Experimental Autoimmune Uveoretinitis Research Articles

Constitutive expression of IL-27 in neuro-retina negatively regulates THIL-17 cell development (95.25)

Abstract Ocular immune privilege is maintained by constitutive expression of inhibitory molecules such as TGF-b, FAS and FAS-ligand. Here, we describe a model of immune privilege for the eye based on IL-27-induced suppression of Th17 responses. We show that Th17 cells are elevated in retina of mice during experimental autoimmune uveoretinitis (EAU) and treatment with anti-IL17 Abs reduces EAU severity. It is of note that the increase and subsequent trafficking of Th17 cells into the retina during EAU is accompanied by corresponding increases in IL-27 and IFN? in the retina. We further show that IL-27 is constitutively expressed in human or mouse retina and that IFN? upregulates its expression in retinal cells. Of particular interest is inhibition of the proliferation of uveitogenic Th17 cells by IL-27, suggesting that IFN? may mitigate uveitis by antagonizing Th17 expansion through induction of IL-27 in the target tissue. In a co-culture system the expansion of Th17 cells (propagated under Th17 polarizing conditions) is inhibited by primary retina cells. However, addition of anti-IL-27 antibody into the culture overrides the inhibitory effects of the retinal cells on the expansion of Th17 cells. The data presented suggests that inhibitory effects on inflammatory cells by the retina are mediated in part by endogenously produced IL-27 and establishes mechanistic links between IL-27 expression and anti- Th17 effects in the eye.

Treatment of experimental autoimmune uveoretinitis with atorvastatin and lovastatin

Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are approved for cholesterol reduction and are commonly used to treat atherosclerosis and coronary artery disease. Statins may also be potent immunomodulatory agents and may be beneficial in the treatment of autoimmune diseases. In this study, we investigated therapeutic effects of atorvastatin and lovastatin on experimental autoimmune uveoretinitis (EAU). EAU was induced in Lewis rats using bovine S-antigen (S-Ag) peptide. Atorvastatin was suspended in 0.5% aqueous methylcellulose and was administered orally at a dose of 10 mg/kg and at a low-dose of 1 mg/kg. Lovastatin was dissolved in DMSO:PBS (1:1) and was administered by intraperitoneal (i.p.) injection at a dose of 2 mg/kg. Both statin treatments were initiated after the clinical onset once daily for 14 days. The rats were examined every other day for clinical signs of EAU. The histological scores and delayed-type hypersensitivity (DTH) were evaluated on day 28 post-immunization. Morphologic and immunohistochemical examinations were performed with light and confocal microscopy, respectively. Lymphocyte proliferation was measured by [ 3H]thymidine incorporation into antigen-stimulated T cells from inguinal lymph nodes. After 72 h, supernatants were collected and assayed for IFN-γ by ELISA. Clinical and histological scores of EAU were decreased in both the atorvastatin (10 mg/kg)- and lovastatin (2 mg/kg)-treated groups. The invasion of T cells and macrophages, and Müller cell proliferation, were inhibited in both atorvastatin- and lovastatin-treated groups. DTH was significantly inhibited in both groups, compared with vehicle-treated groups (controls). Lymphocyte proliferation assay demonstrated decreased proliferation in the presence of 25 μg/ml S-Ag peptide in both groups, compared with controls. In the supernatants of lymph node cells stimulated with S-Ag peptide (5 μg/ml), 77 or 87% inhibition of IFN-γ production was observed in rats treated with atorvastatin or lovastatin, respectively, compared with controls. The current results indicate that atorvastatin administrated orally following the clinical onset has therapeutic effect in EAU as well as lovastatin administrated intraperitoneally. Statins may be useful for treating intraocular inflammation.

Effects of Heme Oxygenase-1 Inducer and Inhibitor on Experimental Autoimmune Uveoretinitis

PurposeExperimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation.MethodsForty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once aily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization.ResultsHemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1.ConclusionsWe suggest that HO-1 plays an important protective role in EAU.

Eye-concentrated distribution of dexamethasone carried by sugar-chain modified liposome in experimental autoimmune uveoretinitis mice

Corticosteroid is generally accepted as a standard therapeutic agent for active inflammatory (and) autoimmune eye diseases. In an attempt to develop a system to deliver corticosteroid most efficiently to the target eye, a sialyl-Lewis X (sLe(x))-conjugated liposome was adopted as a candidate for a carrier of dexamethasone (Dexa) and tissue distribution of intravenous Dexa with the modified liposome as well as Dexa alone as control was studied in normal and experimental autoimmune uveoretinitis (EAU) mice. Intravenous Dexa (1 mg) was widely distributed in all the tissues (eye, brain, heart, lung, liver, kidney, spleen and intestine) examined in similar manner in both mice and Dexa concentration was lowest in the eye except the brain. The tissue concentrations of Dexa in EAU group were all significantly lower than those in the corresponding tissues in normal group. Intravenous Dexa (2 microg) in the modified liposome was almost concentrated to the eye in EAU mice, reaching 13.84 ng/mg tissue in contrast to 2.34 ng/mg tissue in Dexa (1 mg) alone administered EAU mice. In normal mice, Dexa was undetectable in any tissues examined and thus the effect of the modified liposome was not observed. The result supported the potentiality of sLe(x)-conjugated liposome for target-delivering of corticosteroid to inflamed eye.

Thrombospondin Plays a Vital Role in the Immune Privilege of the Eye

Purpose: The role of thrombospondin (TSP)-1 in TGF-β activation and T-cell suppression was studied in the retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells that line the subretinal space, an immune-privileged site in the eye. Methods: Posterior eyecups were prepared by excising the anterior segment, lens, and retina from enucleated eyes of C57BL/6, thrombospondin-1 knockout (TSP-1KO), and TGF-β2 receptor II double-negative (TGF-β2 RII DN) mice, leaving behind a healthy monolayer of RPE resting on choroid and sclera. Serum-free medium was added to these RPE eyecups, and, after various time intervals, supernatants (SNs) were removed and tested. Results: SNs of an ex vivo culture of RPE cells from C57BL/6 mice were shown to inhibit both antigen and anti-CD3 activation of T cells, partially due to constitutive production of TGF-β and to the ability of RPE to activate the latent form of TGF-β. Activation of TGF-β was entirely dependent on TSP-1, also produced by RPE. SNs of RPE from TSP-1KO mice failed to inhibit T-cell activation. Ovalbumin (OVA)-specific delayed hypersensitivity (DH) was not impaired when OVA was injected either into the subretinal space or into the anterior chamber of TSP-1KO mice before OVA immunization. Moreover, experimental autoimmune uveoretinitis was significantly more intense in eyes of TSP-1KO mice and failed to undergo spontaneous resolution unlike wild-type mice. Conclusions: Production of both TSP-1 and active TGF-β by RPE is essential to the creation and maintenance of immune privilege in the subretinal space and that the immune privilege limits the severity and duration of retinal inflammation due to autoimmunity.

Direct detection of reactive nitrogen species in experimental autoimmune uveitis.

PurposeDemonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)2-Fe2+, as a spin trap.MethodsExperimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer.ResultsThe ESR three-line spectrum (g=2.04; aN=12.5 G) obtained was characteristic of the adduct [(MGD)2-Fe2+-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite.ConclusionsThe study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.

Corticotropin releasing hormone and the immune/inflammatory response

Hypothalamic corticotropin-releasing hormone (CRH) acts as the major physiologic ACTH secretagog. Moreover, CRH is distributed in the brain and spinal cord, adrenal medulla, testes, ovaries, gastrointestinal tract, pancreas, myometrium, endometrium, placenta, and diverse inflammatory sites. Immunoreactive CRH has been found in the cytoplasm of immune accessory cells (macrophages, endothelial cells, and tissue fibroblasts), and in inflammatory sites of both acute and chronic inflammation (synovial lining cell layers and blood vessels from the joints of patients with rheumatoid arthritis and osteoarthritis). Additionally, the local presence of CRH in the uveitic eyes, cytoplasm of inflammatory cells (macrophages, lymphocytes, and polymorphonuclear cells) infiltrating the iris, ciliary body, vitreous, retina, and choroid appears to be of pivotal importance in the process of experimental autoimmune uveoretinitis. Traditionally, hypothalamic CRH has been considered to act indirectly in an anti-inflammatory fashion, since the end product of the hypothalamic–pituitary–adrenal axis is cortisol, a well-known anti-inflammatory compound. However, CRH produced at peripheral inflammatory sites has been shown to participate in an autocrine/paracrine stimulation of inflammation. Thus, CRH may have a peripheral, primarily activating role on the immune system. The mechanisms of the CRH-mediated component of the immune/inflammatory response are still unclear. CRH in inflammatory sites seems to be involved in the activation of the Fas/Fas ligand system. Furthermore, locally produced embryonic and endometrial CRH plays a role in both the aseptic inflammatory process of implantation and the anti-rejection process that protects the fetus from the maternal immune system. There are two types of G-protein-coupled CRH receptors (CRH-R1 and CRH-R2). Pyrrolopyrimidine compounds, such as antalarmin, have been developed as CRH-R1 receptor antagonists. Confirming the peripheral pro-inflammatory actions of CRH, antalarmin has been shown to suppress experimental aseptic inflammation. Thus, antalarmin may represent the first in a new class of anti-inflammatory agents operating through CRH-R1. Studies of CRH genetics have provided new insights on the pathogenesis of rheumatoid arthritis in humans. DNA variation across the CRH gene-containing region has been examined in families with multiple cases of rheumatoid arthritis. Transmission Disequilibrium Test analysis showed significant association at the CRH locus.

The role of the ICOS/B7RP‐1 T cell costimulatory pathway in murine experimental autoimmune uveoretinitis

ICOS/B7RP-1 is a new member of the CD28/B7 family of costimulatory molecules and plays differential roles in autoimmune diseases. In this study, we examined the role of ICOS/B7RP-1 pathway in the pathogenesis of mouse experimental autoimmune uveoretinitis (EAU), an animal model of human autoimmune uveitis. ICOS expression was found on infiltrating CD4+ T cells in the region of the retina in EAU-induced mice. The anti-B7RP-1 monoclonal antibody (mAb)-treated or ICOS-deficient mice showed a substantial reduction of disease scores. Blockade of ICOS/B7RP-1 interaction during the effector phase ameliorated the disease, whereas its blockade during the induction phase exhibited no significant effect. Moreover, administration of anti-B7RP-1 mAb effectively ameliorated the disease induced by adoptive transfer of pathogenic T cells. The anti-B7RP-1 mAb treatment inhibited the expansion and/or effector function of pathogenic T cells, given that proliferative response and IFN-gamma production by lymph node cells were reduced upon restimulation with the antigen peptide in vitro. These results suggest that the ICOS/B7RP-1 interaction plays a critical role in the pathogenesis of uveitis. We also indicated that ICOS-mediated costimulation plays differential roles in EAU and experimental autoimmune encephalomyelitis, which is also a Th1 disease induced in the same manner as EAU.

Roles of CD4+CD25+ T cells in the development of experimental murine allergic conjunctivitis

CD25+ regulatory T (T reg) cells play a suppressive role in experimental autoimmune uveoretinitis as well as experimental airway inflammation but their involvement in the development of allergic conjunctivitis (AC) remains unclear. We therefore investigated whether T reg cells play a role in the development of experimental AC (EC). BALB/c and C57BL/6 mice were actively immunized with ragweed (RW). The mice were treated with an anti-CD25 Ab (PC61) or control normal rat IgG (nrIgG) either 2 days prior to active immunization or during the induction phase (days 0, 2, 4, 6 and 8). Ten days after active immunization, the mice were challenged with RW-containing drops. Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis of eosinophil infiltration, and the spleens were harvested for cell culture for splenocyte transfer. Cultured splenocytes were transferred into syngeneic mice, and 4 days after the transfer, the recipient mice were challenged with RW. Twenty-four hours after the challenge, conjunctivas were collected for histological analysis. Pretreatment with PC61 did not affect EC in either strain of mice; however, treatment with PC61 during the induction phase significantly suppressed EC in C57BL/6 mice. In contrast, transfer of RW-primed splenocytes from mice treated with PC61 induced EC that was significantly more severe regardless of strain and treatment protocol. The finding that T reg cells play a suppressive role in the development of EC in splenocyte transfer experiments suggests that modulation of T reg cells may be a possible therapy for AC.

Association of heat shock protein 70 induction and the amelioration of experimental autoimmune uveoretinitis in mice

Experimental autoimmune uveoretinitis (EAU) serves as a model of human endogeneous uveitis. In the present study we examined whether induction of heat shock protein (HSP) 70 by oral geranylgeranylacetone (GGA) administration had a therapeutic effect on murine EAU. When C57BL/6 mice that had received oral administration of GGA (500 mg/kg) were immunized with interphotoreceptor retinoid-binding protein (IRBP)-derived peptide plus adjuvants, the expression levels of HSP70 mRNA and protein were rapidly and transiently upregulated in eyes of the GGA-treated mice, compared with those from vehicle-pretreated and IRBP-immunized mice. The antigen-specific T cell proliferation was partially suppressed in these mice treated with GGA. The mean EAU scores of the GGA-treated mice on day 21 and 28 (2.4±0.2 and 2.1±0.2, respectively) were significantly lower than those in the controls (3.0±0.1 and 2.6±0.2, respectively p < 0.01 ). The histopathological severity of the GGA-treated mice (average 0.33) was markedly milder than that in the controls (average 1.63, p < 0.05 ) at day 21. The present findings demonstrate that the pharmacological induction of HSP70 may be applicable to the amelioration of ocular autoimmune diseases.

Sialoadhesin Promotes the Inflammatory Response in Experimental Autoimmune Uveoretinitis

Macrophages are a prominent component of the effector cell compartment in a number of CD4+ T cell-mediated organ-specific autoimmune diseases. In this study, we investigated the role of the sialic acid binding Ig-like lectin sialoadhesin (Sn, Siglec-1) in a model of interphotoreceptor retinal binding protein peptide-induced experimental autoimmune uveoretinitis in mice with targeted deletion of Sn. Our data show that compared with wild-type mice, experimental autoimmune uveoretinitis is reduced in severity in the initial stages in the Sn knockout (KO) mice. In addition, there is a reduction in the proliferative capacity of T cells from the KO mice draining lymph nodes after immunization with interphotoreceptor retinal binding protein peptides, which is manifest some days before disease onset and persists for the duration of disease. Furthermore, activated T cells from the draining lymph nodes of Sn KO mice secrete lower levels of IFN-gamma. The data suggest a role for Sn in "fine tuning" the immune response to autoantigens by modulating T cell priming.

Retinal Microglial Activation and Chemotaxis by Docosahexaenoic Acid Hydroperoxide

Peroxynitrite generated during the early phase of experimental autoimmune uveoretinitis (EAU) causes peroxidation of docosahexaenoic acid (22:6), a principal unsaturated fatty acid of the photoreceptor membrane, to its hydroperoxide (22:6HP). During this phase, microglia migrate to the site of photoreceptors. The effect of 22:6HP on the migration of isolated retinal microglia was investigated. Retinal microglia were isolated and cultured from newborn Lewis rats and identified immunohistochemically by OX42 antibody staining. Chemotactic activity of the microglia toward 22:6HP was assayed and compared with 22:6 and other control cultures. The effect of 22:6HP on the organization of actin fibers and on the expression of Rac and Iba1 in the microglia was studied by confocal microscopy. The gene and protein expression of Rac and Iba1 in these microglia was analyzed by real-time PCR and Western blot. Ninety-five percent of isolated microglia stained for OX42 and a chemotactic assay showed that 22:6HP was a potent chemoattractant for these cells. Exposure to hydroperoxide resulted in reorganization of F-actin with intense staining within the lamellipodia. Iba1 and Rac were upregulated in these activated cells and localized in the cell periphery and the lamellipodia. 22:6HP can activate retinal microglia and is a potent chemoattractant for microglial migration in response to the activation of Rac and reorganization of actin cytoskeleton. In EAU, microglial migration toward the photoreceptors may be mediated by 22:6HP formed in the photoreceptors, and these migrating cells could help modulate the inflammation.

4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-dioxygenase-dependent mechanisms

Interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveoretinitis (EAU) is a CD4 + T cell-mediated autoimmune disease. Development of EAU is inhibited by treatment with an agonistic anti-4-1BB mAb. Even established EAU was alleviated by anti-4-1BB mAb. However, inhibition of 4-1BB/4-1BB ligand (4-1BBL) interaction does not suppress the development of EAU. It appears that cross-linking of 4-1BB evokes an active antigen-specific suppression mechanism rather than merely blocking 4-1BB/4-1BBL interaction. We found that administration of anti-4-1BB mAb induced massive clonal expansion of CD11c +CD8 + T cells that produced IFN-γ, resulting in accumulation of a high level of indoleamine 2,3-dioxygenase (IDO) in CD11c + dendritic cells. 4-1BB-mediated suppression of EAU was reversed by the pharmacological IDO inhibitor, 1-methyl-tryptophan (1-MT). These studies demonstrate that suppression of EAU results from antigen-driven, 4-1BB-mediated expansion of novel CD11c +CD8 + T cells that suppress antigen-specific CD4 + T cells via an IDO-dependent mechanism.

