H.J. Winkens

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

&NA; 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 μM. A delay of differentiation was also observed using 1 μM 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 μM retinoic acid the onset of transcription of both genes was delayed by 2–3 weeks. We conclude that alltrans‐, 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.

Light induced shift and binding of S-antigen in retinal rods

S-antigen has been quantitated in bovine and rat retina by electroimmunoassay. The molar ratios S-antigen to rhodops in in photoreceptor cells were close to 1:1. Immunofluorescence studies show that light induces a shift of S-antigen towards the rod outer segments where it concentrates. Assays indicate that S-antigen becomes largely water insoluble but detergent soluble under these conditions. On basis of previous ultrastructural and present results, this has been interpreted as a light induced binding of S-antigen to the rod outer segment membranes. The data support evidence from literature that S-antigen interacts with (rhod) ops in and we conclude that S-antigen plays a major role in the phototransduction process.

Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

ABSTRACT 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.

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.

Retinol Dehydrogenase (RDH12) Protects Photoreceptors from Light-induced Degeneration in Mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12-/- mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage1.

Induction of experimental autoimmune uveoretinitis and pinealitis by IRBP. Comparison to uveoretinitis induced by S-antigen and opsin

Microgram quantities bovine IRBP (interphotoreceptor retinoid binding protein) injected in Freunds complete adjuvant induced severe autoimmune uveoretinitis and pinealitis in Lewis rats. At low doses the onset was accelerated and intensified by co-injection of Hemophilus pertussis bacteria. Wistar, BN and PVG rats were less susceptible, while the eyes of athymic, nude rats did not respond. The disease developed similar to but faster than S-antigen-induced uveoretinitis, while its onset was one day earlier and the reactions were slightly more severe. As distinct from these two types of uveoretinitis, opsin (in much higher doses) caused milder reactions in the anterior segment, while retinitis dominated. In each type of inflammation the photoreceptor cell layer was totally destroyed. All three ocular diseases were inhibited by cyclosporine treatment, which indicates that T cell-dependent mechanisms are essential for the development.

Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium.

Retinal pigment epithelial cell fractions have been investigated for their capacity to induce experimental uveitis. Cells of the dark (melanotic) and light areas of the bovine RPE have subsequently been extracted by buffer, Triton X-100, sodium dodecyl sulfate (SDS), and treated with various reagents in order to study some characteristics of the antigen. The SDS-insoluble melanotic fraction, consisting of spindle-shaped, mature melanin granules, proved to be the most uveitogenic preparation. Using pertussis toxin as coadjuvant, 1 microgram of melanin-protein (3.4 x 10(6) granules) was able to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats. The pathogenic activity of the responsible pathogen (PEP-X) was not diminished by SDS, nor eliminated by mildly alkaline SDS or formic acid treatment. However, HCl-deproteinized granules were not uveitogenic. The results show that PEP-X is a highly stable melano-antigen that is probably covalently bound to the granule surface. This is the first time that a melanin-bound antigen has been demonstrated to evoke specific autoaggressive activity. EAAU could adoptively be transferred by sensitized and in vitro stimulated CD4 T-lymphocytes. The evoked inflammation started 3-4 days after injection, was similar to those induced by immunization, and consisted mainly of severe iridocyclitis accompanied by dense flare and cells in the anterior chamber. Choroiditis developed in severe cases of EAAU but no inflammation was detected in the retina, pineal gland or other organs of these rats. EAAU could not be transferred by serum. Immunized PVG rats and guinea-pigs did not develop ocular inflammation. In monkeys a high dose of antigen evoked a very mild EAAU accompanied by choroiditis. In view of its characteristics, EAAU may be a new model for human anterior uveitis.

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‐β‐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.

Lens membranes VII. MIP is an immunologically specific component of lens fiber membranes and is identical with 26K band protein.

Abstract Calf lens MIP ∗ (solvent-extracted from the fiber membranes) and 26K band protein (isolated by SDS-PAGE) have been shown to have very similar amino acid compositions and molecular weights. MIP comprises two components which are immunologically identical with the two antigens detected in 26K band protein. In addition, MIP extracts contain two antigens in very small amounts which are common to urea-treated fiber and epithelial lens membranes. 26K band protein contains one of these antigens. Immunodiffusion, immunofluorescence and SDS-PAGE show MIP to be a specific component of the fiber membranes, which cannot be detected in epithelial membranes. Therefore, MIP may be considered as a marker of differentiation. MIP determinants can neither be found in other ocular tissues than the lens nor in non-ocular tissues, demonstrating the organ specificity of the protein.

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 Freunds 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.