Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats.

PURPOSE: Inducible nitric oxide (NO) synthase (iNOS) has been implicated in the pathogenesis of endotoxin-induced uveitis (EIU). This study was undertaken to localize the cells, in the eye, which express iNOS during EIU in the rat. METHODS: EIU was induced in Lewis rats by a single foot pad injection of 150 micrograms lipopolysaccharide (LPS) from Salmonella typhimurium. At different time intervals after LPS injection, the authors evaluated ocular inflammation (slit lamp observation), iNOS localization by in situ hybridization, and comparison of OX-42- and ED1-positive cell appearance and of glial response by specific immunohistochemistry. RESULTS: iNOS mRNA was not detected in the iris-ciliary body nor in the retina of control rats. It was detected strongly in the epithelial cells of the iris-ciliary body at 6 hours and also in stromal cells of the ciliary processes at 16 hours after LPS injection. In the neuroretina, iNOS mRNA was observed in the inner layers 16 hours after LPS injection. iNOS-positive cells were also present on the vitreous at this time. At 6 and approximately 16 hours after LPS injection, immunohistochemistry experiments revealed a large number of OX-42- and ED1-positive cells (microglia, macrophages, or polymorphonuclear leukocytes) colocalized in part with some iNOS-positive cells in the ciliary body and in the retina. Furthermore, expression of iNOS in Müller cells cannot be excluded. CONCLUSIONS: These observations confirm that subcutaneous injection of endotoxin dramatically induces NOS mRNA expression in the eye, and they demonstrate that epithelial cells of the iris-ciliary body and cells infiltrating the anterior segment of the eye and the retina are the major source of NO. These results support the hypothesis that both inflammatory and resident ocular cells are involved in iNOS expression during EIU.

Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase.

Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory diseases. In the present study, we investigated the potential role of NO in an ocular model of inflammation, endotoxin-induced uveitis (EIU), in Lewis rats. Injection of LPS in one footpad induces severe uveitis after 16 h, which is accompanied by an increase of NO in the aqueous and vitreous humors, as evaluated by nitrite assay. Reverse transcriptase-PCR experiments reveal a large increase of inducible NO synthase (iNOS) mRNA in the iris/ciliary body, from 2 to 24 h after LPS treatment. In the retina, maximal increase of iNOS mRNA was detected 16 h after LPS treatment. Two i.p. injections of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), which inhibits nitrite release in the aqueous and vitreous humors, profoundly reduce clinical and histologic inflammation in EIU rats. These results implicate the NO pathway in the pathogenesis of EIU and demonstrate the possibility of modulating this inflammatory disease by injection of a NOS inhibitor.

Light-induced changes in S-antigen (arrestin) localization in retinal photoreceptors: differences between rods and cones and defective process in RCS rat retinal dystrophy.

The subcellular localization of S-antigen (arrestin), a protein regulating phototransduction in retinal rods, was studied by immunocytochemistry using monoclonal antibodies on sections of Swiss mouse, Lewis, Brown Norway (BN), Royal College of Surgeons (RCS) rdy-p+ (dystrophic) and RCS rdy(+)-p (non-dystrophic) rat retinas. In normal retinas, the topography of S-antigen immunoreactivity in photoreceptor cells varied according to the lighting environment of the animals. In dark-adapted eyes, outer segments did not display any S-antigen immunoreactivity while the inner segments, cell bodies and synaptic terminals were strongly labeled. A few minutes after light exposure, there was an inversion of the pattern of labeling: the label increased in the outer segment but was strongly reduced in the other compartments. After 1 h of light, S-antigen immunoreactivity remained only in outer segments and in a few synaptic terminals. We show that the kinetics of this change is slower in cone than in rod cells, and thus allows the transient visualization of the scarce cone photoreceptors. On the 17th day after birth, photoreceptor cells are well differentiated in all rat strains, including RCS rdy-p+ rats. At this time, the S-antigen shift phenomenon occurred in the non-dystrophic strains, but was not observed in rdy-p+ rats: after light exposure, the intracellular distribution of S-antigen remained the same as in the dark. We suggest that an abnormality in the mechanisms of intracellular protein transport could be a characteristic of this genetic disease.

Ginkgo biloba extract (EGb 761) and a platelet-activating factor antagonist protect the retina in experimental autoimmune uveoretinitis.