Mapping Immune Responses to mRBP-3 1-16 Peptide with Altered Peptide Ligands

Experimental autoimmune uveoretinitis (EAU) can be induced in C57BL/6 mice (I-A(b)) using human retinoid-binding protein-3 (hRBP-3, previously IRBP) residues 1-20. This study of a truncated murine peptide (mRBP-3 1-16) was conducted to determine its pathogenic potential and to characterize partially its interaction with specific T cells. After immunization with mRBP-3 1-16 or hRBP-3 1-20, EAU was assessed by immunohistochemistry. The immune response was assessed by tritiated thymidine incorporation and cytokine production analyzed by enzyme-linked immunosorbent assay (ELISA). T-cell receptor (TCR)- and major histocompatibility complex (MHC)-binding of mRBP-3 1-16 was studied by modeling and by using altered peptide ligands (APLs) and T-cell clones. mRBP-3 1-16 induced EAU in C57BL/6 mice, with severity and kinetics comparable to that after immunization with hRBP-3 1-20. T cells taken from mice immunized with mRBP-3 1-16 had a Th1 phenotype and proliferated in response to reactivation with mRBP-3 1-16, hRBP-3 1-20, or mRBP-3 1-16 APLs. mRBP-3 1-16 APLs elicited at least five distinct patterns of reactivity when tested with the mRBP-3 1-16-reactive T-cell clones. mRBP-3 1-16 immunizes and causes EAU in C57BL/6 mice. The studies using T-cell clones and APLs demonstrate that the immune response to mRBP-3 1-16 is drawn from a diverse population of antigen-specific T cells with a Th1 phenotype. Modeling and analysis of clones indicate that nonpathogenic T cells of an mRBP-3 1-16-reactive T-cell line recognize the peptide in a single register.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis

T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.

Inducible co-stimulator (ICOS) is upregulated in experimental autoimmune uveoretinitis

To study the expression of inducible co-stimulator (ICOS) and its association with T cell effector function in experimental autoimmune uveoretinitis (EAU). Eighteen Lewis rats were immunized by retinal S-antigen (50 microg) emulsified in complete Freund's adjuvant (CFA). Twelve normal rats served as normal controls and 18 receiving injection of CFA and PBS as CFA controls for studying the influence of CFA on the expression of ICOS in CD4+CD25+ T cells. ICOS expression on cells from the spleens, inguinal nodes and retinae on day 0 (normal rats), 7, 13 and 21 was investigated using fluorescent quantitative real-time-PCR and Western blot. Expression of B7RP-1, an ICOS ligand, was also studied by Western blot. The phenotype of the cells from the aforementioned three tissues was identified with flow cytometry using antibodies to ICOS, CD4 and CD25. ICOS+ cells from the lymph nodes, and spleens on day 13 were magnetically sorted and cultured with S-antigen to study the cytokines production with enzyme-linked immunosorbent assay. An obvious uveitis was induced in all the immunized rats on day 13 after S-antigen immunization. The mRNA and protein of ICOS were scarcely detectable in normal rat spleens. In EAU rats, an up-regulation of ICOS could be observed on day 7 and was very pronounced on day 13, followed by a decrease on day 21 in the spleens, draining nodes and retinae. Similarly, B7RP-1 expression seemed to be up-regulated during EAU. Flow cytometry showed that ICOS+ cells were mostly CD4 positive. Kinetics of ICOS+CD4+CD25+ T cells was similar to that of ICOS+ cells. CFA alone was also able to induce increased expression of ICOS in CD4+CD25+ T cells. IFN-gamma was secreted predominantly by ICOS+ T cells. ICOS expression is up-regulated in association with T cell effector capacity in EAU. It is presumed that the ICOS/B7RP-1 costimulatory pathway may play a role in the development of EAU.

Mechanisms for inducing nasal mucosal tolerance in experimental autoimmune uveoretinitis

Delivering soluble (auto) antigenic peptides via the naso-respiratory route induces tolerance to that peptide and suppression of experimental models of autoimmune disease. In the normal lung, respiratory tract dendritic cells (RTDCs) efficiently endocytose soluble antigens, migrate to regional lymph nodes and present peptide to T cells that subsequently become tolerant. This article describes protocols for inducing tolerance via the naso-respiratory tract in experimental autoimmune uveoretinitis (EAU); for the isolation of RTDCs to facilitate definition of, and conditions for, maturation and activation of cells; and to test RTDC ability to induce tolerance in murine EAU when adoptively transferred.

