RESUMEN
The reconstructed skin micronucleus (RSMN) assay was developed in 2006, as an in vitro alternative for genotoxicity evaluation of dermally applied chemicals or products. In the years since, significant progress has been made in the optimization of the assay, including publication of a standard protocol and extensive validation. However, the diverse morphology of skin cells makes cell preparation and scoring of micronuclei (MN) tedious and subjective, thus requiring a high level of technical expertise for evaluation. This ultimately has a negative impact on throughput and the assay would benefit by the development of an automated method which could reduce scoring subjectivity while also improving the robustness of the assay by increasing the number of cells that can be scored. Imaging flow cytometry (IFC) with the ImageStream®X Mk II can capture high-resolution transmission and fluorescent imagery of cells in suspension. This proof-of-principle study describes protocol modifications that enable such automated measurement in 3D skin cells following exposure to mitomycin C and colchicine. IFC was then used for automated image capture and the Amnis® Artificial Intelligence (AAI) software permitted identification of binucleated (BN) cells with 91% precision. On average, three times as many BN cells from control samples were evaluated using IFC compared to the standard manual analysis. When IFC MNBN cells were visually scored from within the BN cell images, their frequency compared well with manual slide scoring, showing that IFC technology can be applied to the RSMN assay. This method enables faster time to result than microscope-based scoring and the initial studies presented here demonstrate its capability for the detection of statistically significant increases in MNBN frequencies. This work therefore demonstrates the feasibility of combining IFC and AAI to automate scoring for the RSMN assay and to improve its throughput and statistical robustness.
Asunto(s)
Aprendizaje Profundo , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Piel/patología , Inteligencia Artificial , Automatización de Laboratorios/instrumentación , Automatización de Laboratorios/métodos , Reacciones Falso Positivas , Estudios de Factibilidad , Citometría de Flujo/instrumentación , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Pruebas de Micronúcleos/instrumentación , Pruebas de Micronúcleos/métodos , Mitomicina/toxicidad , Modelos Biológicos , Pruebas de Mutagenicidad/instrumentación , Pruebas de Mutagenicidad/métodos , Valor Predictivo de las Pruebas , Prueba de Estudio Conceptual , Piel/diagnóstico por imagen , Piel Artificial , Programas Informáticos , Andamios del TejidoRESUMEN
Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/análisis , Caspasa 1/análisis , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inflamasomas/metabolismo , Análisis de la Célula Individual/métodos , Fluorescencia , Células HEK293 , Humanos , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR/agonistas , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células THP-1RESUMEN
Diverse cellular responses to external cues are controlled by a small number of signal-transduction pathways, but how the specificity of functional outcomes is achieved remains unclear. Here we describe a mechanism for signal integration based on the functional coupling of two distinct signaling pathways widely used in leukocytes: the ITAM pathway and the Jak-STAT pathway. Through the use of the receptor for interferon-γ (IFN-γR) and the ITAM adaptor Fcγ as an example, we found that IFN-γ modified responses of the phagocytic antibody receptor FcγRI (CD64) to specify cell-autonomous antimicrobial functions. Unexpectedly, we also found that in peritoneal macrophages, IFN-γR itself required tonic signaling from Fcγ through the kinase PI(3)K for the induction of a subset of IFN-γ-specific antimicrobial functions. Our findings may be generalizable to other ITAM and Jak-STAT signaling pathways and may help explain signal integration by those pathways.
