RESUMO
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.
Assuntos
Conjuntivite Alérgica/imunologia , Rejeição de Enxerto/imunologia , Ativação Linfocitária/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Separação Celular , Conjuntivite Alérgica/complicações , Transplante de Córnea , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Rejeição de Enxerto/complicações , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
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.
Assuntos
Conjuntivite Alérgica/imunologia , Células T Matadoras Naturais/imunologia , Animais , Conjuntivite Alérgica/metabolismo , Conjuntivite Alérgica/fisiopatologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th2/imunologiaRESUMO
BACKGROUND: Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4 T cells. This study examined the role of CD4 T cell-independent mechanisms of corneal allograft rejection. METHODS: BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8 or CD8 T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8 or CD8 T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively. RESULTS.: Both CD8 and CD8 T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8 T cells, but not CD8 T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8 cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function. CONCLUSION: The results support the proposition that CD4 T cell-independent rejection of corneal allografts can be mediated by either CD8 or CD8 T cells. The CD8 T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Transplante de Córnea/imunologia , Rejeição de Enxerto/etiologia , Transferência Adotiva , Animais , Apoptose , Linfócitos T CD8-Positivos/fisiologia , Epitélio Corneano/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Citotóxicos/imunologia , Transplante HomólogoRESUMO
BACKGROUND: The role of CD4(+) T cells as effector cells in corneal allograft rejection is poorly understood. We investigated the role of CD4(+) T cells as helper cells in the generation of allospecific effector macrophages in corneal graft rejection and the role of CD4(+) T cells as apoptosis-inducing effector cells. METHODS: Corneal allografts were transplanted to CD4 knockout, FasL-deficient, and macrophage-depleted hosts. An Annexin-V binding assay was used to evaluate the susceptibility of corneal cells to both Fas-dependent and CD4 T-cell-mediated apoptosis in vitro. RESULTS: Macrophages were essential for graft rejection, but not as effector cells. Anti-BALB/c CD4(+) T cells from immunized C57BL/6 mice induced apoptosis of BALB/c corneal epithelial and endothelial cells. However, anti-BALB/c CD4(+) T cells from FasL-deficient gld/gld mice did not induce apoptosis of BALB/c corneal endothelial cells. Moreover, gld/gld mice had a reduced capacity to reject BALB/c corneal allografts. Although the initial results suggested a role for Fas-induced apoptosis in corneal graft rejection, additional experiments indicated otherwise. The incidence and tempo of immune rejection of Fas-deficient lpr/lpr corneal allografts were no different than those for corneal grafts from Fas-bearing C57BL/6 donors. Moreover, CD4(+) T-cell-mediated apoptosis of corneal cells could not be blocked with either Fas-Fc fusion protein or anti-FasL blocking antibody. CONCLUSIONS: The results suggest that CD4(+) T cells function directly as effector cells and not as helper cells in the rejection of corneal allografts. Although the corneal endothelium is highly susceptible to Fas-induced apoptosis, this is apparently not the primary mechanism of CD4(+) T-cell-dependent rejection.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/imunologia , Transplante de Córnea/imunologia , Rejeição de Enxerto/etiologia , Receptor fas/fisiologia , Animais , Córnea/patologia , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
PURPOSE: Amniotic membrane has been applied to the ocular surface to restore corneal function. The beneficial effect of amniotic membrane transplantation may be due to the immunosuppressive effects of amniotic epithelial cells. The purpose of this study was to determine whether amniotic epithelial cells (AECs) secrete anti-inflammatory and antiproliferative factors that affect the chemotaxis of neutrophils and macrophages and suppress both T- and B-cell proliferation in vitro. METHODS: Human amniotic cells were isolated from human amniotic membrane and cultured in vitro. The supernatants from AEC cultures were collected after 48 hours of incubation. Neutrophil and macrophage chemotactic activity was tested in the presence of AEC supernatant, using 24-well migration assay chambers. Lymphocyte proliferation was tested by H(3)-thymidine incorporation. Apoptosis was examined by caspase-3 and annexin V assays, and expression of cytokines was assessed by RT-PCR. RESULTS: AEC supernatant significantly inhibited the chemotactic activity of neutrophils and macrophages toward macrophage inflammatory protein (MIP)-2 (P < 0.05). The supernatant significantly reduced the proliferation of both T and B cells after mitogenic stimulation (P < 0.05). Caspase-3 assays revealed that the supernatant induced apoptosis of T and B cells, but not of corneal epithelial cells and liver cells. In contrast to lymphocytes, macrophages and neutrophils were resistant to apoptosis induced by AEC supernatant. The AECs expressed message for TNFalpha, Fas ligand (FasL), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), TGFbeta, and macrophage migration-inhibitory factor (MIF). However, AEC induction of apoptosis was inhibited (50%) by anti-FasL antibody but not by anti-TRAIL or anti-TNFalpha antibodies. Moreover, AEC supernatant inhibited macrophage migration in vitro. CONCLUSIONS: AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems.
Assuntos
Âmnio/citologia , Células Epiteliais/imunologia , Imunossupressores/isolamento & purificação , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
BACKGROUND: Oral administration of alloantigens induces down-regulation of Th1 immune responses and reduces the incidence of corneal graft rejection. This study examined the role of Th1 and Th2 cytokines, accessory cells, and lymphoid organs that are known to be instrumental in other forms of antigen-specific tolerance. METHODS: Allogeneic dendritic cells (DC) were administered orally using a protocol that is known to reduce the incidence of corneal allograft rejection and prevent the generation of allospecific delayed-type hypersensitivity (DTH). Hosts included normal mice and gene knockout (KO) mice, including B cell-deficient (mu)MT, interleukin (IL)-4 KO, IL-10 KO, and interferon (IFN)-gamma KO mice. The requirement for either an intact spleen or thymus was also examined. Orally administered paraformaldehyde-fixed, UVB-treated, or sonicated allogeneic cells were tested to determine if dead cells were capable of inducing tolerance. RESULTS: Studies on gene KO mice indicated that a Th1 cytokine (IFN-gamma) and a Th2 cytokine (IL-4) were needed for the development of oral tolerance to alloantigens. By contrast, IL-10 was not required. Although an intact spleen was necessary for the development of tolerance, removal of the thymus did not affect down-regulation of DTH. CONCLUSIONS: Oral tolerance induced with allogeneic cells shares characteristics with antigen-specific unresponsiveness induced by other routes, yet there are some noteworthy differences. The capacity of killed or sonicated allogeneic cells to induce oral tolerance and enhance corneal graft survival indicates that oral tolerance to alloantigens can occur via the indirect pathway of alloantigen presentation. These results also emphasize the remarkable redundancy in the mechanisms that the immune system employs to produce antigen-specific unresponsiveness.
Assuntos
Transplante de Córnea/imunologia , Células Dendríticas/transplante , Isoantígenos/imunologia , Tolerância ao Transplante , Administração Oral , Animais , Linfócitos B/fisiologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Fixadores/farmacologia , Formaldeído/farmacologia , Interferon gama/genética , Interferon gama/fisiologia , Interleucina-10/genética , Interleucina-10/fisiologia , Interleucina-4/genética , Interleucina-4/fisiologia , Isoantígenos/administração & dosagem , Camundongos , Camundongos Endogâmicos , Camundongos Knockout/genética , Fenótipo , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Sonicação , Baço/fisiologia , Linfócitos T/fisiologia , Timo/fisiologia , Tolerância ao Transplante/fisiologiaRESUMO
PURPOSE: To characterize the role of B cells in the induction of anterior chamber-associated immune deviation (ACAID). METHODS: An in vitro model of the ACAID spleen was used to recapitulate the events that occur when antigen is introduced into the anterior chamber of the eye and culminates in the appearance of antigen-specific, CD8(+) suppressor cells. RESULTS: In vitro-generated suppressor cells mimicked those produced by anterior chamber injection of antigen, as shown by their antigen specificity, surface expression of CD8, and capacity to suppress DTH, which is mediated by previously immunized T cells. B cells were found to be necessary for suppressor cell development. The B cell receptor (BCR) was necessary for the induction of ACAID and conveyed antigen specificity to the suppressor T cells. Lysosomal acidification of internalized antigen was necessary for B cells to induce ACAID; however, transporter of antigen processing (TAP) was not required for the generation of ACAID. CONCLUSIONS: The results suggest that B cells use the BCR to capture and internalize antigen from ACAID-inducing macrophages. Lysosomal acidification of the captured antigen is essential for the processing of the ACAID antigen before TAP-independent presentation to suppressor cells.
Assuntos
Câmara Anterior/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/fisiologia , Macrófagos/imunologia , Baço/citologia , Transferência Adotiva , Animais , Hipersensibilidade Tardia/imunologia , Imunização , Injeções , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
PURPOSE: Interleukin-2 (IL-2) and IL-15 receptors have been detected on some murine neoplasms. Accordingly, the expression of these receptors on human uveal melanoma cell lines was examined, and the effect of exogenous IL-2 and -15 on melanoma cell proliferation, susceptibility to natural killer (NK) cell-mediated cytolysis, and sensitivity to apoptosis were assessed. METHODS: Nine human uveal melanoma cell lines and three cell lines from uveal melanoma metastases were tested by flow cytometry for the expression of human IL-2R and -15Ralpha. Melanoma cells were cultured, with or without recombinant human IL-2 or -15, cell proliferation was determined by tritiated thymidine incorporation, and IL-2 and -15 receptor expression was assessed by flow cytometry. The effect of these cytokines on NK activity was evaluated with a standard (51)Cr-release assay. RESULTS: All the melanoma cell lines expressed IL-2R and -15R. IL-2 induced a three- to eightfold upregulation of IL-2R expression in all the melanoma cell lines. Although IL-2 did not affect the proliferation of six of the seven uveal melanoma cell lines, it induced a 32% and 57% increase in the proliferation of both metastatic cell lines. IL-15 induced proliferation on all tested cell lines (4%-68%). Both IL-2 and -15 reduced melanoma cell sensitivity to NK-cell-mediated cytolysis and cisplatin-induced apoptosis. CONCLUSIONS: The results suggest that IL-2 and -15 elaborated by tumor-infiltrating lymphocytes and macrophages may affect the malignant behavior of human uveal melanoma by stimulating proliferation and reducing uveal melanoma cell susceptibility to NK-cell-mediated cytolysis and cisplatin-induced apoptosis.
Assuntos
Melanoma/metabolismo , Receptores de Interleucina-2/metabolismo , Neoplasias Uveais/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Melanoma/fisiopatologia , Melanoma/secundário , Receptores de Interleucina-15 , Proteínas Recombinantes/farmacologia , Neoplasias Uveais/imunologia , Neoplasias Uveais/patologia , Neoplasias Uveais/fisiopatologiaRESUMO
PURPOSE: To determine the role of tumor necrosis factor receptors (TNFRs) in corneal allograft rejection. METHODS: Corneal epithelial and endothelial cells were examined by flow cytometry for the expression of TNFRI and TNFRII and their susceptibility to TNF-alpha-induced apoptosis. Corneal allografts from normal and TNFRI and TNFRII knockout (KO) C57BL/6 mice were transplanted to BALB/c hosts, and the fate of the allografts was monitored. C57BL/6 spleen cells were injected into the anterior chamber (AC) of BALB/c mice to induce anterior chamber-associated immune deviation (ACAID) and promote corneal allograft survival. The presence of ACAID suppressor cells in corneal allograft recipients was tested using a local adoptive transfer (LAT) assay. RESULTS: Murine corneal epithelial and endothelial cells expressed TNFRI and TNFRII and were susceptible to TNF-alpha-induced apoptosis, yet corneal allografts from either TNFRI or TNFRII donors did not enjoy a lower incidence of rejection or a prolongation in survival time compared to corneal allografts from normal C57BL/6 donors. Moreover, all 31 of the TNFRII KO corneal grafts were rejected by naïve BALB/c hosts. Rejection of TNFRII KO corneal grafts occurred even though suppressor cells developed in the hosts and inhibited the expression of delayed-type hypersensitivity to donor alloantigens. CONCLUSIONS: Expression of TNFRII on corneal cells conveys a degree of protection against immune rejection of corneal allografts by a mechanism that is independent of ACAID. Moreover, induction of ACAID before the application of TNFRII KO corneal allografts fails to improve survival and does not replace the TNFRII-dependent protective mechanism.
Assuntos
Câmara Anterior/imunologia , Antígenos CD/fisiologia , Sobrevivência de Enxerto/fisiologia , Ceratoplastia Penetrante/imunologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transferência Adotiva , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Endotélio Corneano/metabolismo , Epitélio Corneano/metabolismo , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Hipersensibilidade Tardia/imunologia , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Linfócitos T Reguladores , Transplante Homólogo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: The study had two purposes: to examine the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors on uveal melanoma cells and metastases arising from uveal melanoma and to determine the susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. METHODS: Nine human uveal melanoma cell lines and three cell lines derived from uveal melanoma metastases were examined for TRAIL receptor expression by flow cytometry. In vitro apoptosis assays were performed to determine the relative susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. Annexin V staining was also used to determine the capacity of either cycloheximide or interferon-beta to enhance TRAIL-induced apoptosis. RESULTS: Five of the nine uveal melanoma cell lines expressed TRAIL-R2 on more than 60% of the cells. All three of the cell lines derived from uveal melanoma metastases expressed TRAIL-R2 on more than 50% of the cells. Cycloheximide exerted a profound effect in enhancing TRAIL-induced apoptosis in all but two of the uveal melanoma cell lines and in all three of the metastases cell lines. Interferon-beta produced a similar enhancement of TRAIL-induced apoptosis, even in cell lines that were previously shown to be resistant. CONCLUSIONS: TRAIL is a potentially useful therapeutic modality for the management of uveal melanomas and their metastases. Moreover, pharmacological agents and biological response modifiers that independently display antineoplastic properties can enhance TRAIL-induced apoptosis in resistant uveal melanoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma/metabolismo , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias Uveais/metabolismo , Anexina A5/metabolismo , Proteínas Reguladoras de Apoptose , Cicloeximida/farmacologia , Sinergismo Farmacológico , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Interferon beta/farmacologia , Melanoma/secundário , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Membro 10c de Receptores do Fator de Necrose Tumoral , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/secundário , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Receptores Chamariz do Fator de Necrose Tumoral , Neoplasias Uveais/patologiaRESUMO
PURPOSE: To determine whether the Th1 cytokine, interferon (IFN)-gamma, is necessary for corneal graft rejection. METHODS: Full-thickness penetrating keratoplasties were performed in normal mice and in IFN-gamma knockout (KO) mice. RESULTS: Sixty-four percent of the MHC-mismatched corneal allografts were rejected in IFN-gamma KO mice. By contrast, MHC-matched corneal allografts were rejected in 50% to 77% of the wild-type hosts, but were not rejected in any of the IFN-gamma KO mice or the wild-type mice treated with anti-IFN-gamma monoclonal antibody. Corneal graft rejection in IFN-gamma-deficient hosts was characterized by an eosinophilic infiltrate compared with a mononuclear inflammatory infiltrate in normal mice. CONCLUSIONS: IFN-gamma is not necessary for the rejection of MHC-mismatched corneal grafts. However, IFN-gamma and Th1 immune mechanisms are necessary for the rejection of MHC-matched corneal allografts that confront the host with foreign minor histocompatibility antigens. The immune response in atopic patients, as in IFN-gamma KO mice, is characterized by cross-regulation of Th1 cytokines, such as IFN-gamma. The present results indicate that MHC matching dramatically reduces the risk of corneal graft rejection when IFN-gamma is depressed or absent. Thus, MHC matching may reduce the risk of corneal graft rejection in patients with atopic keratoconus.
Assuntos
Córnea/imunologia , Rejeição de Enxerto/imunologia , Ceratoplastia Penetrante , Complexo Principal de Histocompatibilidade/imunologia , Células Th1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Feminino , Hipersensibilidade Tardia/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Camundongos Knockout , Transplante de Pele , Transplante HomólogoRESUMO
PURPOSE: This study determined whether adenovirus-mediated transfer of the murine interferon-beta (AdCMVIFN-beta) gene protects against liver metastases arising from intraocular melanomas in mice. METHODS: A replication-deficient adenovirus vector (AdCMVIFN-beta) was used for the in vivo transfer of the murine IFN-beta gene into intraocular melanoma-bearing mice. AdCMVIFN-beta was injected either intravenously or directly into the intraocular melanomas. The effect of gene transfer on liver metastases was ascertained by histopathologic analysis of the livers and by measuring serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are two enzymes associated with liver metastases in patients with uveal melanoma. RESULTS: Mice treated with two intratumoral injections of AdCMVIFN-beta had a 68% reduction in metastatic liver lesions (P = 0.016) and a 51% reduction in liver enzyme levels compared with control mice (P = 0.02). However, the antimetastatic effect of AdCMVIFN-beta was not directly attributable to the adenovirus vector or virus-mediated cytolysis of tumor cells. Intravenous treatment with AdCMVIFN-beta resulted in an 86% reduction in the number of metastatic foci in the liver (P = 0.014) and a 61% reduction of serum AST levels compared with mice treated with AdCMVLacZ (P = 0.015). AdCMVIFN-beta treatment produced a sharp increase in the NK cell activity that was demonstrable in vivo and in vivo. In vivo depletion of NK cells by anti-asialo GM1 antibody abrogated the antimetastatic effects of AdCMVIFN-beta. CONCLUSIONS: The results support the feasibility of activation of NK cell function through gene transfer as one possible therapeutic strategy for reducing hepatic metastases of uveal melanomas.
Assuntos
Terapia Genética , Interferon beta/genética , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Hepáticas Experimentais/terapia , Melanoma Experimental/secundário , Melanoma Experimental/terapia , Neoplasias Uveais/terapia , Adenoviridae/genética , Alanina Transaminase/sangue , Animais , Antineoplásicos , Aspartato Aminotransferases/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Gangliosídeo G(M1)/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos , Interferon beta/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/genética , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uveais/enzimologia , Neoplasias Uveais/genética , Neoplasias Uveais/patologiaRESUMO
PURPOSE: The precise role of antibodies in corneal transplantation is controversial. Clinical and experimental evidence both supports and refutes the contribution of donor-derived alloantibody in corneal allograft rejection. Accordingly, we prospectively evaluated the presence of donor-derived alloantibody in two high-risk donor-host combinations. We also evaluated the ability of this alloantibody to kill corneal epithelial, keratocytes, and endothelial cells in complement-dependent and complement-independent fashions. METHODS: C3H/Hej (H-2(k)) and Balb/c (H-2(d)) corneal grafts were transplanted orthotopically to CB6F1 (H-2(b/d)) and C57BL/6 (H-2(b)) recipients, respectively. These two donor-host combinations represent disparity at the entire MHC and multiple minor histocompatibility loci. Four objectives were addressed. First, we wished to determine if there was a correlation between the appearance of donor-specific serum IgG antibody and corneal graft rejection. Second, we evaluated the effect of passive transfer of hyperimmune donor-specific antibody on corneal allograft rejection. Third, we examined the capacity of donor-specific alloantibody to mediate complement-dependent cytolysis of corneal cells. Finally, we determined the capability of donor-specific alloantibody to mediate apoptosis of corneal cells. RESULTS: The presence of donor-specific serum IgG alloantibodies did not correlate with corneal graft rejection. One hundred percent of CB6F1 and C57BL/6 hosts rejected their C3H and Balb/c orthotopic corneal allografts, respectively. However, two of these seven CB6F1 hosts and one C57BL/6 host did not produce donor-specific IgG alloantibody that was significantly different from naive donors. Passive transfer of hyperimmune allo-antiserum prior to corneal transplantation did not increase the incidence, severity, or tempo of corneal allograft rejection in either donor-host combination. Hyperimmune allo-antiserum produced complement-mediated lysis of C3H corneal endothelial but not C3H corneal epithelial cells in the C3H-CB6F1 donor-host combination. Interestingly, all three corneal cell layers were vulnerable to complement-mediated cytolysis in the Balb/c-C57BL/6 donor-host combination. Additionally, Balb/c corneal epithelial, keratocytes, and endothelial cells were vulnerable to complement-independent, antibody induced apoptosis. CONCLUSIONS: Corneal graft rejection does not appear to correlate with the production of IgG alloantibody and can occur in the absence of donor-specific IgG alloantibody. Antibody-mediated killing of the corneal endothelium can occur in a complement-dependent or complement-independent fashion.
Assuntos
Anticorpos/imunologia , Transplante de Córnea/imunologia , Rejeição de Enxerto/imunologia , Animais , Córnea/imunologia , Córnea/patologia , Feminino , Antígenos de Histocompatibilidade/imunologia , Camundongos , Coelhos , Transplante HomólogoRESUMO
PURPOSE: Classically, corneal allograft rejection is thought to be a T(H)1-mediated phenomenon. However, T(H)2-mediated allograft rejection has been reported in other transplanted organ systems, including the heart and kidney. We previously reported a form of T(H)2-mediated corneal allograft rejection in a murine model with a T(H)2 immune bias. In this study we sought to determine if there was any evidence for this form of corneal allograft rejection in humans. DESIGN: Experimental study with an interventional case series. METHODS: The clinical records of all keratoconus patients undergoing penetrating keratoplasty at the University of Texas, Southwestern Medical Center from 1994 to 1999 were reviewed. Careful attention was paid to a clinical history of atopy. Atopic patients were selected, because these patients have been shown to have a "T(H)2 immune bias." The corneal graft rejection rate in these patients and the number of repeat corneal transplants performed was determined. The experimental group consisted of patients with a clinical history of atopy and keratoconus who had at least one repeat penetrating keratoplasty for an immunologically rejected corneal transplant. Any patient with evidence of primary allograft failure was excluded from this study. Tissue specimens from these patients were embedded in paraffin, serially sectioned, stained with Giemsa stains, and examined histologically. The control group consisted of patients without a clinical history of allergy (and therefore no T(H)2 immune bias) who underwent corneal transplantation for Fuch corneal endothelial dystrophy, or aphakic/pseudophakic bullous keratopathy. Failed grafts from these control patients were also paraffin embedded, serially sectioned, stained, and examined histologically. The human experimental and control corneal specimens were compared with data obtained in a murine model of T(H)2-mediated corneal allograft rejection. Briefly, full-thickness penetrating C57BL/6ByJ corneal allografts were transplanted onto Balb/cByJ and Balb/c-IFN-gamma(tm1Ts) (Balb/c-IFN-gamma knockout) mice. Additionally, full-thickness Balb/cByJ corneal allografts were transplanted onto C57BL/6ByJ and C57BL/6ByJ-IFN-gamma(tm1Ts) mice. Corneal allograft rejection rates and mean rejection times were calculated and compared between wild-type and interferon gamma (IFN-gamma) knockout hosts. The rejected allografts were examined histologically by the same methods used in the human tissue. RESULTS: There were 84 penetrating keratoplasties performed from 1994 to 1999 for keratoconus. Seven of these 84 patients rejected their corneal grafts. Of the 7 patients who rejected their corneal allografts, 4 had repeat penetrating keratoplasty. Of these 4 repeat corneal allografts, 3 showed eosinophilia when compared with rejected grafts in control patients. Atopic keratoconus patients had a mixed inflammatory cellular infiltrate in the rejected corneal tissue specimen with a significantly greater density of eosinophils (P =.001) compared with patients who did not have a pre-existing T(H)2 bias. The inflammatory infiltrate in these patients without a T(H)2 immune bias was mononuclear. In the murine model, corneal allograft rejection did occur in the absence of IFN-gamma, a critical T(H)1 cytokine in both fully allogeneic donor-host combinations. Histologically, rejection in these ("T(H)2 mice") was characterized by a predominant eosinophilic infiltrate in the rejected graft bed when compared with wild-type animals ("T(H)1 mice") that had a predominantly mononuclear infiltrate in the rejected corneal graft bed. CONCLUSIONS: Preliminary findings show that corneal allograft rejection in patients with a pre-existing T(H)2 phenotype is similar to what is seen in the murine model of T(H)2-mediated corneal allograft rejection. Based on this small sample, it appears that eosinophils may play a role in corneal allograft rejection in this group of patients. However, further study is necessary to determine the importance of these cells in allograft rejection.
Assuntos
Córnea/imunologia , Eosinófilos/imunologia , Rejeição de Enxerto/imunologia , Ceratocone/cirurgia , Ceratoplastia Penetrante/imunologia , Células Th2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Córnea/patologia , Citocinas/metabolismo , Eosinofilia/etiologia , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/patologia , Citometria de Fluxo , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Humanos , Imunofenotipagem , Interferon gama/fisiologia , Ceratoplastia Penetrante/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Reoperação , Estudos Retrospectivos , Transplante HomólogoRESUMO
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.
Assuntos
Conjuntivite Alérgica/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Linfócitos T/imunologia , Transferência Adotiva , Ambrosia , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Pólen , Células Th2/imunologiaRESUMO
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.
Assuntos
Neoplasias Oculares/patologia , Neoplasias Hepáticas/secundário , Melanoma Experimental/secundário , Células T Matadoras Naturais/imunologia , Neoplasias Esplênicas/patologia , Corpo Vítreo/patologia , Animais , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Neoplasias Oculares/imunologia , Feminino , Citometria de Fluxo , Gangliosídeo G(M1)/imunologia , Interleucina-10/fisiologia , Fígado/imunologia , Neoplasias Hepáticas/imunologia , Masculino , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esplenectomia , Neoplasias Esplênicas/imunologia , Fator de Crescimento Transformador beta/fisiologia , Corpo Vítreo/imunologiaRESUMO
The enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes degradation of tryptophan, an essential amino acid required for lymphocyte activation and proliferation. Many tumors express IDO which implies that it acts as a mechanism to evade T cell-mediated immune attack, and also to establish an immunosuppressive tumor microenvironment. The purpose of this study was to determine whether primary and metastatic uveal melanoma expressed the IDO gene and whether uveal melanoma cells could deplete tryptophan. In situ expression of IDO in primary uveal melanoma from tumor bearing eyes and metastatic uveal melanoma liver tissues was determined by immunohistostaining with IDO-specific antibody. Reverse transcription PCR was used to assess IDO gene transcription by primary and metastatic uveal melanoma cell lines. IDO protein expression was determined by Western blot of uveal melanoma cell protein lysate. IDO catalytic activity was assessed by measuring the presence of kynurenine, a product generated by tryptophan degradation, in uveal melanoma culture supernatants. Primary uveal melanoma from tumor-bearing eyes and metastatic uveal melanoma from the liver did not express IDO in situ. IDO was not constitutively expressed in either primary or metastatic uveal melanoma cell lines. However, stimulation of primary and metastatic uveal melanoma cell cultures with interferon-gamma (IFN-gamma) universally upregulated both IDO gene and protein expression. Culture supernatants from IFN-gamma treated primary and metastatic uveal melanoma cell cultures contained elevated levels of kynurenine. Addition of the IDO inhibitor 1-methyl dl-tryptophan significantly diminished kynurenine levels in IFN-gamma treated uveal melanoma cell cultures. The results from this study suggest that IFN-gamma inducible IDO upregulation by primary and metastatic uveal melanoma may generate a local immune privileged microenvironment to promote escape from T cell-mediated immune surveillance.
Assuntos
Tolerância Imunológica , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Melanoma/enzimologia , Triptofano/deficiência , Neoplasias Uveais/enzimologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/imunologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Melanoma/genética , Melanoma/imunologia , Melanoma/secundário , Metabolismo/efeitos dos fármacos , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , Triptofano/análogos & derivados , Triptofano/farmacologia , Células Tumorais Cultivadas , Regulação para Cima/imunologia , Neoplasias Uveais/genética , Neoplasias Uveais/imunologiaRESUMO
A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils.
Assuntos
Conjuntivite Alérgica/imunologia , Transplante de Córnea/imunologia , Rejeição de Enxerto/imunologia , Isoantígenos/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Ambrosia/imunologia , Animais , Conjuntivite Alérgica/epidemiologia , Transplante de Córnea/patologia , Suscetibilidade a Doenças/imunologia , Feminino , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Hipersensibilidade Tardia/imunologia , Incidência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Pólen/imunologia , Células Th1/imunologia , Células Th1/patologia , Células Th2/patologiaRESUMO
An important factor in the establishment of ocular immune privilege is the dynamic down regulation of T helper 1 (Th1) immune responses that occurs in response to antigens delivered intraocularly; a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). ACAID is characterized by the generation of splenic regulatory cells that inhibit the expression of delayed-type hypersensitivity. Previous studies have shown that antigens introduced into the anterior chamber of the eye induce the generation of a CD4+ T-cell population that suppress the induction of Th1 immune responses and the appearance of a second population of CD8+ T regulatory cells that suppresses the expression of Th1 inflammatory responses (= efferent suppressor cells). Experiments described here characterized the function of the CD4+ ACAID suppressor cell population and its effect on the generation of CD8+ efferent suppressor cells that inhibit the expression of DTH in situ. Both in vivo and in vitro experiments demonstrated that CD4+ T cells are required for the generation of CD8+ efferent suppressor cells. CD4+ T cells do not require cell contact with CD8+ T cells; instead they produce soluble IL-10 that is sufficient for the generation of ACAID suppressor cells. Finally, the CD4+ afferent T suppressor cells are not natural killer T cells, but do express the CD25 cell surface marker.
Assuntos
Câmara Anterior/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-10/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Comunicação Celular/imunologia , Células Cultivadas , Regulação para Baixo/imunologia , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/imunologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-2/análiseRESUMO
Acanthamoeba cysts are not eliminated from the corneas of human subjects or experimentally infected animals. The persistence of Acanthamoeba cysts in the cornea indicates that either the cysts escape immunological elimination or are not recognized by the host's immunological elements. The aim of this study was to determine the immunogenicity and antigenicity of the Acanthamoeba cyst. Mice were immunized intraperitoneally and serum anti-Acanthamoeba IgG was measured by ELISA. Lymphoproliferative assay and delayed type hypersensitivity (DTH) responses to Acanthamoeba castellanii cyst and trophozoite antigens were used to determine the cell mediated immune responses against Acanthamoeba cysts. A. castellanii cysts were both immunogenic and antigenic, producing anti-Acanthamoeba serum IgG, T lymphocyte proliferation, and delayed type hypersensitivity responses. These results indicate that Acanthamoeba cysts are recognized by the immune system. The persistence of the organism in the human cornea means that these adaptive immune responses fail to kill Acanthamoeba cysts.