Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis.

In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.

Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis.

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken ß-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU).

PURPOSE: SWAP 70-like adaptor of T cells (SLAT; aka Def6) is a recently discovered guanine nucleotide exchange factor for Rho guanosine triphosphate (GTP)ases that has been previously shown to play a role in cluster of differentiation(CD)4+ T cell activation, T-helper (Th)1/Th2/Th17 differentiation and development of experimental autoimmune encephalomyelitis. Here, we investigated the role of SLAT/Def6 in the development of experimental autoimmune uveitis (EAU), an animal model for several uveitic conditions in humans. METHODS: SLAT/Def6 deficient ("KO") mice and C57BL/6 controls were immunized with interphotoreceptor retinoid-binding protein (IRBP), along with pertussis toxin. The development of ocular inflammation was determined by both fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure lymphocyte proliferation and release of cytokines. Purified dendritic cells were tested for their capacity to present antigen to responding lymphocytes. In addition, the lymphoid cells were tested for the expression of forkhead box P3 (FoxP3), using conventional methods, and the activity of T-regulatory cells was determined by their capacity to inhibit in vitro proliferative responses. Serum anti -IRBP antibody levels were measured by enzyme-linked immunosorbant assay (ELISA). quantitative polymerase chain reaction (qPCR) was used to determine the transcript levels of cytokines in inflamed eyes. RESULTS: SLAT/Def6 KO mice had significantly reduced EAU compared to controls. Cells isolated from draining lymph nodes of SLAT/Def6 KO mice exhibited impaired proliferation and production of Th1 and Th17 signature cytokines (interferon [IFN]-γ and interleukin [IL]-17, respectively) when compared with cells isolated from control mice. qPCR of inflamed eyes detected similar levels of IFN-γ transcript in control and SLAT/Def6 KO mice, whereas the IL-17 transcript levels in eyes of the SLAT/Def6 KO mice were lower than in eyes of the controls. The SLAT/Def6 KO mice resembled their wild type (WT) controls, however, in the levels of their serum antibody against IRBP, the antigen presenting capacity of their dendritic cells, the proportion of cells expressing Foxp3 and the immunosuppressive activity of their T-regulatory cells. CONCLUSIONS: SLAT/Def6 KO mice exhibit reduced capacity to develop ocular inflammation and cellular activity when immunized with IRBP. Our study provides new data showing that SLAT/Def6 plays a major role in the T cell-mediated autoimmune processes that bring about the inflammatory eye disease, EAU.

Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

Major role of gamma delta T cells in the generation of IL-17+ uveitogenic T cells.

We show that in vitro activation of interphotoreceptor retinoid-binding protein (IRBP)-specific T cells from C57BL/6 mice immunized with an uveitogenic IRBP peptide (IRBP(1-20)) under TH17-polarizing conditions is associated with increased expansion of T cells expressing the gammadelta TCR. We also show that highly purified alphabeta or gammadelta T cells from C57BL/6 mice immunized with IRBP(1-20) produced only small amounts of IL-17 after exposure to the immunizing Ag in vitro, whereas a mixture of the same T cells produced greatly increased amounts of IL-17. IRBP-induced T cells from IRBP-immunized TCR-delta(-/-) mice on the C57BL/6 genetic background produced significantly lower amounts of IL-17 than did wild-type C57BL/6 mice and had significantly decreased experimental autoimmune uveitis-inducing ability. However, reconstitution of the TCR-delta(-/-) mice before immunization with a small number of gammadelta T cells from IRBP-immunized C57BL/6 mice restored the disease-inducing capability of their IRBP-specific T cells and greatly enhanced the generation of IL-17(+) T cells in the recipient mice. Our study suggests that gammadelta T cells are important in the generation and activation of IL-17-producing autoreactive T cells and play a major role in the pathogenesis of experimental autoimmune uveitis.

Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses.

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.

Differential roles for IFN-gamma and IL-17 in experimental autoimmune uveoretinitis.

IL-17-producing CD4(+) T cells, so called T(h)17 cells, constitute a newly identified inflammatogenic cell population, which is critically involved in some inflammatory diseases. To explore the role of T(h)17 cells in murine experimental autoimmune uveoretinitis (EAU), a model of human autoimmune uveitis where T(h)1 responses predominantly participate in the pathogenesis, IL-17(-/-) mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 for disease induction. Funduscopic examination revealed that EAU was induced in IL-17(-/-) mice just like in wild-type (WT) mice at early phases of the disease. However, at later/maintenance phases, the severity was significantly reduced in IL-17(-/-) mice. Expression of IFN-gamma and MCP-1 was comparable between WT and IL-17(-/-) mice during the time course. In vivo blockade of IFN-gamma and IL-4 resulted in exacerbation of EAU at later phases with augmented IL-17 production. Taken together, our data demonstrated that IL-17/T(h)17 participates in the late phases of EAU and also that T(h)1 and T(h)17 responses are differentially required for EAU.

Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

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.

Chronic recurrent autoimmune uveitis with progressive photoreceptor damage induced in rats by transfer of IRBP-specific T cells.

Recurrent uveitis is a common cause of vision blindness. Using a rat model of chronic recurrent uveitis, we examined the relationship between clinical expression, pathological changes, and the heterogeneity of the disease. Chronic recurrent uveitis was induced by adoptive transfer of interphotoreceptor retinoid-binding protein (IRBP)-specific T cells in a total of more than 60 Lewis rats. In about 75% of cases recurrent uveitis was pathologically a chronic and progressive disease. The major pathological changes included the gradual loss of photoreceptor cells. However, disease progression did not always parallel the severity of ocular inflammation and clinical recurrent disease, with about a quarter showing no pathological damage in the eye.

Methimazole protects from experimental autoimmune uveitis (EAU) by inhibiting antigen presenting cell function and reducing antigen priming.

Methimazole (methyl-mercapto-imidazole, MMI), a compound used clinically in therapy of Graves' thyroiditis, was found to inhibit development of several autoimmune diseases in animal models. It was suggested on the basis of in vitro data that inhibition is through down-regulation of interferon-gamma (IFN-gamma)-induced expression of major histocompatibility complex class I and class II molecules. Here, we investigate the effect of MMI on experimental autoimmune uveoretinitis (EAU) and study its mechanism(s). Treatment of EAU with MMI administered in drinking water inhibited induction of the disease and associated antigen (Ag)-specific proliferation and cytokine production by draining lymph node cells (LNCs). The treatment was protective only if administered during the first but not during the second week after immunization, suggesting an effect on the induction phase of EAU. It is interesting that MMI inhibited disease in IFN-gamma knockout mice, indicating that the in vivo protective effect is IFN-gamma-independent. Flow cytometric analysis of draining LNCs extracted 5 days after immunization showed that MMI partly to completely reversed the increase in Mac-1(+)/class I(+)/class II(+) cells induced by immunization and reduced the proportion of B7-1 and CD40-positive cells, suggesting a deficit in the Ag-presenting cell (APC) population. APC from untreated mice largely restored antigen-specific proliferation of MMI-treated LNCs. We suggest that MMI inhibits EAU at least in part by preventing the recruitment and/or maturation of APC, resulting in reduced generation of Ag-specific T cells.

Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

Immunomodulation of experimental autoimmune uveoretinitis by intravenous injection of uveitogenic peptides.

Intravenous (IV) injection of antigenic proteins induces specific unresponsiveness, as shown by the diminished response to a challenge with these proteins in complete Freund's adjuvant. This study examined the effect of IV treatment with uveitogenic peptides on the development of experimental autoimmune uveoretinitis (EAU). The peptides used were derived from the sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and included R16 (sequence, 1177-1191), which is immunodominant and highly uveitogenic, and R4 (sequence, 1158-1180), which is nondominant and weakly uveitogenic. The efficacy of this treatment was found to depend on both the dose used for the IV injection and that used for the challenge. Thus, EAU induced by R16 at a dose of 0.2 nmol/rat was inhibited completely in all rats treated with the peptide at doses of 400 or 133 nmol and partially by the low dose of 5 nmol/rat. However, the EAU induced by a R16 challenge of 40 nmol/rat was inhibited only partially by the high treatment dose of 400 nmol/rat. The IV treatment was found to be effective in inhibiting the EAU induced by peptide R4. A large dose of R4 was needed to induce EAU (40 nmol/rat), and the disease was inhibited completely in all rats treated IV with this peptide at doses of 800, 400, or 133 nmol. In most animals injected with the 44-nmol dose, also, inhibition was complete. These data show that there is a correlation between the doses needed for achieving inhibition and those used for the challenge. The ratios between these doses in all experiments were found within the range 1-20.(ABSTRACT TRUNCATED AT 250 WORDS)

Serial adoptive transfer of experimental autoimmune uveoretinitis in rats.

