Interleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon gamma, nitric oxide, and apoptosis.

Pathogenic effector T cells in experimental autoimmune uveitis (EAU) are T helper type 1-like, and interleukin (IL)-12 is required for their generation and function. Therefore, we expected that IL-12 administration would have disease-enhancing effects. Mice were immunized with a uveitogenic regimen of the retinal antigen interphotoreceptor retinoid-binding protein, treated with IL-12 (100 ng/d for 5 d), and EAU was assessed by histopathology. Unexpectedly, IL-12 treatment failed to enhance EAU in resistant strains and downregulated disease in susceptible strains. Only treatment during the first, but not during the second, week after immunization was consistently protective. High levels of interferon gamma (IFN-gamma) were present in the serum during IL-12 treatment, but subsequent antigen-specific IFN-gamma production in protected mice was diminished, as were IL-5 production, lymph node cell proliferation, and serum antibody levels. Treated mice had fewer cells and evidence of enhanced apoptosis in the draining lymph nodes. Unlike wild-type mice, IFN-gamma-deficient, inducible nitric oxide synthase (iNOS)-deficient, and Bcl-2(lck) transgenic mice were poorly protected by IL-12, whereas IL-10-deficient mice were protected. We conclude that administration of IL-12 aborts disease by curtailing development of uveitogenic effector T cells. The data are compatible with the interpretation that IL-12 induces systemic hyperinduction of IFN-gamma, causing activation of iNOS and production of NO, which mediates protection at least in part by triggering Bcl-2 regulated apoptotic deletion of the antigen-specific T cells as they are being primed.

Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP).

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific for the retina and pineal gland, induces inflammatory changes in these two organs in immunized animals. We report here on the identification of an immunodominant determinant of bovine IRBP that is highly immunogenic and immunopathogenic in the Lewis rat. The peptide, which comprises the sequence 1169-1191 of bovine IRBP, was shown to be immunodominant by its capacity to stimulate lymphocytes sensitized against whole IRBP. A comparison was made between peptide 1169-1191 and another peptide, 1158-1180, which is nondominant but is immunogenic and immunopathogenic in the Lewis rat. Peptide 1169-1191 was found to be superior in its immunological capacities; the minimal dose of 1169-1191 needed to induce cellular immune response or disease in Lewis rats (0.02-0.1 nmol/rat) is congruent to 1,000 times smaller than that of 1158-1180. In addition, unlike the ocular disease induced by 1158-1180, the disease produced by 1169-1191 resembled that induced by whole IRBP in its kinetics and histopathological features. The immunological activity of 1169-1191 in the Lewis rat was localized to the 10 residues at the COOH terminus; no such activity was exhibited by the truncated peptide 1169-1188, which comprises the 20 residues at the NH2 terminus of the full peptide. The usefulness of this unique experimental system in analyzing the role of immunodominance in peptide immunogenicity and immunopathogenicity is underscored.

Interleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoimmunity.

The present study addresses the feasibility of potentiating oral tolerance by immunomanipulation, using the murine model of experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP). Three feedings of 0.2 mg IRBP every other day before immunization did not protect against EAU, whereas a similar regimen of five doses was protective. However, supplementing the nonprotective 3x regimen with as little as one injection of 1,000 U of human recombinant interleukin-2 (IL-2) resulted in disease suppression that was equal to that of the protective 5x regimen. The protective effect was maintained across a range of IL-2 doses and times of administration; none of the IL-2 regimens tested resulted in disease enhancement. Peyer's Patch cells of 3x-fed and IL-2-treated mice showed greatly increased production of TGF-beta, IL-4, and IL-10 compared with animals given the nonprotective 3x regimen and to animals given the protective 5x regimen. We propose that IL-2 treatment enhances protection from EAU at least in part by stimulating production of antiinflammatory cytokines by regulatory cells in Payer's Patches. Moreover, the observed lymphokine production patterns suggest that whereas protection induced by the 3x + IL-2 regimen is likely to involve antiinflammatory cytokines, protection induced by the 5x regimen might involve anergy or deletion of the uveitogenic T cells. These results could have practical implications for use of IL-2 as a safe and effective way of potentiating oral tolerance.

Visual cycle and its metabolic support in gecko photoreceptors.

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Separation of retinoid receptors from cultured retinoblastoma cells.

Receptor proteins for [3H]retinol and [3H]retinoic acid in cultured human retinoblastoma cells have been separated rapidly and reproducibility by two different methods. By isoelectric focusing, the isoelectric point of the retinol receptor is at pH 4.0; the retinoic acid receptor has a higher isoelectric point of 4.3. Polyacrylamide slab gel electrophoresis revealed a slower migration rate for the [3H]retinoic acid receptor compared to the [3H]retinol receptor. The separate nature of the two proteins has thus been established in this unique human cell line.

Vitamin A receptors of the retina: differential binding in light and dark.

Vitamin A receptors of the retina appear to be differentially extractable in light and in dark suggesting that they could function as inter- or intra-cellular transport vehicles in the visual cycle.

Vitamin A receptors. II. Characteristics of retinol binding in chick retina and pigment epithelium.

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [3H]retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [3H]retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of KD about 1 - 10(-9) and 4 - 10(-8).

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.

Retinol receptors in corneal epithelium, stroma and endothelium.

Specific receptors for retinol are present in the cytosol fraction of corneal epithelium as demonstrated by sucrose density gradient centrifugation. These appear to be (1) protein in nature (2) of small molecular size (2 S) (3) specific for retinol and (4) present in several species. Assuming a receptor molecular weight of 15 000 and a single mole of retinol bound/mole of receptor protein, the association constant value is 5.26-10(7) with deltaG degrees = -8.53 kcal/mol. 2-S receptors are also observed in stroma and endothelium along with another binding species of approximately 8 S. Binding of [3H]retinol in bovine epithelial cytosol can also be demonstrated by disc gel electrophoresis and gel filtration. Immunodiffusion techniques demonstrate that monkey corneal epithelial and stromal cytosol samples do not contain contaminating serum retinol binding-protein.

Interphotoreceptor retinoid-binding protein (IRBP). Molecular biology and physiological role in the visual cycle of rhodopsin.

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retina pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP).

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.

Copolymer 1 inhibits experimental autoimmune uveoretinitis.

Copolymer 1 (Cop 1) inhibits experimental allergic encephalomyelitis induced by a variety of myelin proteins, but has been found ineffective so far in inhibiting other experimental autoimmune diseases such as diabetes or arthritis. Here, we report for the first time that Cop I inhibits the development of experimental autoimmune uveoretinitis, induced in mice by interphotoreceptor retinoid-binding protein (IRBP). Pooled data of three experiments showed that treatment with Cop 1, at 0.5 mg/mouse, reduced the disease severity by 53% ( p = 0.0002). Cop 1 treatment also inhibited the proliferation and the production of cytokines by lymph node cells in response to IRBP and moderately reduced the antibody response to this antigen. The possible mechanisms of EAU inhibition by Cop 1 are discussed.

Heterologous epitopes of IRBP protect against autoimmune uveitis induced by the autologous epitope.

Peptide 161-180 of human interphotoreceptor retinoid-binding protein (IRBP) contains a major uveitogenic epitope for mice of the H-2r haplotype. The human and bovine homologs differ from the autologous murine homolog by three and four amino acid residues, respectively. We compare the immunogenicity and pathogenicity of the three homologs, and investigate their ability to induce oral tolerance to experimental autoimmune uveoretinitis (EAU) induced by the autologous peptide. All three 161-180 homologs were pathogenic, with a hierarchy: human > murine > bovine. All crossreacted with each other and with IRBP. Feeding any of the three homologs (6 x 200 microg over 2 weeks) lowered antigen-specific responses and protected from EAU induced by the autologous homolog, and reduced EAU induced with whole IRBP. Peptide-fed mice had a reduced frequency of peptide-reactive T cells, suggesting a mechanism involving anergy and/or deletion. The results indicate that non-identical, but crossreactive, heterologous epitopes can protect against EAU induced by the corresponding autologous epitope, and even by the whole multi-epitope protein. These findings may impact on clinical trials in which uveitis patients are undergoing oral immunotherapy with bovine retinal antigens.

Effects of experimental ocular inflammation on ocular immune privilege.

PURPOSE: To determine whether the inflammation of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) alters key in vivo and in vitro parameters of ocular immune privilege. METHODS: For EIU induction, C3H/HeN mice received 200 microg lipopolysaccharide (LPS). For EAU induction, B10.A mice were immunized with 50 microg interphotoreceptor retinoid-binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at periodic intervals and assayed for leukocyte content and the ability to suppress or enhance T-cell proliferation. Eyes with EAU were assessed for the capacity to support anterior chamber (AC)-associated immune deviation (ACAID) induction after injection of ovalbumin (OVA). RESULTS: Inflammation within the anterior segment in EIU peaked at 12 to 24 hours and was detected from 10 days onward in EAU. In AqH of EIU, protein content rose within 4 hours, followed by infiltrating leukocytes. EIU AqH promptly lost its capacity to suppress T-cell proliferation and became mitogenic for T cells. In AqH of EAU, protein and leukocyte content rose at 11 days and continued to remain elevated thereafter. Whereas 11-day EAU AqH failed to suppress T-cell proliferation, AqH at later time points reacquired immunosuppressive properties. Injection of OVA into the AC of eyes of mice with EAU failed to induce ACAID. CONCLUSIONS: The intraocular inflammation of EIU and EAU disrupted important parameters of immune privilege, ranging from breakdown of the blood- ocular barrier, to loss of an immunosuppressive microenvironment, to abrogation of ACAID. Because AqH from inflamed EAU reacquired the ability to suppress T-cell proliferation, the authors conclude that the capacity to regulate immune expression and inflammation can be a property even of inflamed eyes.

Differential retinoid binding in chick pigment epithelium and choroid.

A receptor for retinol but not for retinoic acid was demonstrated to be present in fresh chick embryo pigment epithelium and in pigment epithelial cells grown in tissue culture. In contrast, chick choroid was found to possess receptors for both retinoic acid and retinol.

The requirement for pertussis to induce EAU is strain-dependent: B10.RIII, but not B10.A mice, develop EAU and Th1 responses to IRBP without pertussis treatment.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) in mice is an important model for elucidating basic mechanisms in autoimmune eye disease. The need for pertussis toxin (PTX) as an additional adjuvant to elicit EAU has limited the usefulness of this model in some types of studies by introducing a pleiotropic factor with confounding effects on the immune response. METHODS: In the present study the authors examined the ability of B10.RIII mice, the most susceptible strain known so far, to develop EAU in response to the retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), and to a major uveitogenic epitope of IRBP, peptide (p)161-180, in the absence of PTX treatment. RESULTS: The data indicate that high disease scores in response to IRBP and p161-180 were found in B10.RIII mice, without the need for PTX as part of the immunization protocol. Unlike the B10.A strain in which appreciable disease did not develop without PTX, B10.RIII mice mounted a high IFN-gamma response to IRBP in the absence of PTX treatment. Interestingly, and unlike the effect with IRBP, in vitro recall response to p161-180 was low in IFN-gamma, despite good EAU scores. CONCLUSIONS: The data indicate that an important mechanism through which PTX facilitates induction of cell-mediated autoimmunity is by promoting a Th1 polarization of the immune response. The propensity of B10.RIII mice to mount a more polarized Th1 response to IRBP than other strains may contribute to their ability to develop EAU without pertussis adjuvant. Nevertheless, the induction of EAU by p161-180 in the context of a relatively limited IFN-gamma production indicates that non-Th1- and Th-related mechanisms are likely to act in concert to determine the outcome of disease.

Circadian-dependent retinal light damage in rats.

PURPOSE: To determine the relative susceptibility of rats to retinal light damage at different times of the day or night. METHODS: Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure. RESULTS: When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light. CONCLUSIONS: These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.

Unusual immunologic properties of the uveitogenic interphotoreceptor retinoid-binding protein-derived peptide R23.

Peptide R23, consisting of residues 1091-1115 of bovine interphotoreceptor retinoid-binding protein (IRBP), had some unusual immunologic properties in Lewis rats. The peptide induced experimental autoimmune uveoretinitis and pinealitis in these rats, but only at high doses. The minimal immunopathogenic dose was found to be 100 nmol/rat. On the other hand, R23 was highly immunogenic in Lewis rats, producing cellular immunity, as measured by the lymphocyte proliferation assay, with a minimal dose of 1 nmol/rat. This unusual dissociation between the uveitogenic and immunogenic activities of R23 was attributed to different sites on the peptide, stimulating either the lymphocytes which induce disease or those which vigorously proliferate in culture. The potent immunogenicity of R23 was consistent with the peptide being immunodominant, as demonstrated by its capacity to be recognized by lymphocytes sensitized against whole IRBP and to stimulate these cells in culture to proliferate and acquire uveitogenic capacity. Likewise, lymphocytes sensitized against R23 are stimulated in culture by whole IRBP. Unexpectedly, peptide R23 was inferior to whole IRBP in its capacity to stimulate uveitogenicity in R23-sensitized lymphocytes. This finding also was attributed to the preferential stimulation by R23 of the lymphocytes specific for the putative "nonuveitogenic" site on the peptide. Peptide R23 also differs from the other tested bovine IRBP-derived peptides in the specificity of its antibodies. Unlike antibodies to the other peptides, those to R23 showed a strong cross reactivity toward whole IRBP.

Redistribution and reduction of interphotoreceptor retinoid-binding protein during ocular coronavirus infection.

Inoculation of the neurotropic coronavirus mouse hepatitis virus strain JHM intravitreally or into the anterior chamber causes acute infection of the retinal pigment epithelium (RPE) and neural retina. Weeks later, many retinas have foci of moderate to severe atrophy. The effect of coronavirus infection (after intravitreal inoculation) was examined on interphotoreceptor retinoid-binding protein (IRBP), the glycolipoprotein in the interphotoreceptor matrix (IPM) thought to transport retinoids between the photoreceptors and the RPE. Changes in IRBP distribution accompanied virus-associated retinal pathology, including photoreceptor loss and RPE abnormalities. Immunohistochemistry on days 3 and 6 showed that IRBP had diffused into the neural retina away from the IPM. The IRBP became localized abnormally in the same areas as virus-induced lesions, shown by staining adjacent sections with a monoclonal antibody specific for the viral nucleocapsid protein. Moreover, the level of IRBP in isolated retinas, measured in an immunoslot-blot assay, decreased significantly by day 3 and remained low through day 23. This decrease was confirmed in eyecups isolated on day 6. It may be caused in part by loss of photoreceptors and diffusion of IRBP through the retina into the vitreous. These studies show that a virus may induce an acute, limited infection in the retina that can be cleared by the host. However, the infection initiated a series of events resulting in long-term reduction and redistribution of a critical photoreceptor protein.

Interphotoreceptor retinoid-binding protein in retinal rod cells and pineal gland.

Immunoelectron microscopic staining demonstrates Interphotoreceptor Retinoid-Binding Protein (IRBP) in monkey rod cell cytoplasm with virtually none in cone cells. The pineal also contains significant amounts of IRBP demonstrating a similarity of pinealocytes to rod but not cone photoreceptors.