Treatment of experimental autoimmune uveoretinitis with poly(lactic acid) nanoparticles encapsulating betamethasone phosphate

We have developed nanoparticles (NPs), which are capable of targeting a specific lesion and gradually releasing the agent at the site over a prolonged time period after a single intravenous administration. In this study, we evaluated the effects of intravenously administered poly(lactic acid) nanoparticles encapsulating betamethasone phosphate (BP-PLA NPs) on experimental autoimmune uveoretinitis (EAU) in Lewis rats. To determine the localization of NPs within the retina and choroid of rats with EAU, rhodamine (Rh)-encapsulated PLA NPs were injected intravenously and visualized by confocal microscopy. After the disease onset of EAU induced by S-antigen peptide in Lewis rats, either BP-PLA NPs, BP, or saline was injected intravenously, and the eyes were obtained 7 days following treatment and the histological score was determined. The clinical course of EAU was examined using pathological findings and the expression of the glial fibrillary acidic protein, rod opsin, and the surface markers of inflammatory cells (ED1 and pan T-cell) were immunohistochemically determined. Furthermore, T-cell proliferation and delayed-type hypersensitivity (DTH) to S-antigen were assessed. Intravenously injected Rh-PLA NPs accumulated in the retina and choroid of rats with EAU within 3 hr and remained over the succeeding 7-day-period. Furthermore, systemically administered BP-PLA NPs reduced the clinical scores of rats with EAU in 1 day, which were maintained for 2 weeks and decreased the histological scores. In addition, the ocular infiltration of activated T-cells and macrophages in addition to the hypertrophy of Müller cells were markedly reduced with this treatment. Meanwhile, T-cell proliferation and DTH of BP-PLA NPs-treated rats against S-antigen peptide were not significantly different from those of saline-treated rats. Systemically administered BP-PLA NPs inhibit the development of EAU due to the targeting and the sustained release of steroids in situ. The results of these studies suggest that the systemic administration of BP-PLA NPs may lead to a new therapeutic strategy in controlling intraocular inflammation.

A Selective Role for the TNF p55 Receptor in Autocrine Signaling following IFN-γ Stimulation in Experimental Autoimmune Uveoretinitis

IFN-gamma stimulates macrophage activation and NO production, which leads to destruction of the retina in experimental autoimmune uveoretinitis. In this study, we investigate the mechanism of disease resistance in TNF p55 receptor-deficient animals. We show that although T cell priming is relatively unaffected, macrophages lacking the TNF p55 receptor fail to produce NO following IFN-gamma stimulation because of a requirement for autocrine TNF-alpha signaling through the TNF p55 receptor. In contrast to the impaired activation of NO synthesis, MHC class II up-regulation was indistinguishable in wild-type and TNFRp55-/- mice stimulated with IFN-gamma. These defects could be overcome by stimulating macrophages with LPS. Together, these results show that selected aspects of IFN-gamma activation are controlled by autocrine secretion of TNF-alpha, but that this control is lost in the presence of signals generated by pathogen-associated molecular patterns recognizing receptors.

Microarray Analysis of Cytokine and Chemokine Gene Expression after Prednisolone Treatment in Murine Experimental Autoimmune Uveoretinitis

The purpose of this study was to investigate changes in gene expression of cytokines and chemokines and their receptors after systemic prednisolone treatment in experimental autoimmune uveoretinitis (EAU). EAU mice received one intravenous injection of 7.5 mg/kg prednisolone (PDS) sodium phosphate at the peak of inflammation. EAU mice treated with only solvent served as the control. Total RNA was extracted from the whole eyes 1, 2, and 3 days after treatment. Gene expression analysis was conducted with a cDNA microarray, which contains 117 individual transcripts encoding the genes of the cytokines and chemokines and their receptors (29 cytokines and 34 cytokine receptors; 33 chemokines and 21 chemokine receptors). Comparisons of expression between PDS-treated and placebo-treated EAU mice at each time point were performed. The genes were sorted into clusters based on expression profiles, to clarify the gene regulation pattern after treatment. Forty-seven genes had a significant decrease in expression 1 day after treatment, 10 genes on day 2, and 46 genes on day 3. Ten genes were upregulated on day 1, but no gene was upregulated thereafter. Hierarchical cluster analysis of the microarray demonstrated that gene expression changes in EAU after treatment with PDS showed four patterns: flat, mountain, steep downhill, and less steep downhill. The implications of the clusters after treatment with PDS remain unclear. The results showed that hierarchical cluster analysis based on comprehensive gene expression profiles may provide a powerful tool for identifying genes not previously associated with the therapeutic targets in ocular inflammation.