Asunto(s)
Motivo de Activación del Inmunorreceptor Basado en Tirosina/inmunología , Janus Quinasa 2/metabolismo , Listeriosis/inmunología , Macrófagos/inmunología , Receptor Cross-Talk/inmunología , Factor de Transcripción STAT1/metabolismo , Animales , Carga Bacteriana , Células Cultivadas , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Motivo de Activación del Inmunorreceptor Basado en Tirosina/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagocitosis/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ingeniería de Proteínas , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Activación Transcripcional/efectos de los fármacos , Receptor de Interferón gammaRESUMEN
Genes are regulated at the single-cell level. Here, we performed RNA FISH of thousands of cells by flow cytometry (flow-RNA FISH) to gain insight into transcriptional variability between individual cells. These experiments utilized the murine adenocarcinoma 3134 cell line with 200 copies of the MMTV-Ras reporter integrated at a single genomic locus. The MMTV array contains approximately 800-1200 binding sites for the glucocorticoid receptor (GR) and 600 binding sites for the pioneer factor Foxa1. Hormone activation of endogenous GR by dexamethasone treatment resulted in highly variable changes in the RNA FISH intensity (25-300 pixel intensity units) and size (1.25-15 µm), indicative of probabilistic or stochastic mechanisms governing GR and cofactor activation of the MMTV promoter. Exogenous expression of the pioneer factor Foxa1 increased the FISH signal intensity and size as expected for a chromatin remodeler that enhances transcriptional competence through increased chromatin accessibility. In addition, specific analysis of Foxa1-enriched cell sub-populations showed that low and high Foxa1 levels substantially lowered the cell-to-cell variability in the FISH intensity as determined by a noise calculation termed the % coefficient of variation. These results suggest that an additional function of the pioneer factor Foxa1 may be to decrease transcriptional noise.
Asunto(s)
Adenocarcinoma/genética , Citometría de Flujo , Factor Nuclear 3-alfa del Hepatocito/genética , Hibridación Fluorescente in Situ , Virus del Tumor Mamario del Ratón/genética , ARN/genética , Activación Transcripcional , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Sondas de ADN , Dexametasona/farmacología , Procesamiento de Imagen Asistido por Computador , Ratones , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Glucocorticoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Eosinophils (Eos) are found at increased numbers within necrotic areas of tumors. We show that necrotic material from cell lysates containing damage-associated molecular pattern molecules induce eosinophil degranulation (release of major basic protein and eosinophil peroxidase) and enhance their oxidative burst while the stimulatory capacity of cell lysates is significantly diminished following oxidation. High mobility group box 1 (HMGB1), a prototypic damage-associated molecular pattern molecule, released following necrosis but not apoptosis, induced a similar effect on Eos. Additionally, we demonstrate that HMGB1 enhances eosinophil survival and acts as a chemoattractant. Consistently, we show that Eos express an HMGB1 receptor, the receptor for advanced glycation end product, and that anti-receptor for advanced glycation end product could diminish the HMGB1-mediated effects. Of all tested biologic activities, Eos respond most sensitively to the presence of necrotic material including HMGB1 with generation of peroxide. We postulate that Eos "sense" necrotic cell death, migrating to and responding to areas of tissue injury/necrosis. Oxidation of cell lysates reduces their biologic activity when compared with native lysates. We postulate that eosinophil-associated modulation of immunity within tumor and other damaged tissues may be primarily by promoting oxidative degradation of necrotic material. Novel therapeutic strategies may be considered by advancing oxidative denaturation of released necrotic material using Eos or other aerobic strategies.
Asunto(s)
Eosinófilos/inmunología , Proteína HMGB1/metabolismo , Neutrófilos/inmunología , Estallido Respiratorio/inmunología , Anticuerpos/farmacología , Apoptosis/inmunología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Neoplasias Colorrectales/inmunología , Proteína Mayor Básica del Eosinófilo/inmunología , Proteína Mayor Básica del Eosinófilo/metabolismo , Peroxidasa del Eosinófilo/inmunología , Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Proteína HMGB1/farmacología , Humanos , Necrosis/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oxidación-Reducción , Receptor para Productos Finales de Glicación Avanzada/inmunología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Estallido Respiratorio/efectos de los fármacosRESUMEN
Signals from stressed cells and the enteric microbiota activate macrophages and dendritic cells and mediate intestinal inflammation. HMGB1 serves as an immunogenic stimuli causing release of inflammatory cytokines by myeloid cells. Ethyl pyruvate inhibits secretion of HMGB1 and improves survival in models of endotoxemia and hemorrhagic shock. We reasoned that ethyl pyruvate may be protective in colitis, which involves similar inflammatory pathways. In IL-10(-/-) mice with established chronic colitis, ethyl pyruvate administration ameliorated colitis and reduced intestinal cytokine production. IL-10(-/-) mice demonstrated increased intestinal HMGB1 expression and decreased expression of RAGE compared with wild-type mice. Fecal HMGB1 levels were decreased in ethyl pyruvate-treated mice. Furthermore, ethyl pyruvate induced HO-1 expression in intestinal tissue. In TNBS-induced colitis, intrarectal administration of ethyl pyruvate resulted in amelioration of colitis and reduced intestinal cytokine production. In LPS-activated murine macrophages, ethyl pyruvate decreased expression of IL-12 p40 and NO production but did not affect IL-10 levels. Ethyl pyruvate did not inhibit nuclear translocation of NF-kappaB family members but attenuated NF-kappaB DNA binding. Additionally, ethyl pyruvate induced HO-1 mRNA and protein expression and HO-1 promoter activation. Moreover, ethyl pyruvate prevented nuclear-to-cytoplasmic translocation of HMGB1. In conclusion, the HMGB1/RAGE pathway has pathophysiologic and diagnostic significance in experimental colitis. Ethyl pyruvate and other strategies to inhibit HMGB1 release and function represent promising interventions in chronic inflammatory diseases.
Asunto(s)
Colitis/metabolismo , Proteína HMGB1/metabolismo , Piruvatos/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/secundario , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/secundario , Citocinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Etiquetado Corte-Fin in Situ , Interleucina-10/deficiencia , Interleucina-10/genética , Subunidad p40 de la Interleucina-12/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Metástasis de la Neoplasia , Trasplante de Neoplasias , Óxido Nítrico/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Piruvatos/farmacología , Distribución Aleatoria , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , TransfecciónRESUMEN
BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.
Asunto(s)
Citocinas/fisiología , Neoplasias Pulmonares/patología , Células Madre/patología , Células Tumorales Cultivadas , Antígenos CD/análisis , Antineoplásicos/farmacología , Automatización , Supervivencia Celular/efectos de los fármacos , Quimiocinas/biosíntesis , Quimiocinas/fisiología , Citocinas/biosíntesis , Citometría de Flujo , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/fisiología , Humanos , Metástasis de la Neoplasia , Células Madre/efectos de los fármacosRESUMEN
TRAIL is a death ligand that induces apoptosis in malignant but not normal cells. Recently the ability of TRAIL to induce proliferation in apoptosis-resistant normal and malignant cells was reported. In this study, we analyzed TRAIL effects in apoptosis sensitive MCF7, OVCAR3 and H460 human tumor cell lines. TRAIL at low concentrations preferentially induced cell proliferation. At 100 ng/ml, apoptotic death was readily observed, however surviving cells acquired higher proliferative capacity. TRAIL-stimulated production of several cytokines, IL-8, RANTES, MCP-1 and bFGF, and activation of caspases 1 and 8 was essential for this effect. Antibodies to IL-8, RANTES, and bFGF blocked TRAIL-induced cell proliferation and further stimulated apoptosis. For the first time, we report that high TRAIL concentrations induced cell senescence as determined by the altered morphology and expression of several senescence markers: SA-beta-gal, p21Waf1/Cip1, p16INK4a, and HMGA. Caspase 9 inhibition protected TRAIL-treated cells from senescence, whereas inhibition of caspases 1 and 8 increased the yield of SLP cells. In conclusion, in cultured human carcinoma cells, TRAIL therapy results in three functional outcomes, apoptosis, proliferation and senescence. TRAIL-induced proapoptotic and prosurvival responses correlate with the strength of signaling. TRAIL-induced cytokine production is responsible for its proliferative and prosurvival effects.
Asunto(s)
Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Neoplasias/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Inhibidores de Caspasas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Biológicos , Neoplasias/enzimología , Pruebas de Neutralización , Fenotipo , Transducción de Señal/efectos de los fármacosRESUMEN
We have recently demonstrated that cytolysis of human melanoma cells by immune effectors (both NK and T cells) is associated with release of the nuclear chromatin protein, high mobility group box I (HMGB1). Extracellular HMGB1 mediates a number of important functions including endothelial cell activation, stromagenesis, recruitment and activation of innate immune cells, and also dendritic cell maturation that, in the setting of cancer, lead to a chronic inflammatory response. This reparative inflammatory response promotes tumor cell survival, expansion, and metastases. Release of HMGB1 after chemotherapy-induced cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines. Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin, retained HMGB1 within the nucleus for significantly longer periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, release of HMGB1 from dying tumor cells treated with chemotherapy or cells with lymphokine activated killer cell activity is not dependent solely on the mode of cell death. Sequestration of the damage associated molecular pattern molecule, HMGB1, may play a role in the clinical efficacy of platinating agents and suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness.
