transforming-growth-factor-beta and Retinitis

IRBPp-specific regulatory immunity is found in the spleens of mice recovered from experimental autoimmune uveoretinitis (EAU). Induction of this regulatory immunity is dependent on the expression of the melanocortin 5 receptor (MC5r). Therefore, the authors investigated whether dependence on the expression of MC5r was with the T cells or with the APCs mediating protective regulatory immunity in the EAU-recovered mouse spleen.. Wild-type and MC5r-/- mice were immunized to induce EAU. The IRBPp-stimulated T-cell response in spleens of wild-type and MC5r-/- mice were compared for surface markers and cytokine production. Spleen APC were isolated and used to stimulate cytokine production and regulatory activity in IRBP-specific T cells from wild-type or MC5r-/- mice assayed in culture by ELISA, by flow cytometry, and in vivo by adoptive transfer into EAU mice.. IRBPp-specific CD25+CD4+ T cells from spleens of EAU-recovered wild-type mice express a Treg cell phenotype of FoxP3 and TGF-β compared with the effector T-cell phenotype of IFN-γ and IL-17 production in EAU-recovered MC5r-/- mice. APCs from the spleens of wild-type mice recovering from EAU promoted regulatory T-cell activation in IRBP-specific effector T cells from the spleens of EAU-recovering MC5r-/- mice. Spleen APCs from EAU-recovering wild-type, but not MC5r-/-, mice induced TGF-β expression by primed IRBP-specific effector T cells.. Dependence on MC5r expression is with an APC that promotes or selectively activates IRBP-specific FoxP3+ TGF-β+ CD25+CD4+ Treg cells in the spleens of EAU-recovered mice.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Cells, Cultured; Disease Models, Animal; Eye Proteins; Forkhead Transcription Factors; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Receptors, Melanocortin; Retinitis; Retinol-Binding Proteins; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uveitis

The role of thrombospondin (TSP)-1 in TGF-beta activation and T-cell suppression was studied in the retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells that line the subretinal space, an immune-privileged site in the eye.. Posterior eyecups were prepared by excising the anterior segment, lens, and retina from enucleated eyes of C57BL/6, thrombospondin-1 knockout (TSP-1KO), and TGF-beta2 receptor II double-negative (TGF-beta2 RII DN) mice, leaving behind a healthy monolayer of RPE resting on choroid and sclera. Serum-free medium was added to these RPE eyecups, and, after various time intervals, supernatants (SNs) were removed and tested.. SNs of an ex vivo culture of RPE cells from C57BL/6 mice were shown to inhibit both antigen and anti-CD3 activation of T cells, partially due to constitutive production of TGF-beta and to the ability of RPE to activate the latent form of TGF-beta. Activation of TGF-beta was entirely dependent on TSP-1, also produced by RPE. SNs of RPE from TSP-1KO mice failed to inhibit T-cell activation. Ovalbumin (OVA)-specific delayed hypersensitivity (DH) was not impaired when OVA was injected either into the subretinal space or into the anterior chamber of TSP-1KO mice before OVA immunization. Moreover, experimental autoimmune uveoretinitis was significantly more intense in eyes of TSP-1KO mice and failed to undergo spontaneous resolution unlike wild-type mice.. Production of both TSP-1 and active TGF-beta by RPE is essential to the creation and maintenance of immune privilege in the subretinal space and that the immune privilege limits the severity and duration of retinal inflammation due to autoimmunity.

Topics: Animals; Autoimmune Diseases; Enzyme-Linked Immunosorbent Assay; Immune System; Interferon-gamma; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pigment Epithelium of Eye; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Thrombospondin 1; Transforming Growth Factor beta; Transforming Growth Factor beta2; Uveitis

TGF-beta exerts suppressive effects on immunity, but its potential applications in therapy of ocular autoimmunity have not been widely explored. In the present study, the effects of TGF-beta on uveitogenic T cells were examined.. The effects of TGF-beta on newly primed cells from mice given a uveitogenic regimen of interphotoreceptor retinoid-binding protein (IRBP) were compared with the effects on fully polarized Th1 cells from a long-term uveitogenic T-cell line. The parameters measured were T-cell proliferation, IFN-gamma production, induction of IL-12R expression, triggering of pathogenicity, and expression of costimulatory molecules on antigen-presenting cells (APCs) during in vitro exposure to antigen.. TGF-beta suppressed B7.1 expression on APCs in cultures of lymph node cells from immunized mice. It also suppressed T-cell proliferation, IFN-gamma production, IL-12 receptor accumulation, and the IL-12-promoted acquisition of uveitogenic function. In contrast, the polarized Th1 cells were either resistant to suppression or were enhanced by TGF-beta.. The results suggest that TGF-beta suppresses acquisition of effector functions by autopathogenic T cells, in part by interfering with their response to IL-12 through downregulation of IL-12R expression and in part through inhibition of APC function. The data suggest that although TGF-beta may effectively inhibit activation and recruitment of new T cells into the effector pool, it may be less effective in suppressing the reactivation of already polarized memory T cells that are less dependent on IL-12 and costimulation.

