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.

Experimental autoimmune anterior uveitis. The preparation of uveitogenic ocular melanin.

PURPOSE: The purpose of this study was to develop a rapid procedure for the preparation of melanin with a specific, highly uveitogenic activity. METHODS: A crude melanosome fraction was isolated from bovine choroids (containing remnants of adhering retinal pigment epithelium). The fraction was extracted with hot 2% sodium dodecyl sulfate, and Lewis rats were immunized with the purified melanin, using pertussis toxin as coadjuvant. RESULTS: The purified melanin was free from pathogenic photoreceptor antigens and other accompanying or adsorbed proteins. It was able to evoke severe, acute, anterior uveitis with the typical characteristics of experimental autoimmune anterior uveitis (EAAU; without retinitis or pinealitis), even at the level of 1 micrograms melanin protein. CONCLUSIONS: The rapidly prepared ocular melanin exhibits the same qualities as purified choroidal or retinal pigment melanins obtained by much more laborious procedures (which also deliver other subcellular fractions for investigation). It is suitable for the study of the immunopathogenesis of EAAU, which is a new model for human acute anterior uveitis.

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.

Opsin for immunological studies.

Opsin for immunological studies can be prepared free from S-antigen by affinity chromatography on Con A-Sepharose. This preparation, however, contains 2-6% Con A originating from the affinity medium. Con A as impurity disturbs lymphocyte transformation tests carried out with opsin as test antigen. We describe a method for the removal of Con A by immuno-adsorption to Protein A/anti-Con A/IgG in detergent. In addition, we have selected some detergents and detergent concentrations in which opsin can be purified and added to the lymphocyte culture medium.

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 Freund's 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.

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.

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.

Uveitogenic 28/30 kD and 43 kD polypeptides in pigment epithelial membranes of the retina.

UNLABELLED: The purpose of this study was the search for new intrinsic polypeptides of the retinal pigment epithelium (RPE) capable of evoking experimental uveitis. A membrane fraction was prepared from purified bovine RPE cells. The Triton X-100 soluble protein fraction was separated into polypeptide fractions by preparative gel electrophoresis, and the pathogenicity of the main isolated polypeptides was investigated in Lewis rats. After immunization, two hitherto unknown pigment epithelial polypeptides with M(r) 28/30 kD (doublet) and 43 kD (PEP-28/30 and PEP-43, respectively) elicited progressive pigment epitheliitis and choroiditis accompanied by extending plaque-shaped macrophage accumulations along the RPE-Bruch's membrane layer. No inflammatory foci were found within the neural retina. Polypeptide fractions with M(r) 14-17, 25 and 32/34 kD (doublet) (PEP-14/17, PEP-25 and PEP-32/34, respectively) appeared to be non-uveitogenic at the tested dose. PEP-28/30 and PEP-43 exhibited a partial antigenic relationship to PEP-65. PEP-28/30 exhibited marked reactivity to a monoclonal antibody previously raised to a 32 kD RPE-specific protein. IN CONCLUSION: in addition to the previously described main RPE-specific membrane polypeptide PEP-65, the RPE appears to contain two more uveitogenic components, the intrinsic pigment epithelial membrane polypeptides PEP-28/30 and PEP-43. Like PEP-65, these antigens are able to evoke experimental autoimmune pigment epithelial protein-induced uveitis (EAPU) in rats.

Multiple recurrences in melanin-protein-induced uveitis in the rat.

The purpose of the present study was to investigate the occurrence and sequelae of multiple recurrences in experimental melanin-protein-induced uveitis (EMIU). Lewis rats were immunized with purified bovine choroidal melanin granules, and the development of EMIU was studied during six months. Multiple recurrences were observed in virtually all rats that developed primary EMIU. The spontaneous recurrences exhibited an increasingly mild character and a decreasing frequency over time. They occurred one to four times per eye with intervals of five to six weeks. If the inflammations were more severe or chronic the uveal tissues were more seriously damaged. The anterior uvea became slender by loss of cells and stroma during the process suggesting a role as target. Unlike in primary EMIU, the retina finally exhibited areas with damage of the photoreceptor and pigment epithelial cells. Mononuclear cells were the predominant inflammatory cell type in the entire uvea in eyes with serious recurrences or chronic uveitis. The number of recurrences per se did not correlate with the extent of tissue damage but the overall severity of the disease over six months did. In rats recovered from mild recurrences, a single injection of pertussis toxin, or melanin granules emulsified in Freund's incomplete adjuvant was sufficient to reinduce severe EMIU with extensive damage of the uvea. Hence, specific as well as aspecific stimulation of the immune system caused severe recurrences of this type of uveitis. EMIU resembles non-infectious human anterior uveitis in several respects, even in its multiple recurrences.

Assay of S-antigen immunoreactivity in mammalian retinas in relation to age, ocular dimension and retinal degeneration.

The immunoreactivity of S-antigen was assayed in the developing and adult retinas of a variety of mammals, including man. An electroimmunoassay was used with bovine S-antigen (in Triton X-100) as a standard and rabbit antiserum to this antigen was used for precipitation. In the newborn mouse, rat and rabbit retina no S-antigen was detected. During the second to fifth postnatal week a rapid increase in the immunoreactivity of this protein was found, which ran largely parallel to the development of photoreceptors and the increase in retinal rhodopsin content. In the rat, rabbit and guinea pig the adult level of retinal S-antigen remained constant for a long period of life. In many mammalian species the amount of retinal S-antigen immunoreactivity appeared to increase proportionally to the square of the radius of the eye globe, which is closely related to the retinal surface area. Possible implications of this relationship are discussed. Specific anatomical and morphological characteristics of the eye and its tissues, rods/cones ratio, retinal degeneration and anomalous crossreactivity of S-antigens cause deviations from the relationship. For the S-antigen content of adult human retina a value of 950 micrograms was found if purified human S-antigen was used as a standard. Very low values were found in the retina of a mouse strain homozygous for retinal degeneration-slow.

Experimental melanin-protein induced uveitis (EMIU) is the sole type of uveitis evoked by a diversity of ocular melanin preparations and melanin-derived soluble polypeptides.

Experimental melanin-protein induced uveitis (EMIU) is a CD4 T cell-mediated disease involving the choroid and iris, but sparing the retina. The present study was designed to solubilize uveitogenic antigen from melanin granules without enzymatic digestion, and to investigate some of its elements by comparison with different purified melanin preparations. Many melanin surface-derived polypeptides with molecular weights ranging from 1 to > 100 kDa were obtained by extractions of the prepurified granules with hot lithium dodecyl sulfate (LDS). The mixture was electrophoretically separated into seven subfractions, each containing many components and capable of evoking the typical features of EMIU after footpad immunization of Lewis rats. The five low-molecular-weight fractions between M, 1 kDa and 30 kDa exhibited most pathogenicity which was evenly distributed among the fractions. Highly uveitogenic material remained in the melanin preparations even after multiple exhaustive extractions with LDS, and represented about 70% of the detectable protein. The uveal pathogen (UP-X) thus proved to be antigenically stable, and the major part of the pathogenic material was strongly bound to the granule surface layer. Concentrated urea solution was also capable of extracting many uveitogenic melanin polypeptides, but in a different composition than LDS did, and less effectively. Human choroidal melanin provided an LDS-soluble fraction with low pathogenicity. A single intraperitoneal injection of bovine melanin polypeptides together with pertussis toxin, but without footpad immunization in Freund's complete adjuvant, evoked EMIU as well. In all experiments, no uveitis except EMIU was observed, indicating that only one type of uveitogenic epitope was present in a wide variety of carrier molecules. An explanation for this phenomenon is discussed.

Gamma-crystallin-dodecyl sulfate complexes show discrete precipitation in non-ionic detergents.

Gamma-crystallin-sodium dodecyl sulfate (SDS) complexes are precipitated by Triton X-100 and other non-ionic detergents. This is accomplished as discrete lines by double diffusion, radial diffusion and other immunotechniques. Many other protein-SDS complexes including those of alpha- and beta-crystallins remain soluble. Immunoglobulin G tends to precipitate under similar conditions, a reason why the use of detergents in immunoprecipitation reactions has to be carefully controlled. The phenomenon can be used for the estimation of SDS or non-ionic detergents in the presence of gamma-crystallin.

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.

Experimental autoimmune posterior uveitis accompanied by epitheloid cell accumulations (EAPU). A new type of experimental ocular disease induced by immunization with PEP-65, a pigment epithelial polypeptide preparation.

Purified retinal pigment epithelial cells of bovine eyes have been fractionated by a series of buffer and detergent extractions. The electropherogram of the buffer-insoluble, Triton X-100-soluble fraction (RPE-TS) exhibits a major polypeptide band of M(r) 65 kDa and a variety of minor components. Electrophoretically purified 65 kDa-band protein (PEP-65) is immunologically unrelated to the known uveitogenic photoreceptor proteins, to other neural retina proteins, and to PEP-X, the RPE-melanin-bound uveitogenic antigen. An immunocytochemical study of eye tissues suggests that it is exclusively located in the RPE. Immunization of Lewis rats with PEP-65 or affinity-purified RPE-TS induces a new type of ocular disease: experimental autoimmune posterior uveitis accompanied by epitheloid cell accumulations (monocytes) adjacent to the RPE (EAPU). The disease starts 9 days after immunization, provided that pertussis toxin is used as co-adjuvant. The first clinical signs are transient flare and cells in the anterior chamber. Choroiditis develops, and epitheloid cells accumulate focally along one or both sides of the Bruch's membrane-RPE layer. Such foci resemble, in some respects, Dálen-Fuchs nodules which occur in human sympathetic ophthalmia. Areas of inflammation are frequently localized in the chorioretinal periphery adjacent to the pars plana. Vitreous cell infiltration is the most prominent clinical feature of EAPU. During at least 2 months, extending chorioretinal areas containing epitheloid cell collections remain while the adjacent photoreceptor cells sometimes disappear without being invaded by these cells. Retinal vasculitis is seldomly observed and pinealitis is absent. EAPU has the latter feature in common with PEP-X-induced experimental autoimmune anterior uveitis (EAAU). The two diseases differ from the various photoreceptor antigen-induced forms of EAU where pinealitis and inflammation of the neural retina are prominent features. However, just as in EAU and EAAU, EAPU can be adoptively transferred, and is inhibited by cyclosporin treatment suggesting T-cell dependency.

Experimental autoimmune anterior uveitis (EAAU). III. Induction by immunization with purified uveal and skin melanins.

The pathogenicity of uveal tissue and melanin has been a controversial subject for a long time. The present new approach has elucidated some of the problems. Melanin granules have been extracted from bovine choroid, iris, hair and skin, and from human, monkey and rabbit choroid. The melanin granules have further been purified by detergent extractions, and are free from pathogenic retinal antigens. Lewis rats immunized with microgram doses bovine choroidal or iris melanin-protein (in Freund's complete adjuvant or Hunter's adjuvant, combined with pertussis toxin) develop severe experimental autoimmune anterior uveitis (EAAU). No retinitis or pinealitis is found. The other melanins are weakly uveitogenic or inactive. The relative pathogenicity of the various melanins seems to be related to tissue and species specificity. The responsible hypothetic pathogenic structure UP-X (uveal pathogen X) is highly stable and resists proteolytic digestion by various enzymes. Its pathogenic activity is destroyed by hot 6 N HCl or longlasting 0.5 N NaOH treatment. In view of its chemical and immunological features it is probably identical to the pathogen PEP-X of bovine retinal pigment epithelial melanin. UP-X-induced EAAU can be transferred by spleen cells, and is suppressed by cyclosporin showing that a T-cell-mediated pathogenic mechanism predominates. It resembles human anterior uveitis by its specific location, its transient nature, and sparing of the retina. In these respects EAAU differs from retinal photoreceptor antigen-induced forms of EAU where retinitis with photoreceptor damage is a main feature. The involvement of melanin in human ocular diseases is discussed.

Experimental autoimmune anterior uveitis (EAAU): induction by melanin antigen and suppression by various treatments.

The uveitogenicity of melanin has been a controversial subject for a long time, presumably as a result of the use of ill-defined preparations in the experiments. We have developed procedures for the preparation of purified uveitogenic melanins from the retinal pigment epithelium and choroid that are free from pathogenic retinal photoreceptor proteins. The active melano-antigen is located at the surface of the melanin granules and is probably identical in both tissues. It retains its pathogenicity in hot polar detergent and during in vitro proteolysis, but it is inactivated by macrophage phagocytosis and hydrolysis in hot hydrochloric acid. Lewis rats immunized with microgram doses of bovine retinal pigment epithelial or choroidal melanin develop severe experimental autoimmune anterior uveitis (EAAU) about 10 days later. Retinitis and pinealitis are not observed. Skin melanin prepared in a similar way evokes EAAU as well, but it is only weakly pathogenic. EAAU cannot be transferred by serum, and its development can effectively be inhibited by antibodies to the inciting antigen and by cyclosporin. Vitamin E treatment of the animals causes a delay in its onset. The results indicate that cell-mediated immunity plays a dominant role in the pathogenesis of EAAU. This is the first time it has been shown that purified ocular and skin melanins are able to induce an autoimmune disease. The relevance of this finding for the study of melanin-related immunopathology in man is discussed.

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.

Differential effect of macrophage depletion on two forms of experimental uveitis evoked by pigment epithelial membrane protein (EAPU), and by melanin-protein (EMIU).

The purpose of the present study was to clinically and histologically investigate the influence of macrophage depletion on the development of experimental autoimmune pigment epithelial membrane protein-induced uveitis (EAPU), and experimental melanin-protein induced uveitis (EMIU) in the Lewis rat. EAPU is mainly characterized by pigment epitheliitis. Posterior mononuclear cell accumulations enclose and destroy the retinal pigment epithelium (RPE). In EMIU the inflammation is specifically localized in the uvea. EAPU was induced by immunization with RPE membrane protein, and EMIU was evoked by immunization with purified choroidal melanin. Systemic treatment with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes just before the expected beginning of the clinical signs of EAPU (at day 7 and 9 after immunization) resulted in a considerable delay of the uveitis process. In the treated animals the typical plaque shaped cell accumulations (containing many macrophages) along the RPE were lacking. Two weeks after the treatment, severe rebound EAPU developed. Local treatment by subconjunctival liposome injections did not exert any effect on EAPU. In EMIU, macrophage depletion by systemic treatment did not noticeably influence the clinical and histological development of the inflammation. Systemic treatment at the peak stage of EAPU (at day 12 and 14 after immunization) resulted in the rapid disappearance of the clinical signs of uveitis. Vitreous and anterior chamber cells were virtually absent two days later. This situation remained unchanged until the experiment was terminated two weeks later. Already deposited cell accumulations along the RPE did not regress but stopped their progression. Hematogenous macrophages thus appear to play a crucial role in the development of EAPU but the effect of early macrophage depletion on EAPU appeared to be temporary due to blood repopulation. A possible explanation for the differential influence of macrophage depletion on EAPU and EMIU is discussed, and is based on differences in immunopathogenesis.

Macrophage subpopulations and RPE elimination in the pathogenesis of experimental autoimmune pigment epithelial protein-induced uveitis (EAPU).

Experimental autoimmune pigment epithelial protein-induced uveitis (EAPU) is a new type of disease that destroys the retinal pigment epithelium (RPE), and exhibits a hitherto unknown form of progressive chorioretinal dystrophy in which neuroretinal inflammatory foci are absent. The present study was aimed at studying the expression of adhesion molecules, and the kinetics of the appearance of the main types of macrophages and other intraocular immunocompetent cell populations in the various stages of this disease. EAPU was evoked in Lewis rats by immunization with the membrane protein from bovine RPE cells containing PEP-65 as main constituent. In the uvea, increased expression of intercellular adhesion molecule-1, of class II major histocompatibility complex antigen, and of ED2 macrophage reactivity were observed closely before the onset of EAPU. Expression of these reactivities was also slightly elevated by injections of the applied adjuvants alone. The onset of EAPU was mainly characterized by initial uveal infiltrations of ED1+ macrophages and a minor population of CD4 T cells, and an increase in ED3, ED7 and perivascular ED2 reactive macrophages. This was followed by the development of focal accumulations of ED1+ cells at both sides of the Bruch's membrane-RPE layer (Dálen-Fuchs nodules) which was permeated and disintegrated at these sites. The outer choroidal layer, the anterior iridal surface, and the base of the ciliary body more frequently contained active inflammatory cells than the other uveal areas. Lymphoid cells were found scattered through the uvea, aqueous and vitreous. The sites of increased activity of ED2+ and ED3+ cells in the uvea were rather similar to those of ED1 macrophages in the various stages of EAPU. Starting from multiple foci, the process of the formation of plaque-shaped cell accumulations in severe EAPU progressed along the RPE and exhibited a chronic character. The results of this study show that ED1+, ED2+, ED3+ and ED7+ subpopulations of macrophages are actively involved in an immunopathological process in which the RPE is the target. The thickening of the plaque-shaped cell accumulations stops if the integrity of all RPE cells at that site has been affected. We postulate that this is the result of antigen elimination while additional influence of the abrogation of RPE cytokine production is presumed.

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.