Spontaneous Ocular Autoimmunity in Mice Expressing a Transgenic T Cell Receptor Specific to Retina: A Tool to Dissect Mechanisms of Uveitis.

The "classical" EAU model induced by immunization of mice with the retinal protein IRBP or its peptides has been very useful to study basic mechanisms of ocular inflammation, but is inadequate for some types of studies due to the need for active immunization in the context of strong bacterial adjuvants. We generated transgenic (Tg) mice on the B10.RIII background that express a T cell receptor (TCR) specific for IRBP161-180. Three strains of TCR Tg mice were established. Spontaneous uveitis developed in two of the three strains by 2-3 months of age. Susceptibility correlated with a higher copy number of the transgenic TCR and a higher proportion of TCR Tg T cells in the peripheral repertoire. Even in mice with uveitis, peripheral IRBP-specific CD4(+) T cells displayed mostly a naïve phenotype. In contrast, T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells. These mice thus provide a new and distinct model of uveitis from the "classical" EAU, and may represent some types of uveitis more faithfully. Importantly, this new transgenic model of uveitis can serve as a template for therapeutic manipulations, and as a source of naïve retina-specific T cells for a variety of basic and pre-clinical studies. Several examples of such studies will be discussed.

Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules.

Linker for activation of T cells (LAT) is critical for the propagation of T-cell signals upon T-cell receptor (TCR) activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KR LAT) resulted in decreased LAT ubiquitination and elevated T-cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T-cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KR LAT. On TCR stimulation of T cells from these mice, proximal signaling and cytokine production was elevated in 2KR versus wild-type (WT) LAT mice. Enhanced cytolytic activity as well as T-helper responses were observed on LAT expression, which were further elevated by 2KR LAT expression. Despite greater T-effector function, WT or 2KR LAT expression did not have any effect on clearance of certain pathogens or tumors. Our data support the model that lack of tumor clearance is due to increased differentiation and acquisition of effector phenotype that is associated with suboptimal immunity in an immunotherapy model. Thus, our data further reinforce the role of LAT ubiquitination in TCR signaling and uncovers a novel role for LAT in driving T-cell differentiation.

Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals.

Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others. Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase activity nor the related cholera toxin that ADP-ribosylates G(s) (but not G(i)) proteins blocked EAU induction or migration to chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it does so at least in part by disrupting signaling through G(i) protein-coupled receptors. Thus, the net effect of PTX on autoimmune disease would represent an integration of enhancing and inhibitory effects.

Blockade of costimulation through B7/CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance.

It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells.

Residues 1-20 of IRBP and whole IRBP elicit different uveitogenic and immunological responses in interferon gamma deficient mice.

Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease induced by immunization with uveitogenic retinal antigens, or by the adoptive transfer of uveitogenic T-cells of the Th-1-like phenotype. We have previously shown that IFN-gamma-deficient mice (GKO) on the C57BL/6 background are equally susceptible to interphotoreceptor retinoid binding protein (IRBP)-induced EAU as the wild type (WT). In the present study, we evaluated EAU induction in GKO mice by the newly described H-2(b)epitope contained in residues 1-20 of human IRBP, and compared it to the response to the whole IRBP molecule. Similarly to previous observations with IRBP-induced EAU, delayed type hypersensitivity (DTH) and lymphocyte proliferation responses were elevated in GKO mice, as was production of IL-5 and TNF-alpha. However, unlike the responses induced by whole IRBP, there was no detectable IL-10 production to the peptide. Histopathology on day 21 after immunization, revealed that both GKO and WT mice developed retinal lesions, including damage to the photoreceptor cell layer, vasculitis and inflammatory cellular infiltration, but disease scores were significantly higher in GKO, and retinal detachment was observed only in GKO mice. In contrast to the wild type, the cellular infiltrate in eyes of GKO mice contained a prominent component of eosinophils, although of lower proportion in peptide-induced than in IRBP-induced EAU. We conclude that the cytokine and inflammatory responses to human peptide 1-20 differ perceptibly from the responses to whole bovine IRBP, and may explain the elevated EAU scores of GKO mice compared to wild type.

The role of IL-12 in the induction of late-phase cellular infiltration in a murine model of allergic conjunctivitis.

BACKGROUND: The applied murine model of allergic conjunctivitis mimics human disease, and an immediate hypersensitivity reaction (IHR) and a late-phase cellular reaction typically develop in sensitized mice after topical challenge with the allergen. OBJECTIVE: We investigated the role of IL-4, IFN-gamma, and IL-12 in the early and late phases of ocular allergy with use of cytokine knockout (KO) mice and neutralizing antibodies. METHODS: Ragweed-sensitized wild-type or IL-4KO, IL-12KO, IFN-gamma KO, anti-IL-12 mAb-treated, recombinant murine IL-12-treated, and anti-IFN-gamma mAb-treated mice were challenged with the allergen 10 days after the immunization. IHR, cellular infiltration, lymphoproliferative response, and cytokine production from draining lymph nodes were recorded and compared among groups. RESULTS: We show that IL-12KO mice and anti-IL-12 antibody-treated wild-type animals failed to have a cellular infiltration into the conjunctiva. Treatment with recombinant murine IL-12 also reduced the number of infiltrating PMNs but increased the percentage of mononuclear cells in the conjunctiva compared with controls. IFN-gamma KO mice had a significantly stronger IHR and prolonged infiltration into the conjunctiva after challenge with ragweed than controls. CONCLUSION: Our data suggest that the presence of IL-12, although better known as a T(H)1-inducing cytokine, is important for the development and the regulation of the late-phase pathologic features in ocular allergy. Furthermore, IFN-gamma is a limiting factor in the late phase of allergy and thus may be important in preventing chronic allergic disease.

Identification of a new epitope of human IRBP that induces autoimmune uveoretinitis in mice of the H-2b haplotype.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated disease induced by immunization with interphotoreceptor retinoid binding protein (IRBP). Major uveitogenic sites have been identified for mice of the H-2r and H-2k haplotypes but not for the H-2b haplotype. The present communication describes the characterization of an epitope contained in residues 1 to 20 of human IRBP that induces EAU in H-2b mice. METHODS: H-2b (C57BL/6, 129/J) and H-2r (BIO.RIII) mice were immunized with peptide 1-20 or with whole (bovine) IRBP. EAU (histopathology) and immunologic responses (delayed-type hypersensitivity [DTH], lymphocyte proliferation, and cytokine production) were assessed after 21 days. RESULTS: C57BL/6 mice, 129/J and (129/JxC57BL/6)F1 mice, immunized with 200 to 300 microg of peptide, developed DTH and EAU with scores comparable to those induced by 100 microg IRBP. Their lymphocytes proliferated to the peptide and produced interferon-gamma (but not interleukin-4) and transferred EAU to syngeneic recipients. Lymphocytes of IRBP-immunized mice also responded to the peptide. Peptide 1-20-immunized B1O.RIII mice failed to develop either disease or immunologic responses. CONCLUSIONS. Human IRBP peptide 1-20 contains a major epitope for the H-2b haplotype, which is apparently not presented by the H-2r haplotype.

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.

Mice deficient in inducible nitric oxide synthase are susceptible to experimental autoimmune uveoretinitis.

PURPOSE: Nitric oxide (NO) is an important mediator of inflammatory tissue damage. The present study addresses the question whether inducible nitric oxide synthase (iNOS), and consequently the ability to upregulate NO, is required to effect the pathogenesis of experimental autoimmune uveoretinitis (EAU) in mice. METHODS: Mice with a homologous disruption of the iNOS gene (iNOS KO) were evaluated for their ability to develop EAU and associated cellular responses after immunization with the interphotoreceptor retinoid-binding protein. EAU was determined by histopathology 21 days after uveitogenic immunization, and antigen-specific cellular responses were assessed by delayed type hypersensitivity and lymphocyte proliferation. RESULTS: iNOS knockout (iNOS KO) mice developed EAU with scores similar to wild-type mice and exhibited good cellular responses to the immunizing antigen. CONCLUSIONS: A functional iNOS gene is not necessary for EAU pathogenesis. Therefore, upregulation of NO is not required to mediate autoimmune tissue damage in the eye.

Identification of genomic regions controlling experimental autoimmune uveoretinitis in rats.

The present study attempts to identify specific genetic loci contributing to experimental autoimmune uveoretinitis (EAU) susceptibility in F2 progeny of resistant Fischer (F344/N) and susceptible Lewis (LEW/N) inbred rats. F2 progeny of F344/N x LEW/N inbred rats were immunized with the R16 peptide of interphotoreceptor retinoid-binding protein (IRBP). A genome-wide scan was conducted using 125 simple sequence length polymorphism markers in selected F2 animals that developed severe eye disease or remained unaffected to identify phenotype:genotype co-segregation. The F2 population (n = 1287) demonstrated a wide range of histologically assessed EAU scores (assessed on a scale of 0-4). The disease incidence and severity were not consistent with a simple Mendelian inheritance model. Of the F2 hybrid rats, 60% developed EAU, implying the existence of a potent susceptibility locus with incomplete penetrance associated with the LEW genome or a more complex polygenic model of inheritance. Two genomic regions, on chromosomes 4 and 12, showed strong genetic linkage to the EAU phenotype (P < 0.0016), suggesting the presence of susceptibility loci in these chromosomal regions. In conclusion, we have identified two genomic candidate intervals from D4Arb8 to D4Mit17 on chromosome 4 and from the chromosome end to D12Arb8 on chromosome 12, that appear to influence EAU susceptibility in LEW/F344 rats. Further analysis of these genomic regions may lead to identification of the susceptibility genes and to characterization of their function.

Protective effect of the type IV phosphodiesterase inhibitor rolipram in EAU: protection is independent of IL-10-inducing activity.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is a cell-mediated model of retinal autoimmunity that is negatively regulated by interleukin (IL)-10. The antidepressant drug rolipram, a type IV phosphodiesterase inhibitor, enhances IL-10 production by monocyte/macrophages. The effect of rolipram on induction of EAU and its associated immunologic responses was investigated. METHODS: Mice were challenged for EAU induction by immunization with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP) or by adoptive transfer of uveitogenic T cells and were treated with rolipram. EAU severity and immunologic responses to IRBP were analyzed. In addition, the effect of rolipram added to the culture on antigen-driven responses of primed lymph node cells was tested. RESULTS: Rolipram treatment from days -1 to 7 after immunization (afferent phase) was not protective, but severity of EAU was reduced to 50% by treatment from days 8 to 16 after immunization or when EAU was induced by adoptive transfer (efferent phase). Antigen-specific proliferation and interferon (IFN)-gamma production ex vivo by lymph node cells of protected mice were not reduced. However, the addition of rolipram directly to the culture suppressed IRBP-driven proliferation and IFN-gamma production by primed lymph node cells. Freshly explanted lymph node cells of treated mice showed inhibition of IFN-gamma mRNA but no parallel enhancement of IL-10 mRNA by quantitative polymerase chain reaction. Rolipram inhibited EAU in IL-10 knockout mice equally well compared with controls and suppressed their primed lymph node cells in culture. CONCLUSIONS: Rolipram appears to inhibit the expansion and effector function of uveitogenic T cells, raising the possibility that it may be useful for treatment of established disease. Contrary to expectations based on in vitro studies, the protective effects in vivo appear to be independent of IL-10. The observation that suppression of antigen-specific responses is demonstrable only in the physical presence of the drug suggests that, in a clinical setting, continuous administration of rolipram might be needed to sustain its therapeutic effect.

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.

Acute immunosuppression and syngeneic bone marrow transplantation in ocular autoimmunity abort disease, but do not result in induction of long-term protection.

Acute immunosuppression induced by total body irradiation (TBI) or cyclophosphamide (Cy) treatment, followed by syngeneic bone marrow transplantation (SBMT), was reported to be effective in inducing long-term tolerance in some autoimmune disease models. We examined the efficacy of this approach in the mouse model of experimental autoimmune uveoretinitis (EAU). Development of EAU induced by the interphotoreceptor retinoid binding protein (IRBP) was abolished almost completely by either TBI or Cy treatment, followed by SBMT, instituted one week after priming. In parallel, IRBP-specific delayed-type hypersensitivity (DTH) responses and lymph node cell proliferation were strongly suppressed or abolished. However, when these IRBP-immunized, lymphoablated and BM reconstituted mice were rechallenged with the immunizing antigen seven weeks after the primary immunization, they were not protected from developing disease, despite the fact that DTH and lymph node cell proliferation to the antigen were suppressed relative to controls. TBI treatment appeared somewhat more effective than Cy treatment as judged by its more profound effect on immunological responses. These results demonstrate the ability of acute immunosuppression followed by reconstitution of the immune system to inhibit the development of EAU, although long-term protection from disease was not achieved.

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.

Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis.

Experimental autoimmune uveitis (EAU) has been associated with a Th1 response. However, in IFN-gamma-deficient mice, EAU develops in the context of an effector response having Th2-like elements, and administration of IL-12 to mice immunized for EAU induction can be protective. We, therefore, investigated whether endogenous IL-12 is required for development of EAU. IL-12 p40-deficient mice (12KO) were resistant to EAU induced with the uveitogenic retinal Ag interphotoreceptor retinoid binding protein (IRBP). Delayed hypersensitivity to IRBP was marginally reduced, whereas Ag-specific proliferation was enhanced. Primed lymphocytes of wild-type (wt) mice, cultured with IRBP, produced a Th1-like cytokine profile and transferred EAU to syngeneic wt recipients. Interestingly, the same cells were inefficient in transferring EAU to 12KO recipients, unless IL-12 was included in the culture. Primed cells of the 12KO mice produced a Th2-like cytokine profile and failed to transfer EAU. However, when IL-12 was added to the culture, 12KO cells produced large amounts of IFN-gamma and transferred EAU to naive 12KO recipients. We conclude that resistance to EAU of 12KO mice is not due to an inherent inability of these mice to develop ocular disease. Despite an apparent similarity in Ag-specific cytokine responses to IFN-gamma-deficient mice, 12KO mice have inhibited generation of uveitogenic effector cells, a situation that can be reversed even after priming, by adding exogenous IL-12 ex vivo. Lastly, the diminished ability of primed wt lymphocytes to induce EAU in 12KO mice indicates a role for endogenous IL-12 in the efferent phase of disease expression that is distinct from its role during Ag priming.

Uveitogenicity is associated with a Th1-like lymphokine profile: cytokine-dependent modulation of early and committed effector T cells in experimental autoimmune uveitis.

This study addresses the nature of the pathogenic effector T cell in experimental autoimmune uveoretinitis and the effect of different cytokines on these cells in vitro. Lymph node cells of B10.RIII mice immunized with the uveitogenic peptide 161-180 of interphotoreceptor retinoid binding protein were cultured with the peptide with or without IL-12, IL-4, or anti-IL-4. An antigen-specific T cell line was subsequently derived from these cells. Primary cultures of immune lymph node cells stimulated with the peptide proliferated and produced IL-2 and some IL-4, but no IFN-gamma. The addition of recombinant IL-12 resulted in abundant production of IFN-gamma, which was blocked by the addition of IL-4 and was enhanced by anti-IL-4. Only those cultures that produced IFN-gamma in vitro were uveitogenic in vivo. A long-term uveitogenic T cell line, initially derived in the presence of IL-12, produced IFN-gamma and IL-2, but not IL-4, and was CD4+ (Th1-like). Antigen-specific proliferation and IFN-gamma production of the line were enhanced by exogenous IL-4, TGF-beta, IL-2, IL-6, IL-7, and IL-9 and were inhibited by IL-10 and TNF-alpha. Our results provide support for the hypothesis that the uveitogenic effector T cell has a Th1-like phenotype. Furthermore, the data suggest that the effects of the cytokine milieu on fully differentiated Th1 effectors may differ considerably from their effects on less mature stages of antigen-specific T cells.

T cell mechanisms in experimental autoimmune uveoretinitis: susceptibility is a function of the cytokine response profile.

This study addresses the question whether susceptibility versus resistance to experimental autoimmune uveoretinitis (EAU) is connected to a Th1-type (interferon-gamma high, interleukin-4 low), versus a Th2-type (IFN-gamma low, IL-4 high) response. Primed lymph node cells of susceptible Lewis rats produced IFN-gamma in response to antigen in culture and transferred EAU to syngeneic recipients, whereas lymph node cells of resistant F344 rats made no IFN-gamma and did not transfer disease. Reversal of the disease pattern, by treatment of F344 rats with B. pertussis toxin and immunisation of Lewis rats with antigen in incomplete Freund's adjuvant, resulted in a parallel reversal of these response patterns. Neither strain produced significant IL-4 responses. A study of the response patterns in mice confirmed that high Th1 responders were susceptible, whereas low Th1 responders and Th2 responders were resistant. We conclude that susceptibility to EAU is connected with a Th1-dominant response, but resistance can involve either a 'null', F344-like response (Th1-low/Th2-low) or a Th2-dominant response.

Anti-tumor necrosis factor alpha therapy suppresses the induction of experimental autoimmune uveoretinitis in mice by inhibiting antigen priming.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) serves as a model for several immune-mediated diseases that affect the eye in humans. Previous studies indicated that tumor necrosis factor alpha (TNF-alpha) has an important proinflammatory role in EAU and possibly in human uveitis. In this study, the authors investigated the effect of anti-TNF-alpha therapy on EAU in mice. METHODS: Experimental autoimmune uveoretinitis was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). The mice were treated with 100 or 300 microliters rabbit antiserum or polyclonal antibodies to human TNF-alpha. The treatment spanned either the afferent or the efferent stage of EAU (days -1, 1, 3, 5, 7, or days 8, 10, 12, 14, 16, respectively). Control animals were injected with preimmune rabbit serum at the corresponding times or were not treated. Three weeks after immunization, EAU was assessed by clinical evaluation and by histopathology. Immunologic responses were assessed by delayed-type hypersensitivity (DTH), lymphocyte proliferation to IRBP, and relative abundance of IRBP-primed splenocytes. RESULTS: The treatment with rabbit anti-TNF-alpha serum significantly ameliorated disease when given during the afferent stage but had no effect when given during the efferent stage of EAU. The effect on DTH, lymphocyte proliferation, and abundance of antigen-reactive cells roughly paralleled the effect on disease. CONCLUSIONS: Neutralization of systemic TNF ameliorates EAU. The effectiveness of afferent treatment in comparison to the treatment during the efferent stage, together with the reduced proliferation and the reduced abundance of IRBP-responsive cells, suggest that interference with afferent-acting processes such as antigen priming is important to achieve protection from EAU by anti-TNF treatment.

Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response.

This study examines whether genetic susceptibility vs genetic resistance to experimental autoimmune uveoretinitis (EAU) are connected to a predisposition to mount a Th1-dominated (IFN-gamma high, IL-4 low) vs a Th2-dominated (IL-4 high, lFN-gamma low) response. Lewis rats developed disease with high incidence after immunization with the uveitogenic peptide R16, whereas F344 rats were resistant. Primed lymph node cells from both strains proliferated in culture in response to R16. However, while the Lewis cultures transferred EAU to syngeneic recipients, those of F344 did not. The Lewis cultures produced substantially more IFN-gamma mRNA and protein in response to R16, than did those of F344. Both strains made low levels of IL-10 mRNA and IL-4 mRNA. Unlike the primary cultures, long-term (R16-specific) T cell lines derived from each of the strains transferred EAU equally well to their respective recipients, and produced similar, high levels of IFN-gamma mRNA and protein. Treatment of F344 with Bordetella pertussis toxin concurrently with immunization abrogated its resistance, enhanced Ag-specific IFN-gamma production in culture, and yielded a primed cell population capable of transferring EAU. Conversely, immunization of Lewis rats with R16 in IFA induced little or no disease; the primed cells produced minimal amounts of IFN-gamma and did not transfer EAU. However, addition of IL-12 into the culture resulted in a highly pathogenic, IFN-gamma-producing cell population. We conclude that genetic susceptibility to ocular autoimmunity in this model is connected to an elevated Th1 response. Genetic resistance, however, does not seem to involve an elevated Th2 response, but rather an inhibited development of Th1-like effector cells.