RESUMO
There are no studies so far linking molecular regulation of lymphangiogenesis and induction of adaptive immunity. Here, we show that blockade of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling significantly suppresses corneal antigen-presenting (dendritic) cell trafficking to draining lymph nodes, induction of delayed-type hypersensitivity and rejection of corneal transplants. Regulating the function of VEGFR-3 may therefore be a mechanism for modulating adaptive immunity in the periphery.
Assuntos
Transplante de Córnea/imunologia , Regulação da Expressão Gênica/imunologia , Hipersensibilidade Tardia/imunologia , Ceratite/imunologia , Linfangiogênese/imunologia , Transdução de Sinais/imunologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Células Dendríticas/imunologia , Citometria de Fluxo , Glicoproteínas , Rejeição de Enxerto/imunologia , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Microscopia de Fluorescência , Modelos Animais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte VesicularRESUMO
A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação/fisiologia , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Epitélio Pigmentado Ocular/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-1/fisiologia , Antígeno B7-2 , Antígeno CTLA-4 , Células Cultivadas , Citocinas/biossíntese , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Pigment dispersion syndrome causes iris pigment release and often progresses to elevated intraocular pressure and pigmentary glaucoma (PG). Because melanin pigment can have adjuvant like properties and because the Gpnmb gene, which contributes to pigment dispersion in DBA/2J (D2) mice, is expressed in dendritic cells, we tested the hypothesis that ocular immune abnormalities participate in PG phenotypes. Strikingly, we show that D2 eyes exhibit defects of the normally immunosuppressive ocular microenvironment including inability of aqueous humor to inhibit T cell activation, failure to support anterior chamber (AC)-associated immune deviation, and loss of ocular immune privilege. Histologic analysis demonstrates infiltration of inflammatory leukocytes into the AC and their accumulation within the iris, whereas clinical indications of inflammation are typically very mild to undetectable. Importantly, some of these abnormalities precede clinical indications of pigment dispersal, suggesting an early role in disease etiology. Using bone marrow chimeras, we show that lymphohematopoietic cell lineages largely dictate the progression of pigment dispersion, the ability of the eye to support induction of AC-associated immune deviation, and the integrity of the blood/ocular barrier. These results suggest previously unsuspected roles for bone marrow-derived cells and ocular immune privilege in the pathogenesis of PG.
Assuntos
Células da Medula Óssea/fisiologia , Olho/imunologia , Glaucoma de Ângulo Aberto/etiologia , Tolerância Imunológica , Animais , Câmara Anterior/imunologia , Humor Aquoso/imunologia , Movimento Celular , Feminino , Iris/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pigmentos da Retina/metabolismo , Receptor fas/análiseRESUMO
In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.
Assuntos
Antígeno CD11b/imunologia , Córnea/imunologia , Córnea/patologia , Inflamação/metabolismo , Linfangiogênese/fisiologia , Macrófagos/imunologia , Animais , Córnea/citologia , Córnea/fisiologia , Transplante de Córnea , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicoproteínas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Neovascularização Fisiológica/fisiologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Lymphangiogenesis, an important initial step in tumor metastasis and transplant sensitization, is mediated by the action of VEGF-C and -D on VEGFR3. In contrast, VEGF-A binds VEGFR1 and VEGFR2 and is an essential hemangiogenic factor. We re-evaluated the potential role of VEGF-A in lymphangiogenesis using a novel model in which both lymphangiogenesis and hemangiogenesis are induced in the normally avascular cornea. Administration of VEGF Trap, a receptor-based fusion protein that binds and neutralizes VEGF-A but not VEGF-C or -D, completely inhibited both hemangiogenesis and the outgrowth of LYVE-1(+) lymphatic vessels following injury. Furthermore, both lymphangiogenesis and hemangiogenesis were significantly reduced in mice transgenic for VEGF-A(164/164) or VEGF-A(188/188) (each of which expresses only one of the three principle VEGF-A isoforms). Because VEGF-A is chemotactic for macrophages and we demonstrate here that macrophages in inflamed corneas release lymphangiogenic VEGF-C/VEGF-D, we evaluated the possibility that macrophage recruitment plays a role in VEGF-A-mediated lymphangiogenesis. Either systemic depletion of all bone marrow-derived cells (by irradiation) or local depletion of macrophages in the cornea (using clodronate liposomes) prior to injury significantly inhibited both hemangiogenesis and lymphangiogenesis. We conclude that VEGF-A recruitment of monocytes/macrophages plays a crucial role in inducing inflammatory neovascularization by supplying/amplifying signals essential for pathological hemangiogenesis and lymphangiogenesis.
