RESUMEN
WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common "benign" central nervous system tumor but with significant recurrence rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose-response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.
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Neoplasias Encefálicas , Vesículas Extracelulares , Glioblastoma , Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Humanos , Neuronas/metabolismo , Péptidos/metabolismo , Péptidos/farmacologíaRESUMEN
RATIONALE: Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone positioning are unclear. OBJECTIVES: To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation. METHODS: Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure [PEEP] 0) or low Vt (6 ml/kg; PEEP 3 cm H(2)O; 3 h) in supine or prone position. Dorsal-caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1(-/-) or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6-7 ml/kg; PEEP 3 cm H(2)O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates. MEASUREMENTS AND MAIN RESULTS: Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1(-/-) mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα. CONCLUSIONS: Injurious ventilation induces MAPK in an MKP-1-dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB-dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation.
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Fosfatasa 1 de Especificidad Dual/fisiología , Respiración Artificial/efectos adversos , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Animales , Western Blotting , Diterpenos/uso terapéutico , Regulación hacia Abajo/fisiología , Perfilación de la Expresión Génica , Proteínas I-kappa B/metabolismo , Pulmón/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Posición Prona/fisiología , Ratas , Ratas Sprague-Dawley , Posición Supina/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.
RESUMEN
OBJECTIVES: Circulating levels of the proinflammatory mediator High Mobility Group Box Protein 1 (HMGB1) are increased in septic patients and may contribute to sepsis-induced organ dysfunction. Although HMGB1 has been shown to activate neutrophils from healthy volunteers, the responses of neutrophils from septic patients to HMGB1 have not been reported. In the present study we evaluated gene expression and activation of major intracellular signaling pathways in peripheral blood neutrophils obtained from patients with sepsis-induced acute lung injury after culture with HMGB1 or LPS. DESIGN: Ex-vivo study performed in neutrophils from patients with sepsis-induced acute lung injury. SETTING: Immunology and genetics laboratory at an academic medical center. PATIENTS AND PARTICIPANTS: Twenty-two adult patients with sepsis-induced acute lung injury. MEASUREMENTS AND RESULTS: Using gene arrays, distinct patterns of gene expression were found in neutrophils from septic patients after stimulation with HMGB1 or LPS. While more than three-quarters of the genes upregulated by HMGB1 in neutrophils from septic patients also demonstrated increased expression after culture with LPS, the majority of genes affected by LPS did not show altered expression in neutrophils stimulated with HMGB1. Culture of neutrophils with HMGB1 induced downregulation of its own expression, a finding not present after exposure to LPS. Although HMGB1 and LPS both increased nuclear translocation of NF-kappaB, the magnitude of this effect was greater in LPS stimulated neutrophils from patients with sepsis-induced acute lung injury. CONCLUSION: These findings demonstrate that the patterns of gene expression differ between neutrophils from septic patients stimulated with HMGB1 or LPS, and also that neutrophils from septic patients are not anergic but instead demonstrate intact activation of NF-kappaB after exposure to LPS or HMGB1.
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Perfilación de la Expresión Génica , Proteína HMGB1/fisiología , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Síndrome de Dificultad Respiratoria/metabolismo , Sepsis/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Proteína HMGB1/farmacología , Humanos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Neutrófilos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Síndrome de Dificultad Respiratoria/sangre , Síndrome de Dificultad Respiratoria/etiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily, modulates fibrinolysis by interacting with proteolytic mediators, including urokinase plasminogen activator (uPA). Although the roles of uPA and PAI-1 in plasmin generation and the degradation of fibrin are well known, recent evidence also suggests that they can participate in acute inflammatory conditions that involve neutrophil activation. In the present experiments, we found that the addition of PAI-1 to LPS- stimulated neutrophils resulted in enhanced nuclear translocation of NF-kappaB and increased production of the proinflammatory cytokines IL-1beta, Tnf-alpha, and Mip-2. uPA and the kringle domain (KD) of uPA potentiated cytokine expression and NF-kappaB activation by neutrophils cultured with LPS, and had additive effects when combined with PAI-1. The c-Jun N-terminal kinase (JNK) was activated after exposure of resting neutrophils to PAI-1 or the uPA KD. Enhanced JNK activation, but not that of other kinases induced by LPS, was present in neutrophils cocultured with PAI-1 or uPA KD. Inhibition of JNK activation prevented the potentiation of expression of proinflammatory cytokines induced by PAI-1 or uPA KD in LPS stimulated neutrophils. These results demonstrate that PAI-1 and uPA KD enhance LPS-induced neutrophil responses through their effects on JNK mediated pathways.
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Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , Activación Neutrófila/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/farmacología , Transporte Activo de Núcleo Celular , Animales , Citocinas/biosíntesis , Sinergismo Farmacológico , Humanos , Kringles , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , FN-kappa B/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
BACKGROUND: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS). METHODS: Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed. RESULTS: Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages. CONCLUSION: Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.