Oxygen-free radical toxicity is an important factor of tissue necrosis in the eye, especially in the retina. Activation of synthesis and release of platelet-activating factor (PAF) by ocular inflammatory cells and resident cells initiates cascades of mediators and cytokines which contribute to tissue damage in several ocular pathologies. The authors studied the therapeutic effectiveness of Ginkgo biloba extract (EGb 761), a potent free radical scavenger with anti-PAF activity, and of BN 50730, a specific PAF antagonist, on acute experimental autoimmune uveoretinitis induced in rats by S-antigen immunization. These treatments slightly delayed disease onset but had little effect on the severity of uveal inflammation. However, they significantly reduced inflammatory cell infiltration of the retina and the damage of the outer retinal layers. These drugs should become useful adjuvants in the therapy of posterior uveitis and other disorders that might damage the retina.

[Diagnostic and predictive importance of the blast transformation test and basophil activation test in retinal vasculitis]. / Intérêt diagnostique et prédictif du test de transformation blastique et du test d'activation des basophiles au cours des vascularites rétiniennes.

22 patients suffering from Birdshot retinochoroidopathy (10), idiopathic vasculitis (6) and Behcet disease (6) and treated by cyclosporine were tested at regular intervals during 11 to 38 months by the lymphocyte stimulation test (LST) and basophil degranulation test (HBDT) with S retinal antigen. These two tests were found frequently positive especially during acute inflammation for the LST. However HBDT was positive in all the patients before relapse of acute inflammation which is in favor of its predictive value and its involvement in the triggering of inflammation at the basophiles or at the local mast cell level.

[Experimental autoimmune uveitis and retinitis induced by S-antigen immunization]. / Eksperymentalne autoimmunologiczne zapalenie naczyniówki i siatkówki wywolane immunizacja antygenem S.

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats by a single injection in foot pads of S-antigen in complete Freund's adjuvant. Clinically, during the 2 nd week after immunization, EAU starts by acute anterior, lately--posterior uveitis. Peptide S2, located in the N-terminal region of S-antigen, has a regulatory effect on EAU, while peptide M is a pathogenic site inducing EAU. Antibodies directed at peptide S2 were observed in rats not developing EAU while high levels of antibodies directed at S-antigen and peptide M were present in all rats.

Suppression of experimental autoimmune uveoretinitis by prazosin, an alpha 1-adrenergic receptor antagonist.

S-antigen (S-Ag)-induced experimental autoimmune uveoretinitis (EAU) was suppressed in Lewis and PVG rats by treatment beginning 4 days post immunization with prazosin, a specific alpha 1-adrenergic receptor antagonist. A significant suppression of EAU was observed at clinical and histological levels in both treated groups compared to a severe EAU which developed in controls. Fluorescein angiography showed no leakage of dye from the optic disc of a treated PVG rat presenting no ocular inflammation by clinical examination. The treatment had no effect on the titer of anti-S-Ag antibodies. Perivascular infiltrates of T-lymphocytes and macrophages together with alterations of blood-retinal barrier permeability are early events in EAU. Prazosin, by acting on the vascular alpha 1-adrenoreceptors, inhibits vasospasm, preserves blood-retinal-barrier integrity and prevents vascular edema and early inflammatory cell infiltration observed in EAU.

Retino-choroidal changes in endotoxin-induced uveitis in the rat.

A single injection of 100 micrograms of lipopolysaccharide from Salmonella typhimurium in the foot pads of Lewis rats induced acute inflammation of the eye. Clinically, the disease started as early as 0.5 h and peaked 18 h after the inoculation. The aqueous protein concentration was increased after the inoculation. Histopathologically, cellular infiltrates and proteinaceous exudates were observed in the anterior segment (anterior chamber, iris and ciliary body). In addition to those changes described in previous reports, the examination of the posterior segment showed retinal vasculitis, hemorrhagic exudates, focal destruction of photoreceptor cells and choroidal infiltration.

Early disappearance of alpha-transducin in light-induced photoreceptor degeneration in albino rats.

Progressive degeneration of retinal photoreceptor cells occurs in albino rats when exposed to continuous lighting. Three proteins involved in the phototransduction cascade were immunodetected in these cells after various durations of continuous illumination. We found that S-antigen (arrestin) and rhodopsin immunoreactivities persisted for 1-2 months during the degenerative process, whilst immunoreactivity of the alpha subunit of transducin totally disappeared between day 2 and day 4 of continuous light exposure. This suggests that continuous illumination could impair alpha-transducin synthesis, a possible causal factor of photoreceptor damage.

Prevention of experimental autoimmune uveoretinitis and experimental autoimmune pinealitis in (Lewis x Brown-Norway) F1 rats by HgCl2 injections.

Mercuric chloride (HgCl2) induces in Brown-Norway (BN) and (Lewis x Brown-Norway) F1 hybrid rats a transient autoimmune disease characterized by the production of various antibodies to self and non-self antigens and by a dramatic increase of serum IgE. Experimental autoimmune uveoretinitis (EAU) can be induced in Lewis (LEW) and (LEW x BN) F1 hybrid rats by a single immunization with retinal S-antigen (S-Ag). Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP). We demonstrate in this study that (LEW x BN) F1 hybrid rats, injected with HgCl2 7 days before S-Ag immunization, are quite efficiently protected against EAU and EAP. We also show that HgCl2-induced protection is neither due to a cytotoxic effect of HgCl2 nor to CD8+ T-cell dependent mechanisms nor to the HgCl2-induced increase of serum IgE concentration. The role of other hypothetical mechanisms, such as anti-S-Ag anti-idiotypic antibodies and/or HgCl2-induced unbalance between T-helper cell subsets, is discussed.

Inhibition of experimental autoimmune uveoretinitis by mercuric chloride injections in (Lewis x Brown Norway) F1 hybrid rats.

Experimental autoimmune uveoretinitis (EAU), induced in (LEW X BN) F1 rats by immunization with S antigen (S-Ag) is T cell and antibody (ab) mediated and anti-S-Ag IgE ab have been involved in the occurrence of ocular lesions. (LEW X BN) F1 rats repeatedly injected with HgCl2 develop an autoimmune disease characterized by numerous auto-ab and a high increase of serum IgE level. We hypothesize that large amounts of non anti-S-Ag IgE induced by HgCl2 would compete with anti-S-Ag induced by S-Ag immunization so as to prevent EAU to occur. Indeed (LEW X BN) F1 rats immunized with S-Ag 7 days after the first HgCl2 injection are strongly protected against EAU. The putative role of the different mercury-induced autoimmune phenomena in the protection against EAU are discussed.

Severe retinochoroidopathy: variations of humoral and cellular immunity to S-antigen in a longitudinal study.

Ten patients with birdshot retinochoroidopathy, six with isolated retinal vasculitis and eight with Behçet's disease were treated with cyclosporine for one to three years. Autoantibodies to several retinal proteins, circulating lymphocyte subsets and cellular reactivity to S-antigen were evaluated repeatedly during this period. Autoantibody titers were similar in patients and in controls. However the serum content of antibodies to IRBP or S-antigen was lessened during inflammatory periods in some patients. In some sera, antibodies reacted with enzyme digested S-antigen preparations by immunoblot, whereas the same sera were negative for the native protein. A decrease of the CD4+ subpopulation of peripheral blood lymphocytes was associated with relapses of ocular inflammation in birdshot retinochoroidopathy. In this disease and in idiopathic retinal vasculitis, the positive lymphocyte stimulation test and basophil degranulation test with S-antigen were significantly most frequent in the period preceding a relapse of ocular inflammation. These tests could therefore be of predictive value for relapses occurring within the next few months.

[Ivermectin and human onchocerciasis. A study of 234 onchocerciasis patients in the Republic of Mali]. / Ivermectine et onchocercose humaine. A propos d'une étude portant sur 234 onchocerquiens en République du Mali.

In a double blind study Ivermectin has been compared to a placebo in 234 male and female with onchocerciasis who had more than 20 microfilariae per milligram of skin and moderate ocular involvement. Patient was randomized to receive a simple oral dose of Ivermectin 100, 150 and 200 micrograms/kg or placebo. The following was 12 months. The decrease of microfilarodermia since to the 3rd day was from 72.8 to 79.3% of initial rate. At six months it was more than 91% and more than 87% in 12 months. Ocular microfilariae, initially between 12 and 23 stay lower than 2 at 12 months. Punctuated keratitis disappear and did not recidive still 6 months in patients with persistent microfilariae. Ivermectin produce only few side effects. Negative waves have been observed on ECG but without any clinical signs. The Power efficient dose seen to be 100 micrograms/kg.

Retinal S-antigen epitopes in vertebrate and invertebrate photoreceptors.

Monoclonal antibodies specific for the retinal S-antigen were obtained by hybridization of spleen cells from a BALB/c mouse immunized with bovine S-antigen and NS-1 myeloma cells. Five selected antibodies specifically labeled the photoreceptor cells of the retina by immunofluorescence. Whereas antibody S9E2 only reacted with bovine S-antigen, the other antibodies showed interspecies cross-reactivity. They were used for the characterization of specific epitopes of S-antigen in photoreceptors from a wide range of species representative of various classes of vertebrates and invertebrates. The presence of S-antigen in distant species (vertebrates, Amphioxus, nemerteans, annelids, molluscs) indicates a high phylogenetic stability and suggests an important role for this protein in photoreceptor function.

Experimental autoimmune uveoretinitis in athymic rats: specific IgE response to retinal S-antigen and disease.

Experimental autoimmune uveoretinitis (EAU) is an ocular autoimmune disease induced in rats by immunization with retinal S-antigen. Athymic nude rats (rnu/rnu) have been previously shown to be refractory to EAU induction and antibody production to S-antigen, while heterozygous (rnu/+) are good responders. Increasing the antigen dose and adding pertussis adjuvant produced ocular disease in some nude rats, and antibody response in most of them. Specific IgE antibodies were demonstrated by ELI-SA only in the serum of nude rats presenting the disease. However, most immunized nude rats had evidence of mast cell sensitization to S-antigen (direct degranulation test) and of circulating specific IgE detected by passive sensitization of normal mast cells (indirect degranulation test). This positive response could be explained by an incomplete depletion of the different T lymphocyte subsets.

Production and specificity of monoclonal antibodies to retinal S antigen.

Hybridomas producing monoclonal antibodies to the retinal S antigen were obtained by fusion of spleen cells from a BALB/c mouse immunized with purified bovine S antigen and NS-1 myeloma cells. Six cloned hybridomas were selected and expanded as large scale cultures and as ascites in mice. The specificity of the antibodies produced by these hybridomas was assessed by ELISA and immunofluorescence. All were specific for S antigen, except one which showed slight reactivity with other proteins. One antibody was specific for bovine S antigen, whereas the others showed cross reactivity with purified S antigens from various mammals. Immunofluorescence allowed to demonstrate the presence of common epitopes of S antigen in the retinal photoreceptor cells of species representative of every class of Vertebrates.

Antibody determination by ELISA in rats with retinal S antigen-induced uveoretinitis.

Enzyme-linked immunosorbent assay was applied to the determination of the serum IgG antibody contents in rats immunized with the organ-specific autoantigen (S antigen) of the retina. Optimal conditions (i.e. S antigen concentration, serum and conjugate dilutions, enzymatic reaction time) were determined. The assay required only a single serum dilution and was well reproducible. It was very sensitive, allowing the detection of low antibody contents in animals immunized with 1 microgram of S antigen. The time course of the antibody response and its variations according to the dose and the species of the immunizing S antigen were considered.

S antigen-induced experimental autoimmune uveo-retinitis in rats.

Experimental autoimmune uveoretinitis was induced in rats after one injection of purified retinal S antigen mixed with adjuvants. Lewis and PVG/c rat strains were highly sensitive. S antigens isolated from bovine, human, swine and guinea pig retinas had a high pathogenicity in Lewis rats, whereas allogenic S antigen did not induce the disease. Mycobacterial adjuvant was effect in both disease and antibody production but H. pertussis adjuvant strongly increased the severity of the ocular reaction, giving a hyperacute Arthus-type inflammation, even with low doses of antigen. No disease was found after immunization without bacteria (incomplete Freund's adjuvant or alum). With any bacterial adjuvant, the histological pattern was in agreement with the hypothesis of early reagin-mediated phenomena acting on the blood-retinal barrier, as suggested by previous experiments.

[Experimental uveoretinitis induced in monkeys by retinal S antigen. Induction, histopathology (author's transl)]. / Uvéo-rétinite expérimentale induite par l'antigène S rétinien chez le singe. Induction, histopathologie.

S antigen was isolated from human and bovine retinas and purified by two chromatographic steps. Its purity was controlled by SDS-electrophoresis and immunodiffusion. Seven Cynomolgus monkeys received a single subcutaneous immunization with either 50 or 100 micrograms of human S antigen (5 animals) or 100 micrograms of bovine S antigen (2 animals) with complete Freund's adjuvant. All the monkeys had bilateral uveoretinitis and anti-"S" antibodies were found in their serum. The time course of the disease was followed clinically during one year and pathologic features were described at various time intervals. The disease includes prominent retinal vasculitis and granulomatous chorio-retinitis, with a chronic course, and leads after many months to almost complete destruction of the retina, with persistence of inflammatory infiltrates and pigment of cell migration.