Assuntos
Neovascularização da Córnea/metabolismo , Linfangiogênese/fisiologia , Macrófagos/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The immune privilege of the anterior chamber of the eye has been recognized for over 100 years. However, the unique immunological properties of the pigmented epithelial (PE) cells of the eye and the subretinal space (SRS) have only recently been appreciated. The PE cells of the iris, ciliary body, and retina reside in anatomically disparate locations and serve distinctly different functions, yet share interesting immunomodulatory properties that contribute to ocular immune privilege. PE cells in the ciliary body and retina elaborate a variety of soluble factors that either directly or indirectly dampen immune-mediated inflammation; these include transforming growth factor-Beta, somatostatin, thrombospondin and pigment epithelial derived factor (PEDF). The constitutive expression of the immune co-stimulatory molecule, CD86, on iris PE cells not only inhibits T cell proliferation, but also promotes the generation of regulatory T cells. The SRS is now recognized as an immune-privileged site that shares many, but not all, of the properties ascribed to the anterior chamber, including the induction of systemic immune deviation. The prospect of therapeutic retinal transplantation and the possible immunologic etiology for some forms of age-related macular degeneration provides new impetus for gaining a better understanding of ocular immune privilege in the posterior regions of the eye.
Assuntos
Tolerância Imunológica , Epitélio Pigmentado Ocular/imunologia , Animais , Humanos , Ativação Linfocitária , Linfócitos T/imunologia , Trombospondina 1/fisiologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
PURPOSE: The peripheral tolerance that arises after injection of antigen into the anterior chamber (anterior chamber-associated immune deviation; ACAID) is associated in part with CD8+ T cells that suppress the expression of Th1 and Th2 immunity. The purpose of these studies was to determine the genes and molecules that are critical for CD8+ T regulatory cell (T reg) functions in ACAID. METHODS: Ovalbumin (OVA)-specific CD8+ T cells from T-cell receptor (TCR) transgenic OT-1 mice acquire efferent regulatory properties similar to in vivo-generated CD8+ T regs after stimulation with OVA-pulsed TGF-beta2-treated APCs. Changes in the genetic program associated with acquisition of efferent regulatory function in OT-1 CD8+ T cells in vitro were determined by DNA microarray analyses and confirmed by RT-PCR analyses and biological assays. RESULTS: T regulatory OT-1 T cells acquired a novel transcriptional pattern indicative of their function. Genes for molecules associated with TGF-beta function, resistance to TCR-triggered apoptosis, and localization of cells to antigen deposition in peripheral tissues were upregulated, and genes related to cytolytic function were downregulated. Further study showed that CD103, a cell-adhesion molecule that binds E-cadherin, was highly upregulated in in vivo-generated ACAID T regs and was necessary for their suppression of T-cell activation in vitro. CONCLUSIONS: OT-1 CD8 T cells modulated in vitro by exposure to antigen-pulsed, TGF-beta2-treated APCs expressed genes related to immune suppression. Thus, the necessity for CD103 emerges in the efferent CD8+ T-cell regulatory mechanisms in eye-derived tolerance.
Assuntos
Antígenos CD/genética , Linfócitos T CD8-Positivos/fisiologia , Tolerância Imunológica/genética , Cadeias alfa de Integrinas/genética , Linfócitos T Reguladores/fisiologia , Células Th1/imunologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Hipersensibilidade Tardia/imunologia , Imunidade , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta2 , Regulação para CimaRESUMO
PURPOSE: To study the contribution of murine retinal pigment epithelial (RPE) cells to the innate immune-privilege status of the subretinal space as determined by the ability of pigment epithelial-derived factor (PEDF) and somatostatin (SOM), produced by RPE, to regulate macrophage-mediated inflammation. METHODS: Serum-free medium was added to RPE eyecups (a healthy monolayer of RPE resting on choroid and sclera) and the supernatants were removed after 24 hours (RPE SN). The RPE SN was assayed for the presence of PEDF and SOM and for its ability to regulate interleukin (IL)-12, IL-10, and nitric oxide (NO) production by resting and activated macrophages. A group of mice received intradermal injection of lipopolysaccharide (LPS) and PEDF in one ear and LPS alone in the other ear. Ear thickness was measured before- and 24 hours after ear injections. RESULTS: Soluble factors present in the RPE SN inhibited IL-12 production and substantially increased IL-10 while having minimal effects on NO production by activated macrophages. The message for PEDF, SOM, and IL-10 was detected in RPE cells, and the protein for these factors was found in the RPE SN. The stimulation of IL-10 and suppression of IL-12 production by RPE-SN-treated macrophages was neutralized by anti-PEDF antibodies. Neutralization of SOM in the RPE SN, suppressed NO production by activated macrophages. Intradermal injection of PEDF substantially inhibited LPS-induced inflammatory response. CONCLUSIONS: PEDF inhibits LPS-driven macrophage activation in vitro and in vivo. By producing PEDF, the RPE contributes to innate immune privilege of the eye.