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Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Capilares/patología , Glutamina/farmacología , Hemo-Oxigenasa 1/metabolismo , Interleucina-1/administración & dosificación , Lipopolisacáridos/administración & dosificación , Macrófagos Alveolares/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Superficie Celular/metabolismo , Lesión Pulmonar Aguda/prevención & control , Animales , Líquido del Lavado Bronquioalveolar , Glutamina/administración & dosificación , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Lipopolysaccharide (LPS) induces acute lung injury (ALI) via Toll-like receptor 4 (TLR4)-mediated MAPK activation. The lipid A fraction of LPS is considered to be the active moiety, but whether the lipid A-TLR4 interaction accounts completely for ALI-associated MAPK activation in vivo has not been determined. The lipid A fraction of LPS induces a discrete MAPK activation pattern in murine ALI. Mice (C57BL/6J, C3H/HeJ) were treated with intratracheal instillations of purified lipid A or LPS (10, 30, and 100 microg per mouse) or vehicle. ALI was assessed by histology. Chromogenic myeloperoxidase (MPO) activity was measured in lung homogenates. MAPK expression was quantified by immunoblotting. In vitro ERK inhibitor studies using thioglycollate-elicited macrophages were also performed. MPO increased in a dose- and time-responsive fashion. Notably, MPO was 2.4-fold greater after lipid A compared with LPS and vehicle at 6 h after instillation (lipid A, 0.88 +/- 0.25 vs. LPS, 0.37 +/- 0.21 optical density units.min(-1).mg(-1); P < 0.05). However, ALI scores were comparable at 6 and 24 h between LPS and lipid A. MPO was also comparable in vehicle-treated or C3H/HeJ mice treated with LPS or lipid A at 6 and 24 h. Phospho-ERK activation was pronounced at 6 and 24 h after lipid A but not LPS treatment. In vitro studies confirmed the relationship between phospho-ERK activation and cytokine expression in macrophage stimulated with either LPS or lipid A. Compared with whole LPS, the lipid A fraction is associated with amplified whole lung MPO and ERK activation 6 h after intratracheal instillation in mice.
Asunto(s)
Lípido A/farmacología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neumonía/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Animales , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Pulmón/enzimología , Pulmón/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Peroxidasa/metabolismo , Neumonía/inducido químicamente , Neumonía/inmunología , Síndrome de Dificultad Respiratoria/inmunologíaRESUMEN
RATIONALE: The IL-1 receptor-associated kinase (IRAK-1) plays a central role in TLR2- and TLR4-induced activation of nuclear factor (NF)-kappaB, a critical event in the transcriptional regulation of many sepsis-associated proinflammatory mediators. There are two haplotypes for the IRAK-1 gene in Caucasians, with the variant haplotype consisting of five intron single-nucleotide polymorphisms (SNPs) and three exon SNPs. OBJECTIVES: To examine the functional significance of the IRAK-1 variant haplotype in modifying nuclear translocation of NF-kappaB and affecting outcomes from sepsis. MEASUREMENTS AND MAIN RESULTS: One hundred fifty-five Caucasian patients with sepsis were included. Twenty-one (14%) were homozygous for the IRAK-1 variant haplotype as determined by a SNP in which T is replaced with C at nucleotide 1,595 within exon 12 of the IRAK-1 gene. The IRAK-1 variant haplotype was associated with increased nuclear levels of NF-kappaB in LPS-stimulated peripheral blood neutrophils from patients with sepsis compared with that found in patients with wild-type IRAK-1 haplotype (p=0.0009). There was an increased incidence of shock (p=0.047) (odds ratio [OR], 2.9; 95% confidence interval [CI], 1.1-7.7), greater requirement for more prolonged mechanical ventilator support (p=0.04) (OR, 2.7; 95% CI, 1.05-6.9), and higher 60-d mortality (p=0.05) (OR, 2.7; 95% CI, 1.0-6.8) in patients with the IRAK-1 variant haplotype compared with wild type. CONCLUSIONS: These results indicate that the IRAK-1 variant haplotype is functionally significant in patients with sepsis, being associated with increased nuclear translocation of NF-kappaB, more severe organ dysfunction, and higher mortality.
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Haplotipos/genética , Péptidos y Proteínas de Señalización Intracelular/genética , FN-kappa B/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Interleucina-1/genética , Sepsis/fisiopatología , Adolescente , Adulto , Causas de Muerte , Núcleo Celular/metabolismo , Citosina , Exones/genética , Femenino , Homocigoto , Humanos , Mediadores de Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1 , Intrones/genética , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Polimorfismo Genético/genética , Polimorfismo de Nucleótido Simple/genética , Respiración Artificial , Choque Séptico/sangre , Choque Séptico/genética , Timina , Transcripción Genética/genética , Translocación Genética/genéticaRESUMEN
High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-kappaB-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS.