Asunto(s)
Antineoplásicos/farmacología , Proteína HMGB1/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Compuestos Organoplatinos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias del Colon , Terapia Combinada , Humanos , Inmunoterapia , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/metabolismo , Melanoma , Melfalán/farmacología , Microscopía Confocal , Necrosis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Oxaliplatino , Paclitaxel/farmacología , Neoplasias PancreáticasRESUMEN
High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine-activated killing (LAK) cells, tumor-specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL-2-activated PBMC (LAK activity). HLA-A2 and MART1 or gp100-specific cytolytic T lymphocytes induced HMGB1 release from HLA-A2-positive and MART1-positive melanoma cells (FEM X) or T2 cell-loaded, gp100-specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway-mediated cell death was blocked with the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors.
Asunto(s)
Proteína HMGB1/metabolismo , Melanoma/metabolismo , Linfocitos T/fisiología , Transporte Activo de Núcleo Celular , Clorometilcetonas de Aminoácidos/farmacología , Muerte Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Granzimas/farmacología , Humanos , Células Asesinas Activadas por Linfocinas/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-alpha, IL-6, TNF-alpha, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-gamma-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.
Asunto(s)
Células Dendríticas/inmunología , Proteína HMGB1/fisiología , Receptor Toll-Like 9/inmunología , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Virus ADN , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína HMGB1/farmacología , Humanos , Inmunidad Innata , Inflamación , Oligodesoxirribonucleótidos/farmacología , Linfocitos T/citología , Linfocitos T/metabolismo , Receptor Toll-Like 9/agonistasRESUMEN
High mobility group box 1 (HMGB1) is a NF released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. Here we demonstrate that in contrast to the proinflammatory role of HMGB1, preconditioning with HMGB1 results in protection following hepatic ischemia/reperfusion (I/R). Pretreatment of mice with HMGB1 significantly decreased liver damage after I/R. The protection observed in mice pretreated with HMGB1 was associated with a higher expression of IL-1R-associated kinase-M, a negative regulator of TLR4 signaling, compared with controls. We thus explored the possibility that HMGB1 preconditioning was mediated through TLR4 activation. HMGB1 preconditioning failed to provide protection in TLR4 mutant (C3H/HeJ) mice, but successfully reduced damage in TLR4 wild-type (C3H/HeOuj) mice. Our studies demonstrate that in contrast to the role of HMGB1 as an early mediator of inflammation and organ damage in hepatic I/R, HMGB1 preconditioning can be protective.
Asunto(s)
Proteína HMGB1/administración & dosificación , Hígado/irrigación sanguínea , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Acondicionamiento Pretrasplante/métodos , Secuencia de Aminoácidos , Animales , Proteína HMGB1/genética , Células HeLa , Humanos , Inyecciones Intravenosas , Quinasas Asociadas a Receptores de Interleucina-1 , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/administración & dosificación , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/biosíntesis , Daño por Reperfusión/enzimología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND: Administration of immature dendritic cells (DC) prolongs but does not result in indefinite allograft survival. We attempted to achieve this goal by adding a sub-therapeutic dose of immunosuppression. METHODS: DC propagated from B10 (H-2(b)) mouse bone marrow (BM) were transfected with nuclear factor-kappaB (NF-kappaB)-binding-site-specific oligodeoxyribonucleotide (ODN). The allostimulatory activity of transfected and normal DC were examined in mixed-lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) assays in vitro, and their influence on allograft survival by systemic administration of DC in vivo. RESULTS: Transfection of DC with NF-kappaB ODN resulted in complete abrogation of NF-kappaB activity and inhibition of co-stimulation. Allogeneic (C3H, H-2(k)) T cells stimulated by ODN DC demonstrated impairment in MLR and CTL activity. Administration of ODN DC significantly prolonged B10 allograft survival. In contrast to cyclosporine, which failed to enhance the effect of ODN DC, a combination of ODN DC with sirolimus at 6 mg/kg/day for 6 days achieved long-term survival in all allografts. This was associated with low CTL activity of either graft-infiltrating cells or splenic T cells and increased TUNEL-positive cells in T-cell areas of recipient mesenteric lymph nodes. Analysis of transcription factor nuclear translocation with Cellomics indicated that stimulation with ODN DC showed inhibited T-cell nuclear translocation of signal transducer and activator of transcription (Stat)1 and Stat3, extracellular signal-related kinase (ERK) and activating transcription factor (ATF)-2, but not NF-kappaB and P38, compared with mature DC. The selective inhibition was enhanced by sirolimus, but not cyclosporine. CONCLUSIONS: Sirolimus enhances immature DC tolerogenicity by induction of T-cell apoptosis, and promotes immature DC-induced inhibition of Stat1, ERK and ATF-2 activation.
Asunto(s)
Células Dendríticas/trasplante , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/fisiología , Trasplante de Corazón , Inmunosupresores/administración & dosificación , Sirolimus/administración & dosificación , Linfocitos T Citotóxicos/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Citometría de Flujo , Prueba de Cultivo Mixto de Linfocitos , Ratones , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , TransfecciónRESUMEN
The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.
Asunto(s)
Viroterapia Oncolítica/métodos , Virus Vaccinia/fisiología , Animales , Apoptosis/genética , Línea Celular Transformada , Línea Celular Tumoral , Femenino , Eliminación de Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Replicación ViralRESUMEN
Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r = 0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p < 0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n = 7), correlated with the quality of iDC preparations (p = 0.002), and IL-12 p70 production (p = 0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.
Asunto(s)
Células Dendríticas/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Análisis de la Célula Individual , Transporte Activo de Núcleo Celular , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Transporte de ProteínasRESUMEN
Substantial attention has been paid to the role of the toll-like receptor (TLR) ligands of late and their role in regulating the innate immune response. They serve as exogenous danger signals important in informing and driving the distal adaptive immune response to pathogens. Less clear has been the role of the nominal endogenous danger signals released and recognized in stressed cells following genotoxic or metabolic stress as occurs in progressively growing tumors. HMGB1 (high-mobility group B1) is a nuclear protein well characterized for its ability to modify DNA access to transcriptional proteins that is released from necrotic cells as well as secreted through the endosomal route from hematopoietic cells, serving as a late mediator of sepsis. It interacts with high-affinity RAGE (receptor for advanced glycation end products) and TLR2 receptors. Here we show that HMGB1 enhances interferon gamma release from macrophage (but not dendritic cell)-stimulated NK cells. This is effective only when coupled with other pro-inflammatory cytokines particularly with IL-2 in combination with IL-1 or IL-12. We have used this information to suggest that HMGB1, which also promotes epithelial migration and proliferation, drives repair in the absence or inhibition of other factors but enhances inflammation in their presence. The implications for tumorigenesis and tumor progression are quite important as they may be for other states of chronic inflammation.
Asunto(s)
Comunicación Celular/inmunología , Proteína HMGB1/fisiología , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Proteína HMGB1/farmacología , Humanos , Inmunidad Innata , Interferón gamma/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Monocitos/inmunología , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Receptor Toll-Like 2 , Receptores Toll-LikeRESUMEN
Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p<0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.
Asunto(s)
Núcleo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/fisiología , Microscopía Fluorescente , FN-kappa B/metabolismo , Transactivadores/metabolismo , Antineoplásicos/farmacología , Ligando de CD40/metabolismo , Comunicación Celular/fisiología , Separación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Dendríticas/citología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-12/biosíntesis , Células K562 , Células Asesinas Naturales/citología , Células Asesinas Naturales/fisiología , Lipopolisacáridos/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Control de Calidad , Factor de Transcripción STAT1 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Extract: We all shudder about untimely deaths or those that we were not prepared for. As such we perceive such "unscheduled" deaths as dangerous. Similarly, apoptotic death (literally falling leaves) or the programmed cell death of cells in multicellular organisms ranging from slime mold and simple worms through to mammals, has a level of tidiness and well-orchestrated activities with literally hundreds if not thousands of gene products employed with either the primary or secondary purpose of coordinating the orderly death of cells throughout life. During inflammation of any sort, driven by tissue damage or injury or infection by pathogens (virus, bacteria, and parasites), apoptotic death similarly serves to quickly rid the host of damaged cells, promote removal and digestion of the infected cell, and prepare the way for tissue remodeling and repair. When this goes awry, for example during periods of chronic inflammation, tissues are subjected to the contrasting needs of driving apoptotic death whilst maintaining the barrier function of the epithelia (such as skin cells) as well as the selective permeability of mucosal sites (i.e., areas where mucus is secreted to protect the cells from their surroundings, such as gut cells protecting themselves from the gastric acids). Prudently, they need to limit and husband local resources sufficiently for the maintenance of tissue integrity and renewal. It is our provocative and novel contention that cancer in adults (and not children) most often arises in a setting of chronic inflammation and disordered cell death rather than one associated primarily with disordered cell growth as it is popularly imagined by scientists, clinicians, and the general public.
RESUMEN
High mobility group B1 (HMGB1) and its counter-receptor, receptor for advanced glycation end products (RAGE), represent suitable targets for investigation, integrating many aspects of modern biology, particularly that associated with chronic diseases involving inflammation, dysregulated cell death and cancer. Also known as amphoterin, HMGB1 was initially identified over 25 years ago as a transcriptional regulatory molecule causing DNA bending, and facilitating binding of several transcriptional complexes, in particular members of the nuclear hormone receptor family. Although loosely bound to chromatin, it is released from necrotic cells but not apoptotic cells and is actively secreted by activated macrophages in a partially tumor necrosis factor-dependent manner. It serves as a late mediator of septic death present within the serum and inflammatory sites of patients with arthritis, correlating with the inflammatory response, to signal tissue injury, causes sickness behavior, and acts as an endogenous pyrogen. Although known to interact with RAGE on endothelial cells causing activation and leukocyte recruitment, RAGE itself has most recently been shown to serve as a counter-receptor for leukocyte integrins, suggesting that signaling through this molecule is potentially important for cell adhesion and clustering as well as recruitment of inflammatory cells. Targeting the HMGB1 ligand or its receptor represents an important potential application in cancer therapeutics, given its widespread overexpression, as well as that of its receptor in virtually every tumor type carefully examined. This, coupled with its ability to accelerate tumor growth in immunodeficient murine models, suggests that it is a possible therapeutic target in patients with cancer.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Receptores Inmunológicos/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Productos Finales de Glicación Avanzada , Proteína HMGB1/metabolismo , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Receptor para Productos Finales de Glicación AvanzadaRESUMEN
Screening expressed sequence tag databases for endothelial-specific homologs to human junctional adhesion molecule (JAM) and A33-Ag, we identified a protein of 298 aa that represents the recently described vascular endothelial-JAM (VE-JAM)/JAM 2. We confirmed VE-JAM/JAM 2 expression to be restricted to the high endothelial venule of tonsil and lymph nodes, and we further expanded the localization to the endothelium of arterioles in and around inflammatory and tumor foci. In our functional characterizations of VE-JAM/JAM 2, we discovered that it can function as an adhesive ligand for the T cell line J45 and can interact with GM-CSF/IL-4-derived peripheral blood dendritic cells, circulating CD56(+) NK cells, circulating CD56(+)CD3(+) NK/T cells, and circulating CD56(+)CD3(+)CD8(+) cytolytic T cells. In the course of our studies, we also isolated and characterized the functional VE-JAM/JAM 2 receptor, which, upon cloning, turned out to be a submitted sequence representing JAM 3 (accession number NP 113658). With these understandings, we have characterized a protein-interacting pair that can be important in the role of T, NK, and dendritic cell trafficking and inflammation.