Assuntos
Proteínas do Olho/farmacologia , Inflamação/prevenção & controle , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Serpinas/farmacologia , Animais , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/metabolismo , Immunoblotting , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Óxido Nítrico/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/metabolismo , Somatostatina/metabolismoRESUMO
PURPOSE: Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells. METHODS: Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice. RESULTS: OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells. CONCLUSIONS: Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.
Assuntos
Antígenos CD4/imunologia , Tolerância Imunológica/fisiologia , Receptores de Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Câmara Anterior/imunologia , Células Apresentadoras de Antígenos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Hipersensibilidade Tardia/imunologia , Ativação Linfocitária/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/farmacologiaRESUMO
PURPOSE: To determine whether iris PE (IPE) promotes the generation of regulatory T-cells (Tregs) with cell contact via B7-2/CTLA-4 interactions. METHODS: T cells were cocultured with IPE cells obtained from eyes of normal and B7-deficient mice, x-irradiated, and used as regulators. IPE T regulator cells (IPE Tregs) of normal and CD28- or CTLA-4-deficient mice were established. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by [3H]-thymidine incorporation. Neutralizing anti-B7-1 and/or B7-2 antibodies, anti-CTLA-4 antibodies, CTLA-4-Ig fusion proteins were used to abolish regulatory function. IPE-exposed CD8+ T cells were evaluated for expression of B7, CTLA-4, and Foxp3 by using RT-PCR and flow cytometry. CD8+ IPE Tregs were depleted of B7-2+ and CTLA-4+ T cells and assayed for suppressive activity by adding them to bystander T cells. RESULTS: T cells acquired T regulatory activity when exposed to cultured IPE. Ciliary body PE cells did not promote conversion of T cells into Tregs. IPE converted CD8+, but not CD4+, T cells into Tregs by direct cell contact. In the conversion, IPE and responding T cells must both express endogenously synthesized B7-1 and B7-2, and the T cells must also express CTLA-4. Expression of CD28 molecules was not necessary for Treg generation. In addition, the CD8+ Tregs that fully suppress activation of bystander T cells expressed Foxp3. CONCLUSIONS: IPE cells promote conversion of T cells into Tregs solely through a contact-dependent mechanism. T cells exposed to IPE cells acquire full regulatory capacity.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígeno B7-2/imunologia , Linfócitos T CD8-Positivos/imunologia , Iris/citologia , Ativação Linfocitária/imunologia , Epitélio Pigmentado Ocular/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Diferenciação/fisiologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4 , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To study the time course of angiogenesis and lymphangiogenesis in the cornea after a short inflammatory insult. This might be helpful for the timing of corneal transplantation in high-risk eyes. METHODS: The mouse model of suture-induced inflammatory corneal neovascularization was used. After placement of 3 interrupted 11-0 sutures into the corneal stroma of BALB/c mice (left in place for 14 days), corneas were excised 2, 3, 5, 7, 14, and 21 days as well as 1, 2, 3, 6, and 8 months after surgery. Hem- and lymphangiogenesis were evaluated using double immunohistochemistry of corneas with CD31/PECAM1 as panendothelial and LYVE-1 as lymphatic endothelial marker. RESULTS: Both blood and lymphatic vessels grew into the cornea as early as day 2 after suture placement. The outgrowth was initially parallel. Hem- and lymphangiogenesis peaked around day 14. Thereafter, both vessel types started to regress. Regression of lymphatic vessels started earlier and was more pronounced than that of blood vessels. Whereas at 6 and 8 months (partly) perfused CD31+++/LYVE-1(-) blood vessels and (nonperfused) ghost vessels could still be observed, there were no CD31+/LYVE-1+++ lymphatic vessels detectable beyond 6 months after this short inflammation. CONCLUSIONS: After a temporary inflammatory insult to the cornea, there is initially parallel outgrowth of both blood and lymphatic vessels. But thereafter, lymphatic vessels regress earlier than blood vessels and are completely regressed by 6 months. Earlier regression of pathologic corneal lymph versus blood vessels suggests that corneal graft survival in high-risk eyes might best be delayed for a prolonged interval following an inflammatory insult.