Topics: Animals; Antigen-Presenting Cells; Autoimmune Diseases; B7-1 Antigen; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Female; Flow Cytometry; Immunologic Memory; Interferon-gamma; Lymphocyte Activation; Male; Mice; Receptors, Interleukin; Receptors, Interleukin-12; Retinitis; Retinol-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta; Uveitis

Recently, we have reported that the cytokines alpha-melanocyte-stimulating hormone (alpha-MSH) and transforming growth factor-beta2 (TGF-beta2) work in synergy to induce the activation of regulatory T (Treg) cells. When we used alpha-MSH and TGF-beta2 to generate ocular autoantigen-specific Treg cells and adoptively transferred them into mice susceptible to experimental autoimmune uveoretinitis (EAU), there was suppression in the incidence and severity of EAU. Specificity to a retinal autoantigen was required for the Treg cells to suppress EAU. When stimulated, these Treg cells produced TGF-beta1, and their production of interferon-gamma, interleukin (IL)-10, and IL-4 was suppressed. Also, the Treg cells are suppressed in their proliferative response. Our results demonstrate that alpha-MSH with TGF-beta2 induce Treg cells that can subdue a tissue-specific autoimmune response. This also promotes the possibility of using these immunomodulating cytokines to purposely induce antigen-specific Treg cells to prevent and suppress autoimmune disease.

Topics: Adoptive Transfer; alpha-MSH; Animals; Autoantigens; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cells, Cultured; Eye Diseases; Eye Proteins; Female; Immunosuppressive Agents; Interferon-gamma; Lymphokines; Mice; Retinitis; Retinol-Binding Proteins; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Uveitis

Recently, we have found that the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) not only suppresses IFN-gamma production, but also induces TGF-beta1 production by activated effector T cells. These alpha-MSH- treated effector T cells function as regulatory T cells in that they suppress IFN-gamma production and hypersensitivity mediated by other effector T cells. Experimental autoimmune uveoretinitis (EAU) was suppressed in its severity and incidence in mice that were injected with primed T cells activated in vitro by APC and antigen in the presence of alpha-MSH. Moreover, it appeared that alpha-MSH had converted a population of effector T cells polarized to mediate hypersensitivity into a population of T cells that now mediated immunoregulation. To characterize these alpha-MSH- treated T cells, primed T cells were TCR-stimulated in the presence of alpha-MSH in vitro and their lymphokine profile was examined. Such effector T cells displayed enhanced levels of TGF-beta1 production and no IFN-gamma or IL-10, with IL-4 levels remaining unchanged in comparison with inactivated T cells. In addition, if soluble TGF-beta receptor II was added to cocultures of alpha-MSH-treated T cells and activated Th1 cells, the alpha-MSH-treated T cells could not suppress IFN-gamma production by the Th1 cells. These results suggest that alpha-MSH induces T cells with a regulatory lymphokine pattern, and that through their production of TGF-beta1 these cells suppress other effector T cells. Examination of the alpha-MSH-treated T cells showed that alpha-MSH did not alter the phosphorylation of CD3 molecules following TCR engagement. Primed T cells express the melanocortin 5 receptor (MC5r), a receptor that is linked to an intracellular signalling pathway shared by other cytokine receptors. Blocking the receptor with antibody prevented alpha-MSH from suppressing IFN-gamma production by the activated regulatory T cells, suggesting that alpha-MSH immunoregulation is through the MC5r on primed T cells. Surface staining and cell sorting of the alpha-MSH- treated primed T cells showed that the regulatory T cells are CD25+ CD4+ T cells. From these results we find that alpha-MSH can mediate the induction of CD25+ CD4+ regulatory T cells. These regulatory T cells require specific antigen for activation, but through non-specific TGF-beta1-mediated mechanisms they can suppress other effector T cells.

Topics: alpha-MSH; Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Flow Cytometry; Interferon-gamma; Mice; Mice, Inbred BALB C; Peptide Fragments; Receptors, Corticotropin; Receptors, Interleukin-2; Receptors, Melanocortin; Retinitis; Retinol-Binding Proteins; Transforming Growth Factor beta

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and B10.A mice by immunization with S-antigen (S-Ag) to study the potential roles of vascular endothelial growth factor (VEGF) and the beta1 and beta2 isoforms of transforming growth factor (TGFbeta1 and TGFbeta2) during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFbeta1 and TGFbeta2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier (BRB) failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFbeta1 and TGFbeta2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFbeta1 or TGFbeta2. In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFbeta1 or TGFbeta2. Since TGFbeta can inhibit endothelial cell proliferation, it is likely that an increase in TGFbeta may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors.

Topics: Animals; Blood-Brain Barrier; Endothelial Growth Factors; Eye Proteins; Female; Immunization; Ischemia; Lymphokines; Mice; Mice, Inbred Strains; Neovascularization, Pathologic; Rats; Rats, Inbred Lew; Retina; Retinal Artery; Retinitis; Retinol-Binding Proteins; Transforming Growth Factor beta; Up-Regulation; Uveitis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The authors have previously reported that an injection of S-antigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (designated orchidic tolerance) and protects the animals from experimental autoimmune uveoretinitis (EAU) and that the signal for orchidic tolerance induction emigrates from the testis within a few hours after antigen priming of the testis. In order to understand the mechanism by which the signal or signal carrier is generated, they determined in this study changes in immunoreactivity for transforming growth factor-beta (TGF-beta), IFN-gamma, IL-2, Fas and Fas ligand in the testis following an injection of S-Ag. Immunoreactivity for TGF-beta increased with time, reaching a maximum in six hours and declining thereafter. The time required for the maximum expression of TGF-beta coincided well with the time-dependent profile of orchidic tolerance signal generation within the testis. Little or no immunoreactivity was observed for IFN-gamma and IL-2 in normal (control) and S-Ag-injected testes. Immunoreactivity for Fas and Fas ligand was detected both in control and experimental testes and did not change appreciably with time following Ag-priming of the testis. Fas immunoreactivity was found in spermatids and virtually absent in the interstitial tissue, while Fas ligand immunoreactivity was primarily associated with the interstitial cells such as Leydig cells. Fas ligand immunoreactivity was very weak, if any, in the germ cells and Sertoli cells. These results suggest that TGF-beta and Fas ligand expressed in MHC-positive interstitial cells may play an important role in the generation of orchidic tolerance induction signal. A preliminary study showed that splenocytes preincubated with testis extracts and S-Ag, when transferred to naive rats, induced systemic tolerance in recipient animals. Inclusion of anti-TGF-beta or a carboxyl terminal peptide of Fas in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. These results indicate that generation of the orchidic tolerance signal does not require the anatomical structure of the testis but is mediated by molecular entities such as TGF-beta and Fas ligand.

Topics: Amino Acid Sequence; Animals; Arrestin; Autoimmune Diseases; Fas Ligand Protein; fas Receptor; Immunization; Immunoenzyme Techniques; Immunosuppression Therapy; Male; Membrane Glycoproteins; Molecular Sequence Data; Peptide Fragments; Rabbits; Rats; Rats, Inbred Lew; Retinitis; Testis; Transforming Growth Factor beta; Uveitis

Experimental autoimmune uveoretinitis (EAU) in mice, an organ specific autoimmune disease, has been investigated as an animal model for human endogenous uveitis. In this study, we report on the immunosuppressive effect of transforming growth factor-beta 1 (TGF-beta 1) on the development of EAU in mice. Inhibition by TGF-beta 1 of proliferation of interphotoreceptor retinoid-binding protein (IRBP)-specific T cell lines in B10.A mice against IRBP antigen was dose-dependent. However, when spleen cells used as the antigen presenting cell were first cultured with TGF-beta 1, this anti-proliferation effect was abolished. When IRBP-immunized mice were injected intraperitoneally with TGF-beta 1, dose-dependent suppression of EAU was obtained. The proliferation response of lymph node cells from TGF-beta 1 injected mice with IRBP-induced EAU was suppressed compared with phosphate buffered saline (PBS)-injected mice. These findings suggest that TGF-beta 1 may be a cytokine that plays a role in suppressing IRBP induced EAU in mice.