Experimental autoimmune uveoretinitis (EAU) and pinealitis induced by an interphotoreceptor retinoid-binding protein (IRBP)-derived peptide (R4) was serially transferred into naive recipient rats, using spleen cells from recipients of previous "orders" of transfer. The cells initiating the disease in recipients of the first order were either lymph node cells from rats immunized against peptide R4, or lymphocytes of a cell line specific toward this peptide. The serial transfer was successfully carried out through as many as four orders of sequential recipients.

Antigen-specific suppressor cells induced by FK506 in experimental autoimmune uveoretinitis in the rat.

The authors previously reported that FK506 effectively suppressed the induction of experimental autoimmune uveoretinitis (EAU) in rats with much lower doses than cyclosporine A. This study was aimed at analyzing the immune status of the FK506-treated and EAU-suppressed rats and examining the hypothesis whether the agent could induce antigen-specific suppressor T (Ts) cells. It was found that spleens from S-antigen-immunized and FK506-treated rats contained a population of Ts cells inhibiting the proliferative responses of S-antigen-sensitized lymphocytes to S-antigen, yet these cells did not affect the proliferative responses of interphotoreceptor retinoid-binding protein (IRBP)-sensitized lymphocytes to IRBP. The helper T (Th) cells did not exhibit such suppressor activities. Furthermore, transfer of Ts cells from S-antigen-immunized and FK506-treated rats to naive syngenic rats induced partial inhibition of EAU induction or delay of EAU onset after immunizing the recipient rats with S-antigen. Lymphocytes from the EAU-suppressed recipients showed low proliferative response to S-antigen and low levels of antibody to S-antigen. These data thus indicate that FK506 treatment after S-antigen immunization induces an activation of Ts cells specific to S-antigen and that the Ts cells might contribute, at least in part, to the uniquely prolonged and intensive immunosuppression by FK506.

Experimental autoimmune uveitis in HLA-B27 transgenic mice.

The major histocompatibility complex (MHC) gene, HLA-B27 is strongly associated with auto-immune uveitis and spondyloarthropathies in humans. Experimental mouse models of autoimmune uveitis involve systemic immunization with the retinal autoantigen interphotoreceptor retinoid binding protein (IRBP). To assess possible roles of HLA-B27 in autoimmune uveitis, as well as to investigate a possible new animal model of human uveitis, inbred strains of C57BL/6 and C57BL/6 possessing the human HLA-B27 or HLA-A2 transgene were immunized with IRBP emulsified in complete Freund's adjuvant (CFA). Dilated eye examinations were performed to assess the timing and clinical course of any ensuing uveitis. Mice were sacrificed 3 to 4 weeks postinjection and the eyes submitted for histopathologic analysis. CFA alone did not produce any clinical uveitis. Fifty percent of eyes from the background C57BL/6 strain developed uveitis as early as 10 days postinjection. Of the eyes demonstrating uveitis, an average clinical score of 2.5 was present. Pathologically, a moderate scleritis and anterior uveitis was present. Fifty percent of A2 transgenic eyes developed uveitis as early as 14 days postinjection with an average clinical score of 2.0. Pathologically, a mild vitritis was present. Uveitis developed in only 20% of B27 transgenic mice and reached a peak on day 28. The average EAU score in diseased animals was 4.5. A dense retinitis and panuveitis was associated with severe vitritis. We conclude that the presence of the B27 gene is associated with a decreased incidence and slower rate of onset of EAU following immunization with IRBP; however, EAU may be more severe in the HLA-B27 expressing animals who do develop disease.

Cytokine-dependent modulation of oral tolerance in a murine model of autoimmune uveitis.