Assuntos
Córnea/irrigação sanguínea , Neovascularização da Córnea/metabolismo , Ceratite/metabolismo , Linfangiogênese , Vasos Linfáticos/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glicoproteínas/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos BALB C , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Because guinea pig corneal xenografts are rejected acutely (within 16 days) in mouse eyes by a T-cell-dependent mechanism, the authors wished to determine the functional phenotype of CD4+ effector T cells. METHODS: Orthotopic corneal xenotransplantation was performed from strain 13 guinea pigs to BALB/c mice. Grafted eyes were removed at specified times and examined histologically or subjected to cytokine and chemokine mRNA analysis using a multi-probe ribonuclease protection assay. Draining cervical lymph node cells were harvested at specified times and stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells. Supernatants were assayed by enzyme-linked immunosorbent assay for content of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, and IL-10 and cells were used for mRNA analysis. RESULTS: Rejected corneal xenografts were heavily infiltrated with polymorphonuclear leukocytes, the majority of which were eosinophils. These eyes contained mRNA for IL-4, IL-6, IL-10, IL-13, IL-15, and IFN-gamma. When stimulated with guinea pig spleen cells, T cells from draining cervical lymph nodes secreted primarily IL-4, IL-5, IL-10, and IFN-gamma. Eotaxin was overexpressed in eyes with rejected corneal xenografts. CONCLUSIONS: Acute rejection of corneal xenografts in mice is mediated by T cells that display a mixed T-helper (Th) type 2/Th1 phenotype and secrete eotaxin, an eosinophil chemoattractant. Eosinophil-dependent xenograft rejection bears similarities to immune elimination of parasites.
Assuntos
Transplante de Córnea , Citocinas/metabolismo , Eosinófilos , Rejeição de Enxerto/etiologia , Células Th2/metabolismo , Transplante Heterólogo , Doença Aguda , Animais , Antígenos Heterófilos/metabolismo , Córnea/patologia , Citocinas/biossíntese , Citocinas/genética , Olho/metabolismo , Expressão Gênica , Cobaias , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , PescoçoRESUMO
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Sistema Imunitário/fisiologia , Epitélio Pigmentado Ocular/imunologia , Linfócitos T/imunologia , Trombospondina 1/fisiologia , Animais , Doenças Autoimunes/imunologia , Ensaio de Imunoadsorção Enzimática , Interferon gama/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retinite/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta2 , Uveíte/imunologiaRESUMO
PURPOSE: To determine the extent to which expression of major histocompatibility complex (MHC) class I and II molecules contributes to rejection of orthotopic corneal transplants in mice. METHODS: Full-thickness corneas, prepared from eyes of normal C57BL/6 (B6) and BALB/c mice and from B6 mice in which the class II gene I-A or the beta2-microglobulin (beta2mu) gene was disrupted, were placed orthotopically in low- or high-risk eyes of BALB/c (fully incompatible), BALB.B [minor histoincompatible (H) only], and bm12 (class II only disparate) recipients. BALB/c grafts were placed in low-risk eyes of normal B6 and B6 mice with disrupted H-2 DMalpha genes. Graft survival was judged by clinical examination. RESULTS: Recipient-identical class II, but not class I, molecules on fully allogeneic corneas grafted to low-risk beds promoted graft rejection. Allogeneic class II molecules on fully allogeneic corneas placed in high-risk beds promoted graft rejection more strongly than did allogeneic class I molecules. Neither allogeneic class I molecules nor recipient-identical class II molecules on grafts placed in high-risk beds contributed to graft outcome. Mice deficient in H-2 DMalpha failed to reject fully incompatible cornea grafts. CONCLUSIONS: On corneal allografts, where minor H antigens are the major barriers to acceptance, allogeneic class II molecules promote rejection if the graft is placed in high-risk eyes, whereas recipient-type class II molecules promote rejection if the graft is placed in low-risk eyes. Allogeneic class I molecules make a minor contribution to rejection only if the grafts are placed in high-risk eyes.
Assuntos
Córnea/imunologia , Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Ceratoplastia Penetrante , Animais , Modelos Animais de Doenças , Expressão Gênica , Sobrevivência de Enxerto/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação Puntual , Linfócitos T/imunologiaRESUMO
The delicate visual axis that makes precise vision possible is highly vulnerable to the destructive potential of immunogenic inflammation. Immune privilege of the eye is the experimental expression of the way in which evolution has coped with the countermanding threats to vision of ocular infections and ocular immunity and inflammation. Ocular immune privilege has five primary features that account for its existence: blood:ocular barriers, absent lymphatic drainage pathways, soluble immunomodulatory factors in aqueous humor, immunomodulatory ligands on the surface of ocular parenchymal cells, and indigenous, tolerance-promoting antigen-presenting cells (APCs). Three manifestations of ocular immune privilege that have received the most extensive study are the intraocular microenvironment, which is selectively anti-inflammatory and immunosuppressive; the prolonged acceptance of solid tissue and tumor allografts in the anterior chamber; and the induction of systemic tolerance to eye-derived antigens. Anterior chamber-associated immune deviation is known to arise when indigenous, ocular APCs capture eye-derived antigens and deliver them to the spleen where multicellular clusters of these cells, natural killer T cells, marginal zone B cells, and gammadelta T cells create an antigen-presentation environment that leads to CD4(+) and CD8(+) alpha/beta T cells, which as regulators, suppress induction and expression of T helper cell type 1 (Th1) and Th2 immune expression systems. The ways the eye influences local and systemic immune responses to ocular antigens and pathogens carry risks to and benefits for mammalian organisms. As loss of sight is a powerful, negative-selecting force, the benefits of ocular immune privilege outweigh the risks.
Assuntos
Olho/imunologia , Uveíte Anterior/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Imunidade Celular , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Trombospondinas/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
PURPOSE: To examine the effects of intravitreal Mycobacteria tuberculosa adjuvant (MTA) on ocular immune privilege. METHODS: MTA was injected into the vitreous cavity of BALB/c mouse eyes to induce anterior uveitis. The inflamed eyes were then examined for their capacity to afford immune privilege to injected allogeneic tumor cells and to promote anterior chamber-associated immune deviation (ACAID). Aqueous humor (AqH) was tested for IL-12 content and for its ability to inhibit T-cell activation. RESULTS: AqH removed from MTA-inflamed eyes at 6 and 12 h contained high levels of IL-12, which then fell almost to baseline at 24 h. This is relevant to the finding that the inflamed eye failed to support ACAID induction at an early time period and then regained the ACAID-induction capability at a later time. Nonetheless, AqH removed from MTA-inflamed eyes retained its capacity to suppress T-cell activation, and MTA-inflamed eyes afforded extended survival to alloantigenic tumor cells implanted into the anterior chamber. CONCLUSION: Intraocular inflammation evoked by MTA causes the local accumulation of IL-12 and simultaneously robs the eye of its capacity to promote systemic immune tolerance to eye-derived antigens. However, MTA-inflamed eyes retain immune privilege, as indicated by their support of the progressive growth of allogeneic tumor cells.
Assuntos
Tolerância Imunológica , Uveíte Posterior/imunologia , Adjuvantes Imunológicos/toxicidade , Animais , Câmara Anterior , Humor Aquoso/imunologia , Humor Aquoso/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Tolerância Imunológica/fisiologia , Técnicas In Vitro , Injeções , Interleucina-12/metabolismo , Ativação Linfocitária/imunologia , Mastocitoma/imunologia , Mastocitoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Ovalbumina/administração & dosagem , Ovalbumina/toxicidade , Linfócitos T/imunologia , Tuberculose Ocular/imunologia , Tuberculose Ocular/microbiologia , Tuberculose Ocular/patologia , Uveíte Posterior/metabolismo , Uveíte Posterior/patologia , Corpo VítreoRESUMO
PURPOSE: To determine whether the MHC class Ib gene, Qa-1, is expressed in the tissues that surround the immune-privileged anterior chamber (AC) of the murine eye. METHODS: Transcription of Qa-1 mRNA in BALB/c ocular tissues was analyzed by reverse transcription-polymerase chain reaction. Expression of Qa-1 protein was assessed on ocular frozen tissue sections by immunohistochemistry, and within aqueous humor by western blotting. RESULTS: Transcription of Qa-1 was found in all tissues surrounding the AC of the eye. Immunohistological staining revealed Qa-1 expression on corneal endothelium, corneal epithelium, and lens epithelium. No soluble Qa-1 was detected in aqueous humor. CONCLUSIONS: Qa-1, unlike other MHC class I molecules, is ubiquitously expressed in tissues surrounding the AC of the eye, raising the possibility that Qa-1 plays a role in creating and maintaining ocular immune privilege.
Assuntos
Endotélio Corneano/metabolismo , Epitélio Corneano/metabolismo , Epitélio/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Cristalino/citologia , Animais , Câmara Anterior/imunologia , Western Blotting , Linhagem Celular , Transformação Celular Viral , Corpo Ciliar/metabolismo , Expressão Gênica , Antígenos H-2/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Imuno-Histoquímica , Iris/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To determine the immunopathogenesis of delayed orthotopic corneal xenograft rejection in mice deficient in the xenoreactive CD4+ T cells that mediate acute rejection. METHODS: CB.17 SCID and BALB/c mice were used as recipients of orthotopic cornea grafts obtained from strain 13 guinea pigs. Before transplantation, SCID recipients, which do not normally reject guinea pig cornea grafts, were reconstituted with spleen cells (whole, CD4-depleted, CD4/CD8-depleted) or purified CD8+ T cells from normal BALB/c donors. Graft survival was assessed by clinical examination, and median survival times (MST) were calculated. Lymphocytes from mice that rejected guinea pig cornea grafts were analyzed in vitro for their capacity to respond to guinea pig xenoantigens and to lyse guinea pig target cells. RESULTS: SCID mice reconstituted with whole spleen cells from BALB/c donors rejected guinea pig corneas with a vigor identical with that of normal BALB/c mice (MST = 15 and 14 days, respectively), whereas SCID mice reconstituted with CD4-depleted BALB/c spleen cells rejected guinea pig corneas in a delayed fashion (MST = 27 days), as did SCID mice reconstituted with purified CD8+ T cells from BALB/c donors. Although CD8+ T cells from rejector mice failed to lyse guinea pig target cells in vitro, the T cells proliferated and secreted IFN-gamma in response to in vitro stimulation with guinea pig xenoantigens. CONCLUSIONS: Guinea pig cornea xenografts that avoid acute rejection in CD4+ T cell-depleted mice are vulnerable to rejection by CD8+ T cells. Effector CD8+ T cells destroy corneal xenografts through release of proinflammatory mediators (IFN-gamma) rather than by cytotoxicity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Córnea/imunologia , Transplante de Córnea/imunologia , Rejeição de Enxerto/imunologia , Animais , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Sobrevivência de Enxerto/imunologia , Cobaias , Ativação Linfocitária , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Imunodeficiência Combinada Severa/imunologia , Baço/imunologia , Transplante HeterólogoRESUMO
PURPOSE: In low-risk eyes of mice, most of the composite corneal grafts composed of syngeneic epithelium layered on allogeneic stroma and endothelium are accepted indefinitely. The study was undertaken to determine the fate of similar composite corneal grafts placed in high-risk mouse eyes. METHODS: Epithelium-deprived allogeneic corneas (C57BL/6) were reconstituted in vitro with BALB/c epithelium, and then transplanted orthotopically into high-risk eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. Acquisition of donor-specific delayed hypersensitivity (DH) was also assessed in recipient mice. Recipients bearing healthy composite grafts were immunized subcutaneously with injected C57BL/6 spleen cells at 2 or 8 weeks after grafting, after which the fate of the grafts was evaluated. RESULTS: Virtually all epithelium-deprived corneal allografts reconstituted in vitro with normal BALB/c corneal epithelium survived indefinitely when placed in high-risk eyes of BALB/c mice. Recipients of these composite grafts failed to acquire donor-specific DH when tested at both 2 and 8 weeks after grafting. Moreover, these recipients did not acquire the capacity to actively suppress donor-specific DH. Within 1 to 3 weeks of sensitization of recipient mice with spleen cells of donor origin, healthy composite grafts in residence for 2 or 8 weeks were rejected. CONCLUSIONS: Replacement of donor epithelium with syngeneic epithelium protects orthotopic allogeneic corneal grafts (stroma plus endothelium) placed in high-risk eyes from sensitizing their recipients and from immune-mediated rejection. Recipients of composite corneal grafts containing syngeneic epithelial layers act as though they are immunologically ignorant of the graft's presence.