Topics: Animals; Antigen-Presenting Cells; Autoimmune Diseases; Cells, Cultured; Disease Models, Animal; Eye Proteins; Female; Immunosuppressive Agents; Mice; Mice, Inbred Strains; Retinitis; Retinol-Binding Proteins; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Uveitis

Intratesticular injection of a retinal protein (S-antigen) into Lewis rats induces systemic immunotolerance (designated orchidic tolerance) and renders animals refractory to experimental autoimmune uveoretinitis (EAU) produced by S-antigen immunization. We demonstrated in this work that the immunotolerance could be transferred to syngeneic naive rats by both CD4+ and CD8+ regulatory cells. Attempts were then made to characterize the cytokines involved in the immunosuppressive activity of these regulatory cells. Using the in vitro lymphoproliferation assay, the inhibitory effect of CD4+ cells on effector cells was found to be reversed by antibodies against IL-4 and IL-10 but not by anti-TGF-beta antibody. IL-4 (IC50 = 1.6 ng/10(6) cells) and IL-10 (IC50 = 0.6 ng/10(6) cells) added to the assay medium were potent inhibitors of effector cell proliferation. Increased immunoreactivity and mRNA expression for IL-4 and IL-10 was observed for CD4+ regulatory cells. The inhibitory effect of CD8+ regulatory cells was reversed by anti-TGF-beta antibody but not by antibodies against IL-4 and IL-10. Compared with control CD8+ cells, CD8+ cells from tolerized rats demonstrated higher immunoreactivity for TGF-beta but did not show an enhanced expression of mRNA for TGF-beta. TGF-beta 1 and TGF-beta 2 added to effector cells showed dichotomous effects; both isoforms stimulated cell proliferation at 2.5 ng/10(6) cells and inhibited at lower or higher concentrations. These results led us to conclude that IL-4 and IL-10 are important cytokines for the immunosuppressive effect of CD4+ regulatory cells generated in orchidic tolerance. TGF-beta is an important immunosuppressive cytokine for CD8+ regulatory cells but further studies will determine whether other cytokines are also involved.

Topics: Animals; Arrestin; Autoimmune Diseases; Base Sequence; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; DNA Primers; Immune Tolerance; In Vitro Techniques; Interleukin-10; Interleukin-4; Lymphocyte Activation; Male; Rats; Rats, Inbred Lew; Retinitis; RNA, Messenger; Testis; Transforming Growth Factor beta; Uveitis

Intra-vitreal injection of 300 U of interleukin (IL)-1 beta into the rabbit eye induces an inflammation of the retina characterized by hemorrhage, monocyte and neutrophil infiltration, and an increase in vascular permeability that peaks 24 h post-injection. Since the epiretinal vessels involved in this inflammation form part of the blood-retina barrier, we used this model to investigate the effects of the immunosuppressive cytokine TGF beta 1 on inflammation within the context of the central nervous system. We found that intra-vitreal injection of 1 microgram rh TGF beta administered concomitantly with rh IL-1 beta significantly reduced IL-1 beta-induced hemorrhage by 78%, and monocyte and neutrophil infiltration by 53% and 62%, respectively. In contrast, TGF beta did not reduce the IL-1 beta-induced increase in vascular permeability. However, TGF beta by itself caused a statistically significant increase in serum proteins in perfused tissues of the eye, to give a 3.1 +/- 0.4 fold increase in protein content over control values. No cellular inflammation accompanied this alteration in vascular permeability. These data indicate that whereas the local administration of TGF beta may be an effective inhibitor of cellular inflammation in the CNS, the effects on alterations in vascular permeability and accumulation of serum proteins may be more complex.

Topics: Animals; Blood Proteins; Blood-Retinal Barrier; Capillary Permeability; Chemotaxis, Leukocyte; Female; Immunosuppressive Agents; Interleukin-1; Leukocyte Count; Rabbits; Retinal Hemorrhage; Retinal Vessels; Retinitis; Transforming Growth Factor beta

The immunoreactivity of types I, III and V collagen, fibronectin and transforming growth factor-beta (TGF-beta) was studied by an immunogold labeling method in retinal vessels of rats with experimental autoimmune uveoretinitis (EAU) induced by retinal S-antigen. The basal lamina of retinal capillaries in normal rats showed low immunoreactivities for the extracellular matrix components and TGF-beta. However, at the peak of inflammation (day 13-16 postimmunization), labeling of all three types of collagen and fibronectin increased considerably in the basal lamina of retinal vessels. TGF-beta immunoreactivity was detected mainly in the vascular endothelium and the pericytes. The results suggest that TGF-beta synthesized by the endothelial cells and pericytes may regulate the synthesis and composition of extracellular matrix components in the vascular basal lamina during the course of EAU development.

Topics: Animals; Antigens; Arrestin; Autoantigens; Autoimmune Diseases; Collagen; Endothelium, Vascular; Eye Proteins; Female; Fibronectins; Immunohistochemistry; Membrane Proteins; Microscopy, Immunoelectron; Phosphodiesterase Inhibitors; Rats; Rats, Inbred Lew; Retinal Vessels; Retinitis; Transforming Growth Factor beta; Uveitis