In summary, our data suggest that oral tolerance in the mouse EAU model may occur by anergy/deletion or by suppression, depending on the feeding regimen. Tolerance involving putative regulatory cells appears to require the ability to produce both IL-4 and IL-10, whereas induction of tolerance involving anergy may not require the presence of Il-4 and IL-10. We propose that regulatory cells induced by three feedings of IRBP can be selectively enhanced through the use of cytokines. From the point of view of clinical therapy, it would be worthwhile to explore postimmunization feeding regimens involving administration of IL-4 and IL-10.

Interphotoreceptor retinoid binding protein is a potent tolerogen in Lewis rat: suppression of experimental autoimmune uveoretinitis is retinal antigen specific.

AIMS: Administration of unfractionated retinal antigen(s) (retinal extract, RE) suppresses RE induced experimental autoimmune uveoretinitis (EAU) and offers a potential therapeutic alternative to non-specific immunosuppressive therapies for posterior uveitis and autoimmune diseases. S-Ag and interphotoreceptor retinoid binding protein (IRBP) are two major autoantigens within soluble RE. It was aimed to assess, firstly, as has previously been shown with S-Ag, if IRBP can induce intranasal tolerance and, secondly, the contribution of both these major autoantigens to tolerance induction by whole RE. METHODS: Animals were tolerised by intranasal administration with S-Ag or IRBP, either alone or in combination, or RE before immunisation with either IRBP or RE. Control animals were administered nasally either PBS or MBP. Daily clinical responses were recorded biomicroscopically and histological grades were obtained using a semiquantitative scoring system. Weekly serum antibody levels to retinal antigens were measured by ELISA and delayed hypersensitivity responses (DTH) were assessed by skin reactivity to intradermal inoculation with retinal or non-specific antigens. RESULTS: Microgram doses of IRBP successfully suppressed both clinically and histologically IRBP induced EAU. This suppression was accompanied by reduced antigen specific DTH reactivity but maintained T cell dependent (IgG2a) antibody responses. Furthermore, combined S-Ag and IRBP administration afforded equal suppression of RE induced EAU when compared with RE therapy alone. Suppression of RE induced EAU was not achieved with administration of a non-retinal specific autoantigen, MBP. Although individually, both S-Ag and IRBP suppressed RE induced EAU, whole RE was unable to protect against IRBP induced disease. CONCLUSIONS: Intranasal administration of IRBP suppressed IRBP induced EAU in the Lewis rat. S-Ag and IRBP are the major contributors to the tolerogenicity within RE, despite the known uveogenicity of other retinal antigens within RE and induction of tolerance was retinal antigen specific. Furthermore, suppression induced by single antigen administration is antigen specific although concomitant bystander suppression may also play a role. RE was unable to protect against IRBP induced disease despite tolerogenic levels of antigen within RE. Although this may be due in part to a dose effect of either tolerising or immunising antigen, further investigation into the possible antigen dominance of IRBP or mucosal processing of combinations of antigens is necessary so that the full efficacy of mucosal tolerance therapy can be assessed.

Rodent models of experimental autoimmune uveitis.

The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproducible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from retinal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study of basic mechanisms as well as in clinically relevant interventions.

Identification of multiple associative and dissociative proliferative and uveitogenic T-cell sites in human interstitial retinoid-binding protein.

Human interstitial retinoid-binding protein (HIRBP) is a 136 kDa retinal protein capable of inducing experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis (LEW) rats. In order to identify the T-cell recognition sites of HIRBP responsible for uveitogenic and proliferative responses, 125 overlapping peptides corresponding to its entire 1262 amino acid sequence were synthesized. Individual peptides were tested for their ability to induce EAU in LEW rats and to stimulate lymphocyte proliferation in rats immunized with the native molecule. Our previous results showed the presence of nine uveitogenic peptides in HIRBP with a minimum requirement of eight amino acids needed to induce EAU in LEW rats. Our present studies show nine proliferative peptides, four of which are also responsible for uveitogenicity. Another four peptides known to actively induce EAU were unable to elicit proliferative responses. However, these peptides overlapped or were adjacent to peptides that elicited good proliferative responses. A single, highly proliferative peptide was located on the amino terminus of HIRBP. In addition, EAU was adoptively transferred with lymph node cells (LNC) of LEW rats previously immunized with two synthetic peptides known to be uveitogenic. Our study indicates that human IRBP is a complex molecule containing multiple and spatially distinct T-cell epitopes responsible for its uveitogenicity, adoptive transfer of EAU and proliferative responses.