Inhibition of experimental autoimmune uveitis by retinal photoreceptor antigens coupled to spleen cells.

Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell-mediated inflammatory diseases of the uveal tract and retina of the eye and of the pineal gland. EAU and EAP can be induced by several retinal autoantigens including S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP). In this study we investigated the effect of intravenous administration of S-Ag and IRBP coupled to syngeneic spleen cells on the development of EAU and EAP. Injection of S-Ag or IRBP coupled to spleen cells 5 days prior to immunization with native S-Ag or IRBP, respectively, was effective in preventing the induction of EAU and EAP in LEW rats. Conversely, LEW rats receiving S-Ag-coupled spleen cells and challenged with IRBP or LEW rats receiving IRBP-coupled spleen cells and challenged with S-Ag developed a severe EAU within 10 days to 2 weeks following immunization, as did all control animals receiving sham-coupled spleen cells and challenged with the two retinal antigens. The results show that the administration of retinal autoantigens coupled to spleen cells effectively protects against the development of EAU when animals are subsequently challenged with the tolerizing antigen but not when challenged with another unrelated pathogenic retinal autoantigen.     

Retinal expression of a neo-self antigen, beta-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis.

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of beta-galactosidase (beta-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of beta-gal systemically. Immunization with beta-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to beta-gal, showing that tolerance was not elicited by levels of retinal beta-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with beta-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with beta-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal beta-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that beta-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.     

Uveitogenic potential of lymphocytes sensitized to interphotoreceptor retinoid-binding protein

In our previous study rats immunized with bovine retinal interphotoreceptor retinoid-binding protein (IRBP) were found to develop inflammation in the eye and the pineal gland. This inflammatory disease was distinct in several aspects from experimental autoimmune uveoretinitis (EAU) induced by the retinal S-antigen (S-Ag). The current study examined the adoptive transfer of IRBP-induced EAU. We established that lymphocytes from IRBP immunized donor rats were capable of transferring EAU after in vitro stimulation with either IRBP (lymph node or spleen cells) or concanavalin A (spleen cells only). Recipients of these cells developed uveoretinitis and pinealitis identical to the actively induced disease. As compared with the S-Ag system, recipients of IRBP sensitized cells developed disease earlier, and smaller numbers of cells were needed to transfer EAU. Development of inflammation was directly related to a cellular response to the specific retinal antigen used for sensitization. Moreover, the unique nature of ocular inflammation was reestablished in the IRBP system: high proportions of polymorphonuclear leukocytes were found in the inflamed tissue of certain recipients despite a lack of a humoral response to the specific antigen. In contrast to the eye, only mononuclear leukocytes comprised the inflammation in the pineal gland.     

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.     

Antigen-directed retention of an autoimmune T-cell line

We have used the T-cell-mediated, organ-specific autoimmune disease model of experimental autoimmune uveoretinitis (EAU) in the Lewis rat to study antigen-directed retention of autoimmune T helper cells in the target organ. We have compared the migration into the eye of two T-helper-cell lines: ThS, specific for retinal S antigen (S-Ag), that is uveitogenic to normal syngenic recipients, and ThP, specific to purified protein derivative of tuberculin (PPD), that is non-uveitogenic. The retention of adoptively transferred 51Cr-labeled ThS and ThP was studied up to the stage of disease induction in unprimed animals, during the acute stage of EAU induced by active immunization with S-Ag, and during the acute stage of a uveitis induced by a nonocular antigen (bovine serum albumin, BSA). Low numbers of cells from the two lymphocyte lines were detected in the eyes of unprimed animals, with no obvious increase of ThS over ThP, despite induction of EAU in the recipient animals by the injected ThS cells. In S-Ag-induced EAU many more ThS accumulated in the eye than ThP. In BSA uveitis both T-cell lines accumulated in the eye to the same extent, but more than in control noninflamed eyes. These results demonstrate the presence of increased antigen-specific retention of circulating autoimmune T helper lymphocytes during the acute stage of an ocular antigen-specific, but not ocular antigen nonspecific, inflammation. Since detectable accumulation of ThS cells in the eye was not a prerequisite for the induction of EAU, this phenomenon appears to be the result, rather than the cause, of the autoimmune process.     

Autoimmune uveitis elicited with antigen-pulsed dendritic cells has a distinct clinical signature and is driven by unique effector mechanisms: initial encounter with autoantigen defines disease phenotype

Human autoimmune uveitis is a heterogeneous group of potentially blinding ocular diseases in which most patients who exhibit immunity recognize the same retinal Ag. It is represented by the model of experimental autoimmune uveitis (EAU) induced in mice by immunization with retinal Ag in CFA. Murine EAU is characterized by a Th1/Th17 response pattern, which may not represent all types of human uveitis. We report in this study a new model of EAU induced by injection of matured dendritic cells loaded with a uveitogenic retinal peptide. Dendritic cell-induced EAU demonstrated unique characteristics compared with traditional EAU in terms of clinical manifestations, the nature of the inflammatory infiltrating cells, the cytokine response profile, and a strict requirement for IFN-gamma, whereas IL-17 appeared to play a minor role. Disease was self-limiting, but could be reinduced with the same Ag in CFA, albeit with reduced severity, suggesting post-recovery resistance. Our study demonstrates in a disease setting that the context in which the same autoantigen is initially presented to the immune system precipitates distinct forms of pathology via a distinct pathogenic pathway on the same genetic background. These findings may shed new light on the complex biology and the heterogeneous nature of human uveitis, and provide an alternative model for uveitic diseases of immune origin.     

A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T lymphocyte-mediated autoimmune disease, which serves as a model for several human ocular inflammations of an apparently autoimmune nature. EAU pathology in some rodents and in monkeys can readily be induced by immunization with several different retinal proteins; however, advancing research into the cellular mechanisms of this disease has raised the need for an EAU model in an immunologically and genetically well defined species. We report here the induction of EAU in the mouse, which has hitherto been considered a species refractory to EAU, with two retinal Ag, the retinal soluble Ag and the interphotoreceptor retinoid-binding protein. Although all the mouse strains tested exhibited lymphocyte responses and antibody titers to both retinal Ag, EAU was inducible in only some of the strains, and the uveitogenic responses to retinal soluble Ag and interphotoreceptor retinoid-binding protein appeared to be mutually exclusive. The EAU model in mice was found to differ in several respects from the EAU model in other rodent species. Induction of the disease was achieved with a relatively high dose of Ag and an intensified immunization protocol, and the onset of disease was later, the duration was longer, and the course was less acute. Anterior segment involvement was slight or nonexistent, and damage to the retina and uvea was of a focal rather than of a diffuse nature. Murine EAU appeared to approximate some types of human uveitis more closely than the EAU models described in other rodent species with respect to its pathologic manifestations as well as its more chronic course. The relatively longer duration of the active stage of disease in murine EAU should facilitate therapeutic intervention in established disease, which was not feasible in the more acute models of EAU. The extensive knowledge of the immunologic parameters of the mouse and the availability of genetically defined strains should be of great value in the study of cellular mechanisms and immunogenetics of ocular autoimmune disease.     

Cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation

The retinal pigment epithelial (RPE) cell has long been considered an important regulatory cell, maintaining physiological and structural balance within the retina. We have previously shown that the RPE cell may also be important in autoimmunity and transplantation. These cells can be induced by cytokines to express MHC class II Ag in ocular inflammatory and autoimmune conditions. In this report we show that isolated rat RPE cells can be induced to express class II Ag following incubation with rat rIFN-gamma. The ability of RPE cells to present Ag was determined by both T cell proliferation assays and IL-2 production. Only the Ia-positive RPE cells can present retinal Ag (S-Ag and interphotoreceptor-binding protein) to specifically sensitized rat Th cells. Moreover, the ability of chloroquine to inhibit this activity suggests that the RPE cell is also capable of processing Ag prior to Ag presentation. These studies indicate that cytokine-mediated activation of RPE cells may be a basic component of ocular immunity and an important aspect of RPE cell transplantation.     

T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat

Long-term S-antigen (S-Ag)-specific T lymphocyte lines were derived from the lymph nodes of immunized Lewis rats that had been pretreated with low-dose cyclophosphamide. The protocol consisted of functional selection by alternating cycles of stimulation with S-Ag presented on syngeneic accessory cells and proliferation in IL 2-containing spleen-conditioned medium, coupled with early phenotypic selection for cells bearing the helper/inducer membrane marker (W3/25), by panning on antibody-coated plastic dishes. This protocol consistently resulted in the rapid generation of in vivo functional cell lines capable of mediating experimental autoimmune uveoretinitis (EAU) when transferred into naive rats at 5 to 10 X 10(6) cells/rat systemically or 1 to 2 X 10(6) cells/rat intravitreally. The disease appeared within 6 to 8 days, usually with minimal anterior chamber involvement, and was often unilateral. Pathologic changes resembled those seen in EAU induced by active immunization. The disease could be transferred without concomitant formation of serum antibodies. The uveitogenic line cells were negative for Ia antigen and positive for the W3/25 membrane marker, which appeared to be stable in long-term culture. They proliferated vigorously in vitro and produced IL 2 in response to S-Ag or Con A, but not to unrelated antigens. The establishment of uveitogenic T helper lymphocyte lines will permit the analysis of the cellular mechanisms involved in EAU in a more defined system than has been available.     

Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE40. Inhibition of experimental autoimmune uveoretinitis

A characteristic of activated T lymphocytes is the expression of high affinity IL-2R. We studied a new method of selective immunosuppression directed against activated T cells by using a chimeric recombinant protein (IL-2-PE40) composed of IL-2 fused to a modified Pseudomonas exotoxin lacking its cell recognition domain. As a model of T cell-mediated disease, we used experimental autoimmune uveoretinitis (EAU) produced in Lewis rats by active immunization with the retinal S-Ag. The treatment protocol consisted of i.p. injection of IL-2-PE40 at 0.25 micrograms/g every 12 h. Controls were PBS, PE40, or IL-2-PE40asp553 a mutant form of the molecule with reduced activity. Treatment with IL-2-PE40 resulted in a significant reduction of the incidence and severity of EAU over controls. The analysis of the effect of i.p. injection of IL-2-PE40 on the popliteal draining lymph nodes of immunized animals showed a marked reduction in the lymphocytes content. Transfer experiments demonstrated that IL-2-PE40 prevented the development of EAU effector T cells. Interestingly, although activated B cells were reported to express IL-2R, there was no significant reduction of antibody production against the immunizing Ag under IL-2-PE40 treatment, suggesting sparing of the B cells.     

Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cells expressing V beta 8 and V alpha 2-like genes.

Experimental autoimmune uveoretinitis (EAU) is a predominantly T cell-mediated autoimmune disease induced in susceptible animals by active immunization with human or bovine retinal S-Ag or by passive transfer of activated S-Ag or peptide-specific CD4+ T cells. During the course of studies aimed at the identification of T cell and B cell recognition sites in bovine and human S-Ag, a new potent uveitogenic region, located near the carboxy terminus of the molecule, was identified and characterized. Analysis of several synthetic peptides from this region showed that a 14 amino acid residue peptide, BSAg339-352, was highly uveitogenic when injected with adjuvants into Lewis rats. A uveitogenic T cell line, R737, was raised by in vitro selection of lymphocytes from animals immunized with peptide BSAg333-352. Northern blot analysis of mRNA from the R737 T cell line was positive for the rat homologs of murine V beta 8 and V alpha 2 T cell receptor gene probes. Whereas peptide BSAg339-352 defined the pathogenic site, nonpathogenic, proliferative sites were found in close physical association. This region is immediately adjacent to previously characterized pathogenic and proliferative sites contained in residues BSAg352-364. These results, as well as our previous observations, show S-Ag to be a complex molecule with several highly conserved amino acid sequences that can elicit pathogenic T cells with restricted T cell receptor V gene usage capable of active and passive elicitation of experimental autoimmune uveoretinitis.     

Liposome-Encapsulated Clodronate Retards the Development of Experimental Autoimmune Uveitis

Abstract

A study was undertaken to determine if the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (C1₂MDP; Clodronat), a treatment known to deplete monocytes, as well as liver and spleen macrophages, would reduce the number of macrophages in the retina of animals with experimental autoimmune uveitis (EAU) and decrease the severity of the disease. EAU was induced in Lewis rats by immunization with S-antigen (S-Ag). Monocytes and macrophages were depleted via an intravenous injection of Cl₂MDP encapsulated in liposomes. Control groups included rats that received no S-Ag (n= 18), S-Ag and no treatment (n=23), S-Ag and free drug (n = 20), or empty liposomes (n=14). Treated animals received injections of the Cl₂MDP-liposomes, free drug, or empty liposomes. Animals were sacrificed at 14, 21 and 28 days post-S-Ag administration. Intravenous, Cl₂MDP-liposomes produced a statistically significant reduction in the severity of the EAU when compared to controls at both days 14 and 21 following S-Ag injection. Immunohistochemical staining with the monoclonal antibody EDI demonstrated that the severity of the ocular inflammatory response correlated with the number of EDI-positive cells in the retina. Following the cessation of treatment, treated animals developed disease that was as severe at day 28 as that of untreated animals at day 21. These results confirm the importance of monocytes and macrophages in EAU by demonstrating the correlation between the presence of EDI-positive cells in the retina and the resultant damage to the retina. Although the dosing regimen employed here did not provide a cure, strategies designed to prevent the local recruitment and/or activation of mononuclear phagocytes may prove to be useful in the treatment of EAU.     

Fas and Fas ligand expressed on cells of the immune system, not on the target tissue, control induction of experimental autoimmune uveitis

The Fas-Fas ligand (FasL) interaction is important for maintaining lymphocyte homeostasis by signaling for activation-induced cell death. Mice homozygous for the lpr or gld mutations do not express functional Fas or FasL, respectively, and spontaneously develop progressive autoimmune symptoms. Recent studies implicated expression of FasL on immunologically privileged tissues in protection from immune-mediated damage. Conversely, tissue expression of Fas may facilitate damage. We evaluated the susceptibility of lpr and gld mice to induction of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease induced with retinal Ags, which targets the neural retina. gld as well as lpr mice immunized with a retinal Ag developed disease of lower incidence and severity than wild-type controls. Delayed hypersensitivity responses were not significantly different among immunized gld, lpr, or wild-type mice, although in vitro Ag-specific lymphocyte responses of the mutant mice were lower. To evaluate whether the diminished ability of gld and lpr mice to develop EAU was due to a defect at the level of the tissue or the immune system, radiation bone marrow chimeras constructed between wild-type and mutant mice were immunized to induce EAU. Mutant recipients of wild-type bone marrow, but not wild-type recipients of mutant bone marrow, developed normal disease scores. These results indicate that normal expression of Fas and of FasL on cells of the immune system is important for EAU expression. Unexpectedly, neither lack of Fas nor lack of FasL on the ocular tissues affected expression of EAU.     

Abrogation of anti-retinal autoimmunity in IL-10 transgenic mice due to reduced T cell priming and inhibition of disease effector mechanisms

Experimental autoimmune uveitis (EAU) induced by immunization of animals with retinal Ags is a model for human uveitis. The immunosuppressive cytokine IL-10 regulates EAU susceptibility and may be a factor in genetic resistance to EAU. To further elucidate the regulatory role of endogenous IL-10 in the mouse model of EAU, we examined transgenic (Tg) mice expressing IL-10 either in activated T cells (inducible) or in macrophages (constitutive). These IL-10-Tg mice and non-Tg wild-type controls were immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein. Constitutive expression of IL-10 in macrophages abrogated disease and reduced Ag-specific immunological responses. These mice had detectable levels of IL-10 in sera and in ocular extracts. In contrast, expression of IL-10 in activated T cells only partially protected from EAU and marginally reduced Ag-specific responses. All IL-10-Tg lines showed suppression of Ag-specific effector cytokines. APC from Tg mice constitutively expressing IL-10 in macrophages exhibited decreased ability to prime naive T cells, however, Ag presentation to already primed T cells was not compromised. Importantly, IL-10-Tg mice that received interphotoreceptor retinoid-binding protein-specific uveitogenic T cells from wild-type donors were protected from EAU. We suggest that constitutively produced endogenous IL-10 ameliorates the development of EAU by suppressing de novo priming of Ag-specific T cells and inhibiting the recruitment and/or function of inflammatory leukocytes, rather than by inhibiting local Ag presentation within the eye.     

Identification of a potent new pathogenic site in human retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of the eye which can be induced in LEW rats by immunization with either human or bovine S-antigen (S-Ag). In previous reports, two nonimmunodominant pathogenic sites were found using synthetic peptides corresponding to conserved sequences at amino acid residues 303-314 and 286-297 of the bovine sequence. In this report, a 20-residue synthetic peptide encompassing amino acids 343-362 located near the C-terminus was found to be highly immunopathogenic in LEW rats. The onset of EAU was observed at as early as 8 days when high doses of a peptide-encompassing residues 343-362 were used. EAU was elicited with as little as 0.5 microgram of peptide per animal. Smaller peptides from this region were also tested for uveitogenicity, further refining the site to 13 amino acids. Uveitogenic T cell lines were made to this site in two ways; first, by the in vitro selection of a bulk T cell line raised to human S-Ag with peptide 343-362. Second, by the in vitro selection of a peptide-specific line from an animal immunized with peptide 352-364, which corresponds to the minimal uveitogenic site. Both of these lines adoptively transferred EAU to LEW rats, further establishing the pathogenicity of this site. A proliferative site distinct from, but overlapping, the uveitogenic site was also found. The potent uveitopathogenicity of peptides from this region indicates that it is a major pathogenic site responsible for EAU induced in LEW rats by immunization with human S-Ag.     

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.     

Ocular immune privilege and the impact of intraocular inflammation

Immune privilege, a characteristic of the internal compartments of the eye, is a physiologic mechanism that is designed to provide the eye with protection against pathogens while protecting the delicate visual axis from the sight-destroying potential of immunogenic inflammation. It is assumed that the presence of intraocular inflammation is incompatible with the existence of immune privilege. The validity of this assumption has been tested in four animal models of intraocular inflammation-systemic and local endotoxin-induced uveitis (EIU), mycobacterial adjuvant-induced uveitis (MAIU), and experimental autoimmune uveitis (EAU). Immune privilege was assessed in inflamed eyes by growth of intracamerally injected allogeneic tumor cells, by the capacity to support immune deviation following intracameral injection of antigen (ovalbumin, OVA), by assaying protein, leukocyte, and selected cytokine content of aqueous humor (AqH), and by capacity of inflamed AqH to suppress T cell activation in vitro. The results indicate that, irrespective of the type of inflammation, tumor cells formed progressively growing tumors in inflamed eyes. Moreover, OVA injected into the anterior chamber of eyes inflamed by MAIU and EAU failed to induce immune deviation. AqH from inflamed eyes reflected breakdown of the blood:ocular barrier as well as transient loss of its immunosuppressive properties. Immunosuppressive microenvironments routinely reemerged in inflamed eyes, and the immunosuppressive agent present under these circumstances in AqH was active TGF beta2. It is concluded that immune privilege is surprisingly resistant to abolition by intraocular inflammation, and that maintenance of immune privilege in the face of ongoing inflammation depends upon the emergence of progressive and partially different immunosuppressive mechanisms.     

Multiple, autoreactive TCR V beta genes utilized in response to a small pathogenic peptide of an autoantigen in EAU.

The restricted usage of particular T cell receptor beta chain genes in autoimmune disease was studied in LEW rats using T cell hybridomas specific for an immunodominant sequence of bovine retinal S-Ag, which induces experimental autoimmune uveoretinitis. T cell hybridomas from a pathogenic T cell line, R858, specific for residues 273-289 of bovine retinal S-Ag were analyzed in order to determine the contribution of their TCR V beta to self specificity as determined by recognition of the pathogenic epitope represented in the autologous rat S-Ag sequence. Six different, functional TCR rearrangements were expressed by the panel of hybridomas, including two distinct V beta 8.2 rearrangements and functional V beta 10, V beta 14, V beta 19 rearrangements, and an unidentified V beta gene. All hybridomas were Ag specific and reacted both to nonself-peptide derivatives as well as to self-peptide homologues. No unique pattern of peptide reactivity distinguished V beta 8.2+ hybridomas from V beta 8.2- hybridomas; all of the hybridomas were most reactive to the nonself sequences and reacted to self peptide with one to three orders of magnitude less sensitivity. However, all V beta 8.2+ hybridomas were much better responders overall and were activated by lower concentrations of all peptides than were V beta 8.2- hybridomas. Although V beta 8.2 gene usage is strongly associated with autoimmune pathology, these data show that in LEW rats several different TCR V beta genes are utilized in response to a short pathogenic sequence of this autoantigen and show that V beta 8.2 receptors are not uniquely self-reactive. However, the enhanced reactivity to Ag of V beta 8.2+ hybridomas relative to V beta 8.2- hybridomas specific for the same peptide may help explain the close association of V beta 8.2 TCR gene usage with pathogenicity found in autoimmune disease models.     

Small heat shock protein αA-crystallin prevents photoreceptor degeneration in experimental autoimmune uveitis

The small heat shock protein, αA-crystallin null (αA-/-) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA-/- and wild type mice with EAU as donors and Rag2-/- as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses.     

Essential role of the MyD88 pathway, but nonessential roles of TLRs 2, 4, and 9, in the adjuvant effect promoting Th1-mediated autoimmunity

Induction of tissue-specific experimental autoimmune diseases involves an obligatory adjuvant effect to trigger an innate response of a type that will drive a Th1-biased adaptive response. This is achieved by use of CFA containing mycobacteria (Mycobacterium tuberculosis), whose recognition by cells of the innate immune system depends on TLRs that signal through the adaptor molecule MyD88. We examined the role of selected components of the MyD88 pathway in promoting experimental autoimmune uveitis (EAU). Mice deficient in MyD88, TLR2, TLR4, or TLR9 were immunized with the retinal Ag interphotoreceptor retinoid-binding protein in CFA, and their EAU scores and associated immunological responses were examined. MyD88-/- mice were completely resistant to EAU and had a profound defect in Th1, but not Th2, responses to autoantigen challenge. Surprisingly, TLR2-/-, TLR4-/-, and TLR9-/- mice were fully susceptible to EAU and had unaltered adaptive responses to interphotoreceptor retinoid-binding protein. Examination of IL-1R family members, which share the common adaptor MyD88 with the TLR family, revealed that IL-1R-deficient mice, but not IL-18-deficient mice, are resistant to EAU and have profoundly reduced Th1 and Th2 responses. These data are compatible with the interpretation that TLR9, TLR4, and TLR2 signaling is either not needed, or, more likely, redundant in the adjuvant effect needed to induce EAU. In contrast, signaling through the IL-1R plays a necessary and nonredundant role in EAU and can by itself account for the lack of EAU development in MyD88 mice.