RESUMO
Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.
Assuntos
Ativação de Neutrófilo/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Síndrome do Desconforto Respiratório/enzimologia , Síndrome do Desconforto Respiratório/imunologia , Animais , Células Cultivadas , Endotoxemia/enzimologia , Endotoxemia/etiologia , Endotoxemia/imunologia , Ativação Enzimática/imunologia , Injeções Intraperitoneais , Lipopolissacarídeos/administração & dosagem , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Síndrome do Desconforto Respiratório/etiologiaRESUMO
The transcriptional regulatory factor nuclear factor (NF)-kappaB has a central role in modulating expression of proinflammatory mediators that are important in acute lung injury. In vitro studies have shown that competition between NF-kappaB and cAMP response element binding protein (CREB) for binding to the coactivator CREB-binding protein (CBP) is important in regulating transcriptional activity of these factors. In the present study, we examined in vivo interactions between CBP, CREB, and NF-kappaB in hemorrhage- or endotoxemia-induced acute lung injury. Association of CBP with CREB or the p65 subunit of NF-kappaB increased in the lungs after hemorrhage or endotoxemia. Inhibition of xanