RESUMEN
IL-17 has been associated with selected inflammatory and autoimmune diseases. We characterized the expression of this proinflammatory cytokine following HSV-1 corneal infection and investigated whether IL-17R signaling modulated the host response to the viral pathogen at early time-points postinfection. IL-17 was elevated in the murine cornea 24 h after high-dose virus infection and subsequently persisted at low levels during the first week. Immunofluorescent studies showed that the IL-17R was expressed by cultured mouse corneal fibroblasts. Exposure of corneal cells to IL-17 led to production of IL-6 and MIP-2 in vitro and in vivo, indicating that the IL-17R was functional. Mice lacking IL-17R displayed significantly reduced neutrophil infiltration and corneal opacity. However, this effect was transient, as corneal pathology and neutrophil influx resembled that of wild-type (WT) hosts 4 days postinfection. HSV-1 growth and clearance in IL-17R(-/-) hosts were similar to that of the WT controls. Infection of IFN-gamma gene knockout mice was associated with elevated IL-17 levels and accelerated corneal opacity, suggesting that IFN-gamma negatively regulated IL-17 expression. Collectively, our results establish that IL-17 is rapidly produced in the cornea after HSV-1 infection and is regulated at least in part by IFN-gamma. The absence of IL-17 signaling results in a transient decrease in the expression of proinflammatory mediators, neutrophil migration, and corneal pathology, but control of virus growth in the cornea and trigeminal ganglia is not compromised. Thus, IL-17 actively influences early virus-induced corneal inflammation.
Asunto(s)
Herpesvirus Humano 1 , Interleucina-17/fisiología , Queratitis Herpética/fisiopatología , Receptores de Interleucina-17/fisiología , Animales , Células Cultivadas/metabolismo , Células Cultivadas/virología , Quimiocinas/biosíntesis , Quimiocinas/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Córnea/metabolismo , Córnea/patología , Córnea/virología , Femenino , Fibroblastos/metabolismo , Fibroblastos/virología , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/fisiología , Interleucina-17/biosíntesis , Interleucina-17/farmacología , Interleucina-1alfa/farmacología , Interleucina-6/biosíntesis , Interleucina-6/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/patología , Receptores de Interleucina-17/deficiencia , Receptores de Interleucina-17/genética , Organismos Libres de Patógenos Específicos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The purpose of this study was to characterize the synthesis of alpha-chemokines IP-10, MIG, and I-TAC by human corneal epithelial cells (HCE) following exposure to proinflammatory mediators. Supernatants were collected from HCE cultures stimulated with individual or combinations of TNF-alpha, IL-1alpha, and IFN-gamma, and assayed for alpha-chemokines by ELISA. RT-PCR was used to detect IFN-gamma receptor mRNA. Activation of STAT 1 was determined by Western blotting. Stimulation of HCE with either IL-1alpha or TNF-alpha increased IP-10 protein synthesis up to 6-fold, whereas insignificant levels of MIG and I-TAC were induced. The epithelial cells were found to express IFN-gamma receptors constitutively. Exposure to the ligand resulted in STAT 1 phosphorylation and production of nanogram amounts of IP-10, I-TAC, and MIG. When HCE were stimulated with combinations of TNF-alpha and IFN-gamma, or IL-1alpha and IFN-gamma, the levels of IP-10 and I-TAC secreted were > 150-fold higher than that produced following exposure to a single cytokine. In contrast, MIG protein synthesis was not enhanced upon stimulation with cytokine combinations. The abundant production of ELR(-)alpha -chemokines following appropriate stimulation suggests that HCE may play an important role in the recruitment of effector cells such as activated T-lymphocytes to inflamed corneal tissue. The data also indicate that the synthesis of IP-10, I-TAC, and MIG are differentially regulated in HCE.
Asunto(s)
Quimiocinas CXC/biosíntesis , Epitelio Corneal/metabolismo , Mediadores de Inflamación/farmacología , Antivirales/farmacología , Western Blotting , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/citología , Epitelio Corneal/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-1alfa/farmacología , Fosforilación , ARN Mensajero/genética , Receptores de Interferón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor de Interferón gammaRESUMEN
PURPOSE: Chemokines responsible for recruiting lymphocytes such as activated T cells into the cornea have not been clearly defined. IP-10, I-TAC, and MIG are chemoattractants for these lymphocytes. The goal of this study was to determine whether human corneal keratocyte (HCKs) in culture synthesize these chemokines in response to proinflammatory mediators. METHODS: HCKs grown in vitro were stimulated with IL-1alpha, TNF-alpha, or IFN-gamma. Induction of alpha-chemokine gene expression was quantitated by real-time PCR and ELISA. Activation of the transcriptional activator STAT1 by IFN-gamma receptors expressed on HCKs was determined by Western blot analysis. RESULTS: HCKs incubated with TNF-alpha, IL-1alpha, or IFN-gamma resulted in a >2000-fold increase in IP-10 protein secretion by 36 hours after stimulation. In contrast, stimulation with TNF-alpha, IL-1alpha, or IFN-gamma induced levels of MIG and I-TAC that were not significantly greater than constitutive levels. Treatment of HCKs with IFN-gamma activated STAT1 and, in combination with either TNF-alpha or IL-1alpha, enhanced MIG and I-TAC synthesis >20-fold. CONCLUSIONS: IP-10 synthesis is induced in HCKs by IL-1alpha, TNF-alpha, and IFN-gamma. In contrast, induction of I-TAC and MIG synthesis in HCKs requires costimulation with IFN-gamma and either IL-1alpha or TNF-alpha. The results suggest therefore, that the upregulation of I-TAC and MIG gene expression at sites of corneal inflammation are more tightly regulated than that of IP-10. A role for differential induction of the three alpha-chemokine genes in corneal inflammatory processes at the eye surface is discussed.
Asunto(s)
Quimiocinas CXC/biosíntesis , Córnea/metabolismo , Citocinas/farmacología , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Western Blotting , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Córnea/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1 , Transactivadores/metabolismo , Regulación hacia ArribaRESUMEN
PURPOSE: To investigate the production of monokine induced by gamma-interferon (MIG) during a primary Herpes simplex virus type 1 (HSV-1) infection of the cornea. We hypothesize that multiple CXCR3 ligands are involved in T cell recruitment during HSV-1 corneal infection and that neutrophils have the potential to contribute to their production. MATERIALS AND METHODS: Levels of MIG were evaluated in an in vivo murine model of HSV-1 corneal infection by quantitative ELISA. Cultured murine corneal fibroblast (MCF) cells and purified neutrophils were stimulated in vitro with IFN-γ and IL-1α to determine inducers of MIG. Cellular sources of MIG production in vivo were investigated via cellular depletion studies. Additionally, MIG production resulting from interaction between resident human corneal cells and neutrophils was evaluated in an ex vivo model of human corneal infection. RESULTS: MIG was significantly elevated on days 2-6 and on day 8 following corneal infection. MCF and neutrophils secreted MIG in response to IFN-γ, but not IL-1α stimulation. Co-stimulation with IFN-γ and IL-1α induced a four-fold increase in MIG production by MCF. However, the same combination led to a three-fold decrease in MIG production by neutrophils. In vivo, a 52% reduction in MIG levels was observed in the neutrophil depleted host. In the human ex vivo model, MIG levels were significantly elevated in response to communication between HSV-1 infected corneal tissue and neutrophils. CONCLUSIONS: Here, we report the evidence for the production of MIG, a second CXCR3 ligand, during the primary immune response to HSV-1 corneal infection. Our results support the hypothesis that both neutrophils and resident corneal cells contribute to MIG production in vivo. However, neutrophils produce MIG in response to communication with HSV-1-infected resident corneal cells more efficiently than by direct interaction with virus. In addition, we found that MIG production by neutrophils and resident corneal cells was differentially regulated by IL-1α.
Asunto(s)
Quimiocina CXCL9/genética , Queratocitos de la Córnea/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Herpesvirus Humano 1/fisiología , Queratitis Herpética/genética , Neutrófilos/metabolismo , Animales , Células Cultivadas , Quimiocina CXCL9/metabolismo , Córnea/metabolismo , Córnea/virología , Queratocitos de la Córnea/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Interferón gamma/farmacología , Interleucina-1alfa/farmacología , Queratitis Herpética/metabolismo , Queratitis Herpética/virología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
PURPOSE: To determine whether interleukin (IL)-1alpha- and tumor necrosis factor (TNF)-alpha-stimulated human corneal epithelial cells (HCECs) and human corneal keratocytes (HCKs) produce the alpha-chemokines epithelial cell-derived neutrophil attractant (ENA)-78 and granulocyte chemotactic protein (GCP)-2. METHODS: Cultures of HCECs and HCKs were stimulated with either human recombinant IL-1alpha or TNF-alpha. At selected times after stimulation, culture supernatants were harvested and assayed for ENA-78 and GCP-2 by enzyme-linked immunosorbent assay. RNA was extracted from cell cultures to measure steady state levels of intracellular ENA-78 and GCP-2 pre-mRNA and mRNA by the reverse transcription-polymerase chain reaction. RESULTS: Exposure of HCECs to either IL-1alpha or TNF-alpha stimulated a more than 4.5-fold increase in ENA-78 RNA and protein synthesis without stimulating a significant increase in either GCP-2 RNA synthesis or protein production. Exposure of HCK to IL-1alpha stimulated a 10-fold increase in ENA-78 and GCP-2 RNA synthesis and a more than 300-fold increase in ENA-78 and GCP-2 protein production. In contrast, exposure of keratocytes to TNF-alpha significantly enhanced ENA-78 RNA synthesis, resulting in a more than 68-fold increase in ENA-78 protein synthesis without significantly enhancing either GCP-2 gene expression or protein secretion. CONCLUSIONS: ENA-78 gene expression is significantly enhanced in both HCECs and HCKs in response to either IL-1alpha or TNF-alpha stimulation. In contrast, GCP-2 synthesis is only inducible in IL-1alpha-stimulated HCKs. The results suggest that GCP-2 gene expression is more tightly regulated in diseased or injured corneal tissue than is ENA-78 gene expression.
Asunto(s)
Quimiocinas CXC/genética , Sustancia Propia/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Interleucina-8/análogos & derivados , Interleucina-8/genética , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Quimiocina CXCL5 , Quimiocina CXCL6 , Quimiocinas CXC/metabolismo , Sustancia Propia/citología , Sustancia Propia/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/metabolismo , Fibroblastos , Humanos , Interleucina-8/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Herpes simplex virus (HSV)-1 infection of the murine cornea is known to stimulate a vigorous interleukin (IL)-6 response, but whether this pleiotropic cytokine is an essential participant in corneal inflammation is unclear. This study was designed to compare the early inflammatory response in IL-6 gene-deficient mice to that in wild-type hosts. METHODS: Gene knockout and wild-type mice (C57BL/6 background) were infected intracorneally with HSV-1 (strain RE) and observed through clinical examination and immunohistochemistry for the development of corneal opacity. Virus corneal titers were determined by standard plaque assay on Vero cells. Cytokine and chemokine levels in corneal lysates were measured with commercial ELISA kits. RESULTS: Corneal opacity in IL-6(-/-) mice was substantially diminished in comparison with IL-6(+/+) hosts 24 to 48 hours after intracorneal viral infection, and corneal levels of (MIP)-2 and MIP-1alpha were significantly reduced. Local administration of IL-6 at the time of infection restored corneal opacity and chemokine levels to that of wild-type hosts. Antibody neutralization of endogenous IL-6 in IL-6(+/+) animals reduced corneal opacity scores and MIP-2 levels to that of IL-6(-/-) mice. Ex vivo studies with excised corneal buttons revealed that uninfected IL-6(-/-) corneas injected with IL-6 produced MIP-2 and MIP-1alpha at levels comparable to that seen in IL-6(+/+) hosts. CONCLUSIONS: Collectively, these results suggest that IL-6 promotes corneal inflammation by acting in an autocrine-paracrine fashion to induce resident corneal cells to make MIP-2 and MIP-1alpha, which in turn recruit neutrophils to the virus infection site.
Asunto(s)
Quimiocinas/metabolismo , Córnea/metabolismo , Herpesvirus Humano 1/fisiología , Interleucina-6/fisiología , Queratitis Herpética/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Neutrófilos/fisiología , Animales , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CXCL2 , Córnea/patología , Córnea/virología , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/patología , Opacidad de la Córnea/virología , Ensayo de Inmunoadsorción Enzimática , Técnicas para Inmunoenzimas , Queratitis Herpética/patología , Queratitis Herpética/virología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
PURPOSE: The interleukin (IL)-8 promoter possesses a NF-kappa B-binding site with affinity to p50p65 and p65p65 complexes while the monocyte chemoattractant protein (MCP)-1 promoter's NF-kappa B-binding site has exclusive affinity to p50p65 heterodimers. The purpose of this study was to determine whether the two NF-kappa B sites play a role in the capacity of tumor necrosis factor (TNF)-alpha-stimulated human corneal epithelial cells (HCECs) to produce nanogram amounts of IL-8 in the absence of MCP-1 synthesis. METHODS: IL-8 and MCP-1 promoters were cloned into luciferase reporter vectors. Site-directed mutagenesis of wild-type promoters was used to mutate the NF-kappa B-binding motif in the wild-type IL-8 reporter plasmid into a motif with exclusive affinity to p50p65 and to mutate the NF-kappa B binding motif in the wild-type MCP-1 reporter plasmid into a motif with affinity to p65p65. Luciferase activity was determined after transfection of reporter vector constructs into TNF-alpha-stimulated HCECs. The chromatin immunoprecipitation assay was used to confirm binding of NF-kappa B subunits to IL-8 and MCP-1 promoters in vivo. RESULTS: Promoters with affinity to p65p65 homodimers were active in driving the expression of the reporter gene, whereas promoters with affinity to p50p65 heterodimers did not induce significant reporter gene expression. Incorporation of a CCAAT enhancer-binding protein (C/EBP)-binding site immediately upstream of p65p65-binding sites significantly enhanced promoter activity. CONCLUSIONS: The results suggest that the interaction of p65p65 homodimers and C/EBP transcriptional factors with IL-8 promoters and not MCP-1 promoters account for the capacity of HCECs to produce IL-8 selectively, in the absence of MCP-1 production.
Asunto(s)
Quimiocina CCL2/genética , Epitelio Corneal/efectos de los fármacos , Regulación de la Expresión Génica , Interleucina-8/genética , FN-kappa B/metabolismo , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Bases , Sitios de Unión/fisiología , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Técnicas de Cultivo de Célula , Quimiocina CCL2/biosíntesis , Ensayo de Cambio de Movilidad Electroforética , Epitelio Corneal/metabolismo , Humanos , Interleucina-8/biosíntesis , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción CHOP , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection, claiming millions of lives annually. The virus infects various cells of the respiratory tract as well as resident inflammatory cells such as macrophages. Infection activates a variety of cellular factors such as cytokines and the pro-inflammatory transcription factor, NF-kappa B, all of which are important players in the respiratory disease. However, the exact natural route of RSV infection and its etiology remain relatively unknown. In this paper, we test the hypothesis that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with RSV, and that the infection leads to the activation of proinflammatory macromolecules. RESULTS: Corneal swabs obtained from pediatric patients with acute respiratory disease were found to contain RSV at a high frequency (43 positive out of 72 samples, i.e., 60%). Primary corneal epithelial cells in tissue culture supported robust infection and productive growth of RSV. Infection resulted in the activation of TNF-alpha, IL-6 and sixteen chemokines as well as NF-kappa B. Three proinflammatory CXC chemokines (MIG, I-TAC, IP-10) underwent the greatest activation. CONCLUSIONS: The ocular epithelium is readily infected by RSV. The pro-inflammatory cytokines are likely to play critical roles in the etiology of inflammation and conjunctivitis commonly seen in pediatric patients with respiratory infections. RSV-eye interactions have important implications in RSV transmission, immunopathology of RSV disease, and in the management of conjunctivitis.
Asunto(s)
Citocinas/biosíntesis , Epitelio Corneal/virología , FN-kappa B/fisiología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Infecciones del Sistema Respiratorio/virología , Niño , Preescolar , Conjuntivitis Viral/virología , Epitelio Corneal/citología , Epitelio Corneal/inmunología , Células Gigantes , Humanos , Técnicas In Vitro , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación ViralRESUMEN
BALB/c mice sensitized to herpes simplex virus type 1 (HSV-1) develop a vigorous delayed-type hypersensitivity (DTH) response upon intradermal virus antigen challenge. Although CD4(+) T cells are a key mediator of this response, neutrophils are the most abundant cells at the antigen challenge site both initially and at the peak of the reaction. We investigated what role, if any, neutrophils play in the DTH to a viral antigen. We show here that antibody-mediated depletion of neutrophils 1 day before antigen challenge significantly suppressed ear swelling and markedly reduced cellular influx. Additionally, neutrophil depletion was associated with decreased expression of macrophage inflammatory protein 2 (MIP-2) and MIP-1alpha, as well as with a >60-fold increase in HSV-1 replication. Neutralizing antibodies to neutrophil chemoattractants MIP-2 or MIP-1alpha but not KC significantly suppressed DTH and sharply reduced neutrophil accumulation in the ear pinna. Purified bone marrow-derived neutrophils exposed to interleukin-1alpha (IL-1alpha) produced chemokines in an 8-h assay. Administration of neutralizing antibody to IL-1alpha significantly reduced ear swelling and suppressed the levels of MIP-2, MIP-1alpha, MIP-1beta, and RANTES. We conclude that neutrophils are a critical component of the DTH response to viral antigen. They are recruited to the DTH test site by MIP-2 and MIP-1alpha, where they can be activated by IL-1alpha. The infiltrating cells also help suppress virus replication in immunized mice.