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Proteína HMGB1/metabolismo , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Proteína HMGB1/genética , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Unión Proteica , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/sangre , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.
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Lipopolisacáridos/administración & dosificación , Pulmón/inmunología , Pulmón/patología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Transporte Activo de Núcleo Celular/inmunología , Adolescente , Adulto , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Intubación Intratraqueal , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Masculino , FN-kappa B/sangre , FN-kappa B/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.
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Citocinas/genética , Genómica , Macrófagos Alveolares/fisiología , Neutrófilos/fisiología , Movimiento Celular , Quimiotaxis de Leucocito , Regulación de la Expresión Génica , Humanos , Cinética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-kappaB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-kappaB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKbeta, HMGB1 exposure resulted in activation of both IKKalpha and IKKbeta. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-kappaB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-kappaB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS.
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Proteína HMGB1/farmacología , Activación de Macrófagos/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Expresión Génica , Productos Finales de Glicación Avanzada , Quinasa I-kappa B , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Mutagénesis , FN-kappa B/genética , FN-kappa B/fisiología , Neutrófilos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas/fisiología , Receptor para Productos Finales de Glicación Avanzada , Receptores de Superficie Celular/genética , Receptores Inmunológicos/metabolismo , Factor 6 Asociado a Receptor de TNF , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Neutrophils are critical initiators and effectors of the innate immune system and express Toll-like receptor 2 (TLR2) and TLR4. Although signaling through pathways involving phosphoinositide 3-kinase (PI3-K) and the downstream kinase Akt (protein kinase B) plays a central role in modulating neutrophil chemotaxis and superoxide generation in response to engagement of G protein-coupled receptors, the importance of these kinases in affecting inflammatory responses of neutrophils stimulated through TLR2 has not been examined. In these experiments, we found activation of Akt in neutrophils stimulated with the TLR2-specific ligands peptidoglycan and the lipopeptide tri-palmitoyl-S-glyceryl-Cys-Ser-(Lys)(4) that occurred earlier and was of greater magnitude than that present after exposure to the TLR4 agonist LPS. The release of the proinflammatory mediators TNF-alpha and macrophage inflammatory protein-2 was inhibited in a dose-dependent manner by PI3-K blockade. The IC(50) for inhibition of peptidoglycan-stimulated Akt activation and macrophage inflammatory protein-2 release correlated closely, indicating linkage of these two events. PI3-K blockade did not inhibit nuclear translocation of NF-kappa B, but did prevent Ser(536) phosphorylation of the p65 subunit of NF-kappa B, an event required for maximal transcriptional activity of NF-kappa B. Inhibition of PI3-K also prevented activation of p38 mitogen-activated protein kinase and extracellular receptor-activated kinase 1/2 in TLR2-stimulated neutrophils. These results demonstrate that the PI3-K-Akt axis occupies a central role in TLR2-induced activation of neutrophils.
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Glicoproteínas de Membrana/fisiología , Activación Neutrófila , Neutrófilos/enzimología , Neutrófilos/patología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular/fisiología , Animales , Células Cultivadas , Quimiocina CXCL2 , Quimiocinas/metabolismo , Citocinas/metabolismo , Activación Enzimática , Inflamación/enzimología , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transporte de Proteínas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are generated in increased amounts in pathological, biological processes and can play a role in signal transduction. Neutrophils often accumulate in acute inflammatory reactions, at sites where elevated concentrations of ROS are present. ROS have been demonstrated to participate in the activation of intracellular signaling pathways, including those involved in modulating nuclear accumulation and transcriptional activity of NF-kappaB. However, the role of ROS in affecting such events in neutrophils has not been examined. Using exposure of murine bone marrow neutrophils to H2O2 as a model of oxidative stress, we found both strong and persistent activation of ERK1/2, p38, JNK, and PKB, but not the p21-activated kinase. Stimulating the bone marrow-derived neutrophils with H2O2 did not affect nuclear translocation of NF-kappaB. However, production and secretion of the proinflammatory cytokine TNF-alpha in LPS-stimulated neutrophils were inhibited by H2O2. Exposure of LPS- or TNF-alpha-stimulated neutrophils to H2O2 decreased nuclear translocation of NF-kappaB. LPS-induced activation of the transcriptional factor AP-1 was also inhibited by H2O2. This inhibition of nuclear accumulation of NF-kappaB by H2O2 was not caused by an impaired capacity of LPS to stimulate the IKK pathway or to direct oxidative effects on NF-kappaB but rather reflected diminished degradation of IkappaB-alpha. These results indicate that oxidative stress, despite being able to selectively activate intracellular kinases in bone marrow-derived neutrophils, also inhibits NF-kappaB activation and associated TNF-alpha expression. Such inhibitory effects on neutrophil activation may limit tissue damage produced by oxidative stress.