Genetic, Cellular And Molecular Mechanisms In Autoimmune

National Eye Institute

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Abstract

Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal antigens, and defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal antigens such as IRBP, Arrestin, or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these antigens. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels. Some recent findings are described ahead. The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. Development of tolerance to self during ontogeny is being investigated by using mice that have differential expression of IRBP, all bred onto the EAU-susceptible B10.RIII background. 1) IRBP-transgenic (TG) mice that express the N-terminal half of IRBP extraocularly under control of the MHC class II promoter. 2) IRBP knockout (KO) mice that have no detectable expression of IRBP and 3) wild type (WT) mice expressing IRBP in the eye and pineal. We are showing that extraocular expression of a privileged retinal antigen abolishes the ability to develop EAU in a transgene-specific fashion. Unlike F1 hybrids between the FVBN founders and B10.RIII mice, the backcrossed TGs show only minor reductions in IRBP-specific immunological responses. Interestingly, peripheral leukocytes from the TG mice transferred to WT precipitate reduced susceptibility to EAU in the recipients. The immunological mechanism(s) involved in this transfer of tolerance are being investigated. Thymic expression of IRBP by immunostaining was detectable in WT and TG, but not in KO mice. In keeping with this, IRBP-KO mice have strongly enhanced immunological responses to IRBP suggesting an expanded T cell repertoire that had not undergone negative selection. IRBP KO mice do not develop IRBP-induced EAU, indicating that there is no other immunologically crossreactive antigen in the retina that could serve as target for this apparently expanded IRBP-specific T cell repertoire. The precursor frequency and fine specificity of epitope recognition of IRBP KO mice are being investigated. These studies will help understand how the IRBP specific T cell repertoire is generated and controlled. As a complementary approach, mice transgenic for a uveitogenic T cell receptor are in the process of being developed. These mice will provide a powerful tool to study the selection, differentiation and trafficking of antigen-specific T cells in this model. EAU, similarly to uveitis in humans, is genetically controlled by MHC as well as non-MHC genes. We study genetic control of EAU using two approaches: 1) defining genetic markers associated with susceptibility in genetically defined rodents. This approach identified 3 chromosomal regions affecting EAU susceptibility in rats, which co-localize with a number of immunologically relevant loci. Consomic rat strains are now being bred by marker-assisted selection, in which each of these chromosomes from the susceptible background is transferred to a resistant background, and vice versa. Genes differentially expressed by susceptible and resistant strains are being identified using DNA microarrays. These studies will permit closer identification of the genes and pathways determining susceptibility or resistance to EAU. 2) using HLA-transgenic mice in which murine MHC genes have been replaced by human MHC genes we created a "humanized" model of EAU, demonstrating that presentation of retinal antigens by MHC molecules can lead to retinal disease and providing a tool to identify the epitopes involved. Interestingly, one of the ?humanized? strains is developing strong EAU when immunized with S-Ag, which induces little or no uveitis in WT mice. This provides another important tool to address the development of self tolerance, as expression of the human MHC genes would not by itself be expected to alter the thymic expression of S-Ag that has been postulated to be a major factor determining susceptibility. The type of cytokine response generated to a retinal Ag contributes to susceptibility to EAU and is regulated by both genetic and environmental factors. The susceptible, but not the resistant, individual has a cytokine response pattern polarized towards cellular immunity (Th1). We continue to study the regulation and counter-regulation of the Th1 response and its role in pathogenesis and recovery from disease. We have previously shown that the cytokine IL-12, an important component of the innate immune system known enhance the Th1 and counteract the Th2 response, actually plays a role also in control of the Th1 response. Its excess (leading to high levels of various inflammatory mediators) early on dampens further recruitment of uveitogenic Th1 cells into the effector pool and protects from EAU. We are now showing that the same type of regulation applies to experimental autoimmune encephalomyelitis (EAE). Thus, these mechanisms have important ramifications for the regulation of other autoimmune diseases and self-regulation of Th1-driven immune responses in general. As another contrast to the known role of IL-12 we find that it is also needed to elicit EAU in IFN-g KO mice, whose uveitogenic response is not Th1 driven but rather appears more Th2-like. Our data indicate that whereas IL-12 is needed to sustain an effective Th1 effector response in WT mice, in IFN-g KO mice it may be needed to sustain a TNF response. Related studies are aimed at defining the role of other cytokines as well as chemokines and activation molecules in the pathogenesis of EAU. Recently we have cloned and expressed IL-18 and produced neutralizing antibodies against it. We also have obtained IL-18 TG and IL-18 KO mice and are using them to dissect the role of IL-18 in the pathogenesis of EAU and to assess its potential as a new target for therapeutic intervention. Pertussis toxin (PTX) has been used for years to promote cell mediated autoimmunity in animal models, but the mechanisms are poorly understood. Better understanding of how PTX enhances EAU so can give a handle on the environmental triggers that promote development of autoimmune diseases. In earlier studies, we found that PTX given at the time of immunization promotes development a Th1 response to the retinal antigen. We now find that , paradoxically, PTX can completely prevent development of EAU if given later, at the time of effector cell migration to the target organ. This is due at least in part to blockade of signaling Gi-protein coupled chemokine receptors. The data suggest that the effect of PTX is multilevel and complex, and its net effect on autoimmune disease is an integration of enhancing and inhibitory effects. Interestingly, incubation with PTX physically prevented ingress of ex vivo-activated effector T cells into the eye, indicating for the first time that initial entry of cells activated in the periphery into the healthy target organ is dependent on Gi-protein dependent processes. The data also suggest that chemokine blockade can be a relevant therapeutic approach to uveitis.