserved.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.

PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

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.

Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis. I. Induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium.

The uveitogenicity of several protein fractions of the bovine retinal pigment epithelium (RPE) was studied in Lewis rats, and a major pathogenic fraction was selected. Fresh RPE cells were carefully isolated and purified in order to minimize the presence of rod outer segments (ROS). The buffer-insoluble part of the cells was extracted by Triton X-100. Most uveitogenicity was found in the Triton-insoluble pigment and cytoskeleton-containing fraction of RPE (RPE-TI). The S-antigen and opsin contents of RPE-TI were too low to induce an inflammatory response, while transducin, IRBP and cGMP-phosphodiesterase were absent. Hence, a hitherto unknown uveitogenic RPE protein, called PEP-X, evoked the pathogenic response. A typical dose-dependent experimental autoimmune anterior uveitis (EAAU) developed when the rats were immunized with RPE-TI. Initially, mononuclear cells infiltrated the anterior segment. In subsequent severe stages polymorphonuclear cells predominated in the anterior chamber. EAAU differed in particular from the known forms of EAU induced by photoreceptor proteins in that the inflammation remained exclusively anterior and the photoreceptor cells and the pineal gland were not affected. In immunized rats the immune responses to ROS proteins were very low. In contrast, there were consistently high cellular and humoral immune responses to RPE-TI. As in experimental autoimmune (uveo)retinitis (EAU), the development of EAAU could be inhibited by cyclosporin treatment indicating T-cell-dependency. A combination of histopathological, immunological and biochemical results indicates that PEP-X is an intrinsic RPE protein that is highly pathogenic. In view of its characteristics, EAAU may be a valuable model for human acute anterior uveitis, the most prevalent form of uveitis.

Autoimmune responsiveness to retinal IRBP, S-antigen and opsin in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) was induced in rabbits by intravitreal injection of homologous fibroblasts. During the 8 weeks after injection the immune responsiveness to three purified retinal autoantigens was studied. From 2 weeks after injection, animals that developed serious forms of PVR exhibited definite mitotic responses of their lymphocytes to stimulation by the retinal antigens. These responses could consistently be demonstrated for S-antigen and interphotoreceptor retinoid-binding protein (IRBP) during the subsequent period of examination. Marked responses were also noted to opsin, however, their occurrence was more variable. In mild forms of PVR or in controls the responses were weak or absent. This showed that the elevated cellular reactivities were induced by the development of PVR and not by some other experimental factor. Humoral immune responses to the three antigens were absent (as assayed by ELISA). The control groups did not exhibit any elevated immune responsiveness. There appears to be accumulating evidence that inflammation may play a role in the development of PVR. The present results indicate that cellular autoimmune responses to photoreceptor antigens are a secondary phenomenon in PVR, nevertheless, they may be an important factor in the subsequent development of severe PVR. This autosensitization may consequently be taken into consideration in the treatment of complicated human PVR.

Rhodopsin-induced experimental autoimmune uveoretinitis in monkeys.

We present the first evidence that purified rhodopsin can induce experimental autoimmune uveoretinitis (EAU) in monkeys. Injection of a highly purified lipid-free rhodopsin preparation provokes severe chorioretinitis with concomitant anterior uveitis. The onset of disease is earlier, its frequency is higher, and the inflammation is considerably more severe than in EAU induced under similar conditions by opsin. The first inflammatory cells are observed in the ciliary body and pars plana. Within a few days the inflammation extends into the anterior chamber, choroid, and retina. Retinitis predominates in the central area, while chorioretinitis is observed in the periphery, both accompanied by damage to and elimination of the photoreceptor cells. The monkeys develop high cellular and humoral immune responses against rhodopsin and opsin. The cellular response maximum just precedes the onset of EAU. This may indicate that cellular immunity has an important role in the pathogenesis of rhodopsin-induced EAU.

Assay of cellular immune responses in rabbits sensitized to retinal antigens.

Cellular immune responses in retinal autoimmunity have scarcely been studied in rabbits. An effective isolation of rabbit peripheral blood lymphocytes is difficult to achieve and requires specific conditions. This hampers especially the assay of antigen-induced lymphocyte transformation. We have improved the lymphocyte isolation technique, resulting in consistent recoveries of 65-75% and enabling longitudinal studies of immunopathological conditions in rabbits. The technique was incorporated in a routine version of the lymphocyte transformation test, which has been optimized with respect to culture conditions and retinal antigen concentrations.

Rhodopsin-induced experimental autoimmune uveoretinitis: dose-dependent clinicopathological features.

We have studied the clinicopathological features of experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by injection of different doses of rhodopsin and its illuminated form opsin. Rhodopsin consistently appears to be more pathogenic than opsin. Injected in Freund's complete adjuvant and pertussis adjuvant 50 micrograms of rhodopsin induces a frequency of severe EAU similar to 250 micrograms of opsin. Intensity, frequency and location of ocular inflammation are markedly dose dependent. At high dose (100-250 micrograms), rhodopsin induces severe bilateral uveoretinitis in all animals, which starts with acute inflammation of the anterior eye segment at day 10-12 followed by chorioretinitis (predominantly retinitis) which results in complete elimination of the photoreceptor cells. At low dose (20 micrograms), rhodopsin induces mild transient inflammation in 60% of the animals, mainly consisting of mild posterior retinitis which starts at day 20 and leads to a typical multiple focal destruction of the photoreceptor cells. Intermediate doses cause an intermediate type of disease. Omission of pertussis adjuvant lowers the frequency of severe disease at low doses of rhodopsin, delays its onset and changes its features. The last characteristic has been observed in particular at intermediate doses (50-100 micrograms). In these cases, EAU usually starts by cell infiltration of the vitreous, while the anterior segment is only mildly affected. Without pertussis adjuvant the pathogenicity of opsin is low. Even in both adjuvants severe EAU can only be evoked by a high dose of opsin. Although there exists a marked difference in uveitogenicity between rhodopsin and opsin, the immunogenicity is similar and seems not to be correlated with their pathogenicity.

Immune responsiveness to retinal S-antigen and opsin in serpiginous choroiditis and other retinal diseases.

The immune responsiveness to bovine retinal S-antigen and opsin has been investigated in some retinal disorders by means of in vitro lymphocyte proliferation, leukocyte migration inhibition and enzyme linked immune sorbent assays (ELISA). Sensitisation to S-antigen was observed in serpiginous choroiditis, but not in acute posterior multifocal placoid pigment epitheliopathy (APMPPE) or retinitis pigmentosa. No significant immune responsiveness was detected to opsin in any of the three diseases. Elevated antibody titers to S-antigen were observed in some individual patients and healthy subjects. However, none of the patient groups exhibited an elevated antibody titer as compared to the control group. Although serpiginous choroiditis and APMPPE share some prominent clinical characteristics, the sensitisation in the former disease may perhaps be attributed to more severe and prolonged damage of the photoreceptor cells and blood-retina barrier. A combination of previous and present results suggests that in immunological investigations of retinitis pigmentosa patients it is more effective to use human than bovine S-antigen as test antigen because a species specific epitope seems to be involved.

Experimental autoimmune uveoretinitis in rats induced by rod visual pigment: rhodopsin is more pathogenic than opsin.

The rod visual pigment, rhodopsin, and its illuminated form, opsin, were used to induce experimental autoimmune uveoretinitis in rats. Rhodopsin appears to be more pathogenic than opsin. A dose of 250 micrograms rhodopsin injected in Freund's complete adjuvant and pertussis adjuvant induces nongranulomatous inflammation with higher frequency, which starts earlier and is more severe than that induced by opsin. Two weeks postinjection, the mean score of rhodopsin-injected animals is more than twice as high as that of opsin-injected animals. The high pathogenicity of rhodopsin appears to be related to the biochemical integrity of the protein and depends on its state of illumination. The levels of the immune responses (both cellular and humoral) measured at day 10 postinjection do not account for the pronounced difference in pathogenicity between rhodopsin and opsin. The developmental patterns of severe uveoretinitis induced by rhodopsin or opsin were histologically evaluated and appear to be similar. In both cases we observed dense mononuclear and polymorphonuclear cell infiltrations in the retina and anterior uvea. Only in the severe stages does the choroid become involved. However, rhodopsin causes more pronounced involvement of the ciliary body, pars plana, and anterior chamber. The inflammation finally results in total elimination of the photoreceptor cell layer.

Immunological and immunopathological aspects of opsin-induced uveoretinitis.

In an extension of our previous studies, experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats by injection of very high doses of bovine opsin. The induced reaction consisted predominantly of a mild posterior retinitis. Varying the amount of injected opsin between 300 and 1,000 micrograms did not influence this result, provided that the antigen was injected in Freund's complete adjuvant. Pathogenicity of opsin appeared to be lower than that of interphotoreceptor retinoid binding protein (IRBP) or S-antigen, while EAU induced by the latter antigens was much more dose-dependent than EAU induced by opsin. An increase of the dose strongly accelerated the onset and increased the incidence of EAU from low to moderate. However, severe inflammation and high incidence were only obtained by co-injection of Hemophilus pertussis bacteria. This adjuvant especially increased cellular immune responses to opsin as measured by lymphocyte transformation. No marked effects on humoral responses were detected by ELISA, using different types of opsin preparations. Development of opsin-induced EAU was inhibited by ciclosporin, a suppressor of certain specific T cell functions. Ciclosporin injections lowered the antibody response of the rats and eliminated measurable lymphocyte transformation in vitro. Induction of opsin-EAU therefore appears to be T-cell-dependent. The effect of pertussis adjuvant may be explained by enhancement of the T cell responses to opsin and by increasing the permeability of the blood-retina barriers. Other properties of the adjuvant may be of importance as well. A relationship between change in molecular conformation and uveitogenicity of opsin is discussed.

Opsin-induced experimental autoimmune retinitis in rats.

Experimental autoimmune retinitis has been induced in Lewis rats by injection of opsin in mycobacterial adjuvant and Hemophilus pertussis adjuvant. Clinical, histopathological and immunological parameters of the disease are reported. Two types of opsin were prepared from purified bovine retina outer segments, one type in Triton X-100 and the other in lithium dodecyl sulfate. Both preparations were free from S-antigen. Dodecyl sulfate-denaturated-opsin displayed lower antigenicity and pathogenicity than Triton-opsin. Triton-opsin (250 micrograms) induced moderate to severe non-granulomatous uveitis (predominantly retinitis) in 70% of the Lewis rats at the end of the second week after injection. The photoreceptor cell layer was destructed within a few days. This group displayed high responses to opsin in the lymphocyte transformation test. In view of observed histological features, the possible early involvement of vasoactive factors is discussed. Low opsin doses (50 or 100 micrograms) seldomly induced severe retinitis, while the incidence of mild pathology was low. Lewis rats appeared to be more susceptible for the development of experimental autoimmune retinitis than Wistar rats.