RESUMEN

Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have previously shown that NKT cell-deficient mice develop significantly fewer liver metastases from intraocular melanomas than do wild-type (WT) mice. Here, we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d(-/-) mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. The increased number of metastases in WT mice was associated with reduced liver NK cytotoxicity and decreased production of IFN-γ. However, liver NK cell-mediated cytotoxic activity was identical in non-tumor bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for the suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with production of IL-10 by bone marrow-derived liver cells that were neither Kupffer cells nor myeloid-derived suppressor cells and by increased IL-10 receptor expression on liver NK cells. IL-10(-/-) mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. This impairment of liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases.

Asunto(s)

Antígenos CD1d/metabolismo , Células Asesinas Naturales/fisiología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Melanoma/inmunología , Células T Asesinas Naturales/fisiología , Neoplasias de la Úvea/inmunología , Animales , Anticuerpos/farmacología , Antígenos CD1d/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Neoplasias Hepáticas/patología , Activación de Linfocitos , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Receptores de Interleucina-10/metabolismo , Neoplasias de la Úvea/patología

RESUMEN

PURPOSE: Expression of the chemokine receptor CXCR4 by tumors is associated with metastatic migration and invasion of tumor cells. The importance of CXCR4 expression by uveal melanomas in metastasis to the liver was recently demonstrated when injection of CXCR4-negative uveal melanoma cells into mice resulted in reduced liver metastasis compared with CXCR4-positive uveal melanoma cells. Factors in the eye can induce downregulation of genes by epigenetic mechanisms. This study examined whether epigenetic regulation by the ocular environment induced downregulation of CXCR4 expression. METHODS: LS174T colon cancer cells were injected in the anterior chamber (AC), subcutaneously (SC), or in the spleen capsule to induce liver metastasis in immune-deficient mice. CXCR4 gene transcription was analyzed by RT-PCR, and protein expression was determined by flow cytometry. Methyltransferase and histone deacetylase activities were determined by ELISA. Treatment with either 5-Aza-2-deoxycytidine (5-Aza) or trichostatin A (TSA) was used to induce demethylation or inhibit histone deacetylases, respectively. RESULTS: AC-derived LS174T cells showed lower CXCR4 gene expression compared with SC-, liver-derived, or wild-type tumor cells. AC-derived LS174T tumor cells expressed methyltransferase activity compared with SC-, liver-derived, and wild-type tumor cells. Deacetylase activity was elevated in AC-derived LS174T tumor cells compared with SC-derived, liver-derived, and wild-type tumor cells. Treatment of AC-derived LS174T tumor cells with 5-Aza upregulated CXCR4 expression. TSA treatment did not restore CXCR4 expression. CONCLUSIONS: These studies demonstrate that ocular microenvironment factors induce methylation and downregulation of tumor CXCR4 expression.

Asunto(s)

Cámara Anterior/metabolismo , Epigénesis Genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Animales , Cámara Anterior/patología , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Ciego/metabolismo , Ciego/patología , Línea Celular Tumoral , Decitabina , Regulación hacia Abajo , Citometría de Flujo , Expresión Génica , Regulación Neoplásica de la Expresión Génica/fisiología , Inhibidores de Histona Desacetilasas/administración & dosificación , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ácidos Hidroxámicos/administración & dosificación , Inyecciones , Inyecciones Subcutáneas , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Receptores CXCR4/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Bazo/patología , Regulación hacia Arriba

RESUMEN

Intraocular tumors reside in an immune-privileged site, yet in certain circumstances, they can undergo immune rejection. Ocular tumor rejection can follow one of two pathways. One pathway is CD4(+) T cell-dependent and culminates in ischemic necrosis of the tumor and phthisis (atrophy) of the eye. A second pathway is also CD4(+) T cell-dependent but does not inflict collateral injury to ocular tissues, and the eye is preserved. We isolated two clones of a murine tumor, Ad5E1 that undergo profoundly different forms of immune rejection in the eye. Clone 2.1 tumors undergo an ischemic necrotizing form of rejection that requires IFN-γ, T cells, and ocular macrophages and culminates in destruction of the eye. By contrast, the second clone of Ad5E1, clone 4, undergoes rejection that also requires T cells and ocular macrophages, but leaves the eye in pristine condition (nonphthisical rejection). Here, we demonstrate that nonphthisical tumor rejection of clone 4 tumors is IFN-γ-independent but requires an ocular macrophage population that contains M1 and M2 macrophages. Clone 4 tumor-bearing eyes displayed ten- and 15-fold increases in M2- and M1-associated markers Arg1 and NO2, respectively. This is in sharp contrast to previous results with clone 2.1 tumor rejection, in which M2 markers were undetectable, and the eye was destroyed. These results suggest that the presence of M2 macrophages tempers the immune rejection of intraocular tumors and promotes immune effectors that inflict minimal injury to innocent bystander cells and thereby preserve the integrity and function of the eye.

Asunto(s)

Neoplasias del Ojo/inmunología , Neoplasias del Ojo/patología , Interferón gamma/inmunología , Macrófagos/inmunología , Animales , Modelos Animales de Enfermedad , Ojo/patología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Introduction of alloantigens into the AC induces a form of immune tolerance known as ACAID, which induces antigen-specific CD8+ Tregs, contributing to ocular immune privilege by down-regulating immune responses. Recent evidence suggests IFN-γ is needed for the suppressive function of CD8+ ACAID Tregs. This study tested the hypothesis that IFN-γ is needed for alloantigen-specific ACAID CD8+ Tregs to execute their suppressive function but is not required for the establishment of ACAID CD8+ Tregs. To address this hypothesis, ACAID was induced by injecting BALB/c spleen cells into the AC of WT C57BL/6 mice, IFN-γ(-/-) C57BL/6 mice, or anti-IFN-γ-treated WT C57BL/6 mice. LAT assays using C57BL/6 APCs as stimulators, CD4+ T cells from C57BL/6 mice previously immunized toward BALB/c alloantigens as effector cells, and IFN-γ-competent, IFN-γ(-/-), or IFN-γR(-/-) CD8+ Tregs were used to evaluate the suppressive function of CD8+ ACAID Tregs in response to IFN-γ. IFN-γ(-/-) mice or mice treated with anti-IFN-γ antibody prior to AC injection of alloantigen failed to develop ACAID. The suppressive function of IFN-γ(-/-) ACAID CD8+ Tregs was restored through the administration of exogenous IFN-γ. This suppressive responsiveness toward IFN-γ was CD8+ Treg-intrinsic, as CD8+ Tregs from IFN-γR(-/-) mice, which were primed in the AC with alloantigens, were not able to suppress alloantigen-specific DTH responses. These results indicate that IFN-γ is not needed for the induction of CD8+ ACAID Tregs but is required for ACAID Tregs to exert the suppression of allospecific DTH responses.

Asunto(s)

Cámara Anterior/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/metabolismo , Tolerancia Inmunológica , Interferón gamma/genética , Isoantígenos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/metabolismo

RESUMEN

Although intraocular tumors reside in an immune-privileged site where immune responses are suppressed, some tumors are rejected. An example of this is the rejection of intraocular adenovirus-induced (adenovirus type 5 early region 1 [Ad5E1]) tumors in C57BL/6 mice. We previously identified an Ad5E1 tumor clone in which the rejection is IFN-γ dependent and culminates in the destruction of both the tumor and the eye. Although Ad5E1 tumors are not rejected when transplanted into the eyes of IFN-γ KO mice, they are rejected after s.c. transplantation. Thus, outside of the eye Ad5E1 tumors elicit a form of tumor immunity that is IFN-γ independent. In this article, we demonstrate that IFN-γ-independent s.c. rejection requires both CD4(+) and CD8(+) T cells. Furthermore, s.c. tumor rejection requires IL-17, which is produced by IFN-γ-deficient CD4(+) T cells in response to tumor Ags (TAs). Splenocytes from CD4-depleted IFN-γ KO mice produce significantly less IL-17 compared with splenocytes from isotype-treated IFN-γ KO animals in response to TAs. Furthermore, depletion of IL-17 decreases CTL activity against Ad5E1 tumor cells. In this model we propose that, in the absence of IFN-γ, CD4(+) T cells produce IL-17 in response to TAs, which increases CTL activity that mediates tumor rejection; however, this does not occur in the eye. IL-6 production within the eye is severely reduced, which is consistent with the failure to induce Th17 cells within the intraocular tumors. In contrast, the s.c. environment is replete with IL-6 and supports the induction of Th17 cells. Therefore, IFN-γ-independent tumor rejection is excluded from the eye and may represent a newly recognized form of ocular immune privilege.

Asunto(s)

Ojo/inmunología , Interferón gamma/inmunología , Interleucina-17/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

IL-17A is a proinflammatory cytokine that has received attention for its role in the pathogenesis of several autoimmune diseases. IL-17A has also been implicated in cardiac and renal allograft rejection. Accordingly, we hypothesized that depletion of IL-17A would enhance corneal allograft survival. Instead, our results demonstrate that blocking IL-17A in a mouse model of keratoplasty accelerated the tempo and increased the incidence of allograft rejection from 50 to 90%. We describe a novel mechanism by which CD4(+)CD25(+) regulatory T cells (Tregs) respond to IL-17A and enhance corneal allograft survival. Our findings suggest the following: 1) IL-17A is necessary for ocular immune privilege; 2) IL-17A is not required for the induction of anterior chamber-associated immune deviation; 3) Tregs require IL-17A to mediate a contact-dependent suppression; 4) corneal allograft Tregs suppress the efferent arm of the immune response and are Ag specific; 5) Tregs are not required for corneal allograft survival beyond day 30; and 6) corneal allograft-induced Treg-mediated suppression is transient. Our findings identify IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4(+)CD25(+) Tregs is necessary for survival of corneal allografts.

Asunto(s)

Trasplante de Córnea/métodos , Supervivencia de Injerto/inmunología , Inmunosupresores , Interleucina-17/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos , Rechazo de Injerto/inmunología , Fenómenos del Sistema Inmunológico , Interleucina-17/farmacología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones

RESUMEN

PURPOSE: To explore the role of natural killer T (NKT) cells in the development of liver metastases in mice harboring intraocular melanomas. METHODS: Cells derived from the cutaneous B16 melanoma cell line (B16LS9) were transplanted either into the vitreous body or under the spleen capsules of wild-type C57BL/6 mice and NKT-cell-deficient Jα18(-/-) and CD1d(-/-) mice. The development of liver metastases was evaluated by histopathology. The effect of NK cells on liver metastases was determined by selective depletion with anti-asialo-GM1 antiserum in vivo and NK-cell-mediated cytolysis of B16LS9 melanoma cells in vitro. The role of IL-10 and transforming growth factor (TGF)-ß in the inhibition of liver NK resistance to liver metastases was determined by in vivo and in vitro neutralization with monoclonal antibodies. RESULTS: Liver NKT cells, especially type I NKT cells, enhanced liver metastases arising from intraocular melanomas. NKT-cell-deficient mice developed significantly fewer liver metastases that were NK-cell dependent. Tumor-induced liver NKT cells, especially type I NKT cells, inhibited liver NK-cell cytotoxicity by an IL-10-dependent process. CONCLUSIONS: NKT cells exert protective effects in many murine tumor models. However, the present results reveal that NKT cells exacerbate liver metastases arising from intraocular melanomas. To the authors' knowledge, this is the first report that liver NKT cells, especially type I NKT cells, inhibit liver NK-cell antimetastatic activity by the production of IL-10. These results suggest that hepatic NKT cell activity can have an important effect in the immune surveillance of liver metastases.

Asunto(s)

Neoplasias del Ojo/patología , Neoplasias Hepáticas/secundario , Melanoma Experimental/secundario , Células T Asesinas Naturales/inmunología , Neoplasias del Bazo/patología , Cuerpo Vítreo/patología , Animales , Citotoxicidad Inmunológica , Ensayo de Inmunoadsorción Enzimática , Neoplasias del Ojo/inmunología , Femenino , Citometría de Flujo , Gangliósido G(M1)/inmunología , Interleucina-10/fisiología , Hígado/inmunología , Neoplasias Hepáticas/inmunología , Masculino , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esplenectomía , Neoplasias del Bazo/inmunología , Factor de Crecimiento Transformador beta/fisiología , Cuerpo Vítreo/inmunología

RESUMEN

Although intraocular tumors reside in an immune privileged site, some tumors are rejected nonetheless. For example, intraocular adenovirus-induced (Ad5E1; adenovirus type 5 early region 1) tumors are rejected in syngeneic C57BL/6 mice by one of two pathways. One pathway leads to extensive necrosis of innocent bystander cells and culminates in destruction of the eye, a condition called phthisis. The second pathway is characterized by piecemeal tumor cell death that rids the eye of the tumor while preserving the architecture and function of the eye. To study the mechanisms of phthisical tumor rejection, we isolated a cell clone-designated clone 2.1 that consistently undergoes rejection in a phthisical manner. CD4(+) T cells and macrophages were required for phthisical rejection of intraocular clone 2.1 tumors and M1 macrophages were involved in mediating tumor rejection. In vitro and in vivo inhibition of iNOS (inducible nitric oxide synthase) abolished macrophage-mediated killing of tumor cells and rejection of intraocular tumors. A role for M1 macrophages was further supported by investigations showing that intraocular tumors grew progressively in IFN-γ KO (knockout) mice. Studies in mice deficient in TNF-α, TNF receptor-1, or TNF receptor-2 revealed that although TNF-α was not needed for tumor rejection, it was required for the development of necrotizing inflammation and phthisis of tumor-bearing eyes. Together, our findings suggest new strategies to successfully eliminate ocular tumors while preserving the integrity of the eye.

Asunto(s)

Neoplasias del Ojo/inmunología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Animales , Transformación Celular Viral , Inhibidores Enzimáticos/farmacología , Neoplasias del Ojo/enzimología , Neoplasias del Ojo/genética , Neoplasias del Ojo/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , NG-Nitroarginina Metil Éster/farmacología , Linfocitos T/inmunología , Transfección , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

PURPOSE: To determine the function of γδ T cells in early- and late-phase responses in allergic conjunctivitis. METHODS: Wild-type (WT) C57BL/6 and γδ T cell-deficient (TCR-δ(-/-)) mice were immunized intraperitoneally and challenged topically for 7 consecutive days with short ragweed pollen. Natural killer T (NKT) and γδ T cell-double-deficient mice were generated by treating TCR-δ(-/-) mice with anti-CD1d antibody. Allergic conjunctivitis was evaluated clinically, and the late-phase response was assessed by histopathology. Cytokine profiles were evaluated by ELISA. The afferent and efferent arms of allergic conjunctivitis were assessed by adoptive transfer of CD4(+) T cells from WT or TCR-δ(-/-) mice into naive TCR-δ(-/-) or WT mice. RESULTS: TCR-δ(-/-) mice had decreased clinical manifestations of allergic conjunctivitis compared with WT mice. TCR-δ(-/-) mice had decreased eosinophilic infiltration compared with WT mice. TCR-δ(-/-) mice produced less Th2-associated cytokines interleukin (IL)-4, -5, and -13 compared with WT mice. Clinical manifestations of allergic conjunctivitis were lowest in NKT cell-depleted TCR-δ(-/-) mice. However, late-phase allergic conjunctivitis in NKT cell-depleted, TCR-δ(-/-) mice was the same as TCR-δ(-/-) mice. Adoptive transfer of CD4(+) T cells revealed that γδ T cells are needed for the afferent and efferent arms of allergic conjunctivitis. CONCLUSIONS: γδ T cells are needed for full expression of both the clinical manifestations and the late phase of allergic conjunctivitis. Thus, γδ T cells have an important impact in the expression of allergic conjunctivitis and are a potential therapeutic target in the management of allergic diseases of the ocular surface.

Asunto(s)

Conjuntivitis Alérgica/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Linfocitos T/inmunología , Traslado Adoptivo , Ambrosia , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunología , Polen , Células Th2/inmunología

RESUMEN

Corneal transplantation is the oldest, most common, and arguably the most successful form of organ transplantation. In uncomplicated first-time cases, corneal allografts enjoy a success rate of up to 90% even though the transplants are performed without HLA matching or the use of systemic immunosuppressive drugs. In rodents, corneal allografts transplanted across entire MHC and multiple minor histocompatibility barriers enjoy long-term survival in >50% of the hosts, while skin grafts invariably undergo immune rejection. These observations are the basis for "immune privilege" of corneal transplants. In spite of this immune privilege, immune rejection can occur and remains the leading cause of corneal graft failure. Rodent models of penetrating keratoplasty have facilitated studies that have challenged, and in some cases, refuted prevailing dogmas. The long-held belief that CD4+ T helper 1 (Th1) cells were the sole mediators of corneal allograft rejection has fallen to the wayside based on studies in interferon-gamma (IFN-γ)⁻(/)⁻ mice. The dogma that biasing the alloimmune response down a Th2 pathway would enhance graft survival has also been disproven, and in fact, compelling evidence indicates that Th2-based immune rejection of corneal allografts is swifter and more intense than Th1-based rejection. Animal studies have also pre-empted emerging dogmas including the hypothesis that Th17 cells play a crucial role in allograft rejection. Instead, IL-17A appears to be necessary for corneal allograft survival. Finally, IFN-γ, and IL-17A, which were normally viewed as proinflammatory, exert the opposite effect in the context of corneal transplantation and are necessary for corneal allograft survival.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Trasplante de Córnea , Animales , Antígenos CD/inmunología , Rechazo de Injerto/inmunología , Humanos , Inmunidad Celular , Interleucina-17/inmunología

RESUMEN

Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, it has been reported that corneal allograft rejection soars in IFN-γ(-/-) mice or mice treated with anti-IFN-γ mAb. Th17 is a recently described IL-17A-producing Th cell population that has been linked to renal and cardiac graft rejection, which was originally thought to be Th1-mediated. We tested the hypothesis that Th17 cells mediate corneal allograft rejection in an IL-17A-dependent fashion and unexpectedly found that depletion of IL-17A increased the incidence of rejection to 90%. We demonstrate that the exacerbated rejection following depletion of IL-17A did not result from a loss of cross-regulation of Th1 cells or exaggerated delayed-type hypersensitivity responses. Instead, inhibition of the Th1 or Th17 cell lineages promoted the emergence of a Th2 cell subset that independently mediated allograft rejection. These findings demonstrate that IL-17A is not required for corneal allograft rejection and may instead contribute to the immune privilege of corneal allografts.

Asunto(s)

Trasplante de Córnea , Rechazo de Injerto/inmunología , Interleucina-17/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Inmunología del Trasplante , Traslado Adoptivo , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Supervivencia de Injerto , Inmunohistoquímica , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Trasplante Homólogo

RESUMEN

PURPOSE: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Asunto(s)

Cámara Anterior/inmunología , Trasplante de Córnea , Supervivencia de Injerto/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos/administración & dosificación , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Conjuntivitis Alérgica/inmunología , Ciclofosfamida/farmacología , Femenino , Supervivencia de Injerto/efectos de los fármacos , Hipersensibilidad Tardía/inmunología , Interferón gamma/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Isoantígenos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Polen , Trasplante Homólogo

RESUMEN

PURPOSE: Complement factor H (Cfh) is a key regulator of the alternative complement pathway. A Cfh variant (Y402H) increases the risk for AMD. The purpose of this study was to develop a pathophysiologically relevant animal model of AMD based on this genetic risk factor. METHODS: The authors generated chimeric Cfh transgenic mouse lines using two constructs consisting of the human CFH sequence for SCR6-8 (with either 402Y or 402H), flanked by the mouse sequence for SCR1-5 and SCR9-20. They tested the expression of the transgenic mRNA and protein molecules and examined the mice at 12 to 14 months of age for clinical and histologic retinal changes. RESULTS: Nuclease protection assay and qRT-PCR analysis demonstrated transgenic mRNA expression in the liver and in the posterior segment of the eye. Western blot analysis showed that the transgenic proteins are present in the circulation at levels comparable to those of mouse Cfh. The chimeric proteins were found to be functional, as demonstrated by their ability to restore physiological serum levels of complement component C3 in Cfh KO mice. Clinical examination showed subretinal drusen-like deposits. Histology demonstrated an accumulation of subretinal cells that stained with a macrophage/microglia marker. Basal laminar deposits, long-spaced collagen, and increased numbers of lipofuscin granules were seen on electron microscopy. Immunohistochemistry showed a thicker sub-RPE band of C3d staining. CONCLUSIONS: Chimeric Cfh proteins led to AMD-like characteristics in mice. This may represent a good model for studying the role of complement and other components of the immune system in early AMD.

Asunto(s)

Factor H de Complemento/genética , Modelos Animales de Enfermedad , Degeneración Macular/genética , Animales , Western Blotting , Quimera , Complemento C3/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Genotipo , Hígado/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Segmento Posterior del Ojo/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo

RESUMEN

Allergic conjunctivitis (AC) is elicited by immediate hypersensitivity responses to environmental agents. It is initiated by a T(h)2-dominated immune response that is characterized by production of IgE antibodies and eosinophilic infiltration. By using an experimental mouse model of AC induced by short ragweed (SRW) pollen, we show that sensitized Jalpha18(-/-) mice, which lack type I NKT cells, and CD1d(-/-) mice, which lack type I and type II NKT cells, exhibited a decrease in tearing, lid edema, conjunctival edema and vasodilatation and eosinophil infiltration into the conjunctiva when compared with wild-type (WT) mice in both T(h)1- and T(h)2-prone hosts (C57BL/6 and BALB/c mice, respectively). This demonstrates that NKT cells are needed for both the early and late phases of AC. Adoptive transfer of SRW-primed CD4(+) T cells from Jalpha18(-/-) mice into naive WT BALB/c mice revealed that NKT cells were needed for the maximal induction of allergen-specific T(h)2 cells. Results from adoptive transfer of SRW-primed CD4(+) T cells from WT BALB/c mice to naive Jalpha18(-/-) mice indicated that NKT cells were also needed for the expression of AC produced by allergen-primed CD4(+) T cells. The decreased expression of AC in NKT cell-deficient mice was correlated with significant reduction in the production of T(h)2 cytokines in SRW pollen-sensitized mice compared with WT mice and in the capacity of SRW pollen-sensitized CD4(+) T cells to mediate ocular inflammation when the hosts were confronted with SRW pollen at the ocular surface.

Asunto(s)

Conjuntivitis Alérgica/inmunología , Células T Asesinas Naturales/inmunología , Animales , Conjuntivitis Alérgica/metabolismo , Conjuntivitis Alérgica/fisiopatología , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Th2/inmunología

RESUMEN

Allergic conjunctivitis (AC) and airway hyperreactivity exacerbate corneal allograft rejection. Because AC and airway hyperreactivity are allergic diseases of mucosal tissues, we determined whether an allergic disease of a nonmucosal tissue would affect corneal allograft rejection and whether Th2 cells alone accounted for accelerated graft rejection in allergic mice. Hosts sensitized cutaneously with short ragweed pollen developed cutaneous immediate hypersensitivity but rejected corneal allografts at the same tempo and incidence as naive mice. Th2 immune deviation induced with keyhole limpet hemocyanin and IFA did not affect corneal allograft rejection. Thus, Th2 immune deviation alone does not account for the exacerbation of corneal allograft rejection that occurs in mice with AC. CD4(+) T cells from AC mice elaborated Th1 (IFN-gamma) and Th2 (IL-13) cytokines when challenged with donor alloantigens. Adoptive transfer of Th1 or Th2 cells to nude mice, from AC mice that had rejected corneal allografts, produced graft rejection in 70% and 20% of the hosts, respectively. In contrast, adoptive transfer of a combination of Th1 and Th2 cells produced 100% rejection. Administration of exogenous IFN-gamma could substitute for Th1 cells and produced 100% corneal allograft rejection in recipients of Th2 cells alone. By contrast, IFN-gamma did not significantly enhance corneal allograft rejection mediated by Th1 cells. Thus, exacerbation of corneal allograft rejection in mice with AC is associated with a mixed Th1 and Th2 alloimmune response, and the contribution of Th1 cells is through their production of IFN-gamma.

Asunto(s)

Conjuntivitis Alérgica/inmunología , Rechazo de Injerto/inmunología , Activación de Linfocitos/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Separación Celular , Conjuntivitis Alérgica/complicaciones , Trasplante de Córnea , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Rechazo de Injerto/complicaciones , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante Homólogo

RESUMEN

Mechanisms that maintain ocular immune privilege may contribute to ocular tumor progression by inhibiting tumoricidal immune responses. Consistent with that notion are observations from transplantable tumor models in mice demonstrating that the tumoricidal activity of CD8(+) cytolytic T lymphocytes (CTL) may be inhibited directly by interfering with CTL effector function in the eye or indirectly by abrogating the effector function of CD8+ T cell-activated intratumoral macrophages that are critical for ocular tumor rejection. In addition, epigenetic gene regulation by factors within the ocular tumor environment favors the generation of tumor variants that are resistant to CD8(+) CTL. Intratumoral macrophages may be essential for eliminating these variants because, unlike CTL, their tumoricidal activity is nonspecific. Hence, the inhibition of macrophage effector function within the eye, presumably to preserve immune privilege by minimizing ocular immunopathology, may hasten the outgrowth of tumor escape variants which contributes to ocular tumor progression.

Asunto(s)

Neoplasias del Ojo/inmunología , Melanoma/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Epigénesis Genética , Ojo/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Tolerancia Inmunológica , Macrófagos/inmunología , Ratones , Linfocitos T Citotóxicos/inmunología , Escape del Tumor/inmunología

RESUMEN

PURPOSE: To determine whether blocking the expression of the chemokine receptor CXCR4 using siRNA inhibits chemotactic responses of human uveal melanoma cells to liver-derived factors and prevents liver metastases. METHODS: Human uveal melanoma cells were transfected with CXCR4 siRNA or control siRNA and tested in vitro for chemotactic and invasive behavior in response to soluble factors produced by human liver cells. The effect of CXCR4 siRNA transfection on the formation of liver metastases was tested by injecting transfected melanoma cells into the spleen capsules of NOD-SCID mice, and metastases were quantified by measuring the human housekeeping gene hHPRT in livers. RESULTS: Blocking CXCR4 interaction with its ligand using anti-CXCL12 antibody resulted in a significant reduction in the chemotactic responses of uveal melanoma cells to soluble factors produced by human liver cells. Similarly, blocking CXCR4 gene expression by transfection with CXCR4 siRNA inhibited both the chemotactic and the invasive properties of uveal melanoma cells exposed to factors produced by human livers. Uveal melanoma cells transfected with CXCR4 siRNA produced fewer liver metastases than untreated uveal melanoma cells or uveal melanoma cells transfected with control siRNA. CONCLUSIONS: CXCR4 is a key chemokine receptor that may account for the organ-specific homing of human uveal melanomas to the liver, which contains significant quantities of CXCL2, the only known ligand for CXCR4. CXCR4 is a potential therapeutic target for preventing the initial establishment of liver metastases but has limited application for use in advanced liver tumors.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/secundario , Melanoma/secundario , ARN Interferente Pequeño/farmacología , Receptores CXCR4/genética , Neoplasias de la Úvea/patología , Animales , Movimiento Celular , Quimiocina CXCL12/genética , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/genética , Melanoma/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas , Neoplasias de la Úvea/genética

RESUMEN

PURPOSE: To assess the expression of PD-L1 and PD-L2 on human ocular cells and their potential to regulate ocular inflammation. METHODS: Five categories of human ocular cells were evaluated for PD-L1 and PD-L2 expression by RT-PCR and flow cytometry. Three normal eyes and an inflamed eye from a patient with sympathetic ophthalmia were examined by immunohistochemistry for in situ PD-L1 expression. The immunomodulatory functions of PD-L1 and PD-L2 were tested by coculturing untreated or IFN-gamma-pretreated ocular cells with activated human peripheral blood T cells for 48 hours and assessing T-cell production of IFN-gamma, TNF-alpha, IL-4, and IL-5 by ELISA and T-cell apoptosis by flow cytometry. RESULTS: PD-L1 protein was expressed constitutively in 4 of 5 human ocular cell lines, and its expression was significantly upregulated after stimulation by IFN-gamma. Moreover, in situ expression of PD-L1 in inflamed ocular tissues was remarkably upregulated compared with normal eyes. Although PD-L2 expression was detectable by flow cytometry on 3 of 5 ocular cell lines, immunohistochemical staining did not show expression of PD-L2 on either normal or inflamed ocular tissues. IFN-gamma, TNF-alpha, and IL-5 production by activated T cells cocultured with ocular cells was significantly enhanced in the presence of anti-PD-L1 blocking antibody. However, ocular cell-expressed PD-L1 and PD-L2 did not induce T-cell apoptosis. CONCLUSIONS: PD-L1 expressed on human ocular cells has a presumptive role in controlling ocular inflammation by inhibiting the production of proinflammatory cytokines and a Th2 cytokine by activated T cells. This may represent an important mechanism for maintaining immune privilege in the eye.

Asunto(s)

Segmento Anterior del Ojo/metabolismo , Antígenos CD/genética , Regulación de la Expresión Génica/fisiología , Oftalmía Simpática/inmunología , Segmento Anterior del Ojo/citología , Antígenos CD/metabolismo , Apoptosis , Antígeno B7-H1 , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Activación de Linfocitos/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1 , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología

RESUMEN

PURPOSE: To assess the expression of PD-L1 on human uveal melanomas and its potential to suppress T-cell function. METHODS: A panel of primary and metastatic uveal melanoma cell lines was evaluated for PD-L1 expression by RT-PCR and flow cytometric analysis. Uveal melanoma-containing eyes were examined for PD-L1 expression by immunohistochemistry. PD-L1 function was tested by coculturing IFN-gamma-pretreated uveal melanoma cells with activated Jurkat T cells for 48 hours and assessing T-cell production of IL-2 by ELISA. RESULTS: Five of the nine primary and one of the five metastatic uveal melanoma cell lines tested constitutively expressed PD-L1 protein at various levels. However, all primary and metastatic uveal melanoma cell lines upregulated PD-L1 expression after stimulation with IFN-gamma. Immunohistochemistry demonstrated that PD-L1 was not expressed by primary uveal melanomas in situ. IL-2 production by activated Jurkat T cells was decreased significantly when the cells were cocultured with IFN-gamma-pretreated uveal melanoma cells. More than 70% of IL-2 production was restored by addition of either anti-PD-L1 or anti-PD-1 antibody to the coculture assays (P < 0.01). CONCLUSIONS: Expression of PD-L1 by uveal melanoma cells regulates T-cell function by suppressing IL-2 production. The results imply that the presence of IFN-gamma in the tumor local microenvironment promotes upregulation of PD-L1 expression by uveal melanoma, which may, in part, promote immune escape by impairing T-cell function. The selective blockade of PD-L1 is a potential strategy in T-cell-based immunotherapy for uveal melanoma.

Asunto(s)

Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Melanoma/inmunología , Proteínas de Neoplasias/metabolismo , Linfocitos T/fisiología , Neoplasias de la Úvea/inmunología , Antígenos CD/genética , Proteínas Reguladoras de la Apoptosis/genética , Antígeno B7-H1 , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Interferón gamma/farmacología , Interleucina-2/metabolismo , Células Jurkat , Activación de Linfocitos , Melanoma/patología , Metástasis de la Neoplasia , Receptor de Muerte Celular Programada 1 , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Úvea/patología

RESUMEN

Although intraocular tumours reside in an immune-privileged site, they can circumvent immune privilege and undergo rejection. Ocular tumour rejection typically follows one of two pathways. One pathway involves CD4+ T cells, delayed-type hypersensitivity (DTH), and culmination in ischemic necrosis of the tumour and phthisis (atrophy) of the eye. The second pathway is DTH-independent and does not inflict collateral injury to ocular tissues, and the eye is preserved. In this study, we used a well-characterized tumour, Ad5E1, to investigate the role of CD4+ T cells in the non-phthisical form of intraocular tumour rejection. It has been previously documented that CD4+ T cells and interferon (IFN)-gamma are necessary for rejection of these tumours in the eye. In this study, we demonstrate that CD4+ T cells can circumvent immune privilege and infiltrate intraocular Ad5E1 tumours. Following tumour rejection, CD4+ T cells from tumour rejector mice could be adoptively transferred to severe combined immunodeficiency (SCID) mice and protect them from intraocular Ad5E1 tumour growth. Tumour-specific CD4+ T cells produced IFN-gamma in response to Ad5E1 tumour antigens. Macrophages also contributed to rejection, as they were present in intraocular Ad5E1 tumours, and local depletion of macrophages resulted in progressive tumour growth. Ocular macrophages contributed to Ad5E1 tumour rejection, as Ad5E1 tumour rejection did not occur in macrophage-depleted SCID mice reconstituted with rejector CD4+ T cells. This demonstrates that macrophage and CD4+ T-cell co-operation is needed for non-phthisical rejection of intraocular tumours.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Neoplasias del Ojo/inmunología , Macrófagos/inmunología , Traslado Adoptivo , Animales , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Neoplasias del Ojo/patología , Neoplasias del Ojo/prevención & control , Hipersensibilidad Tardía/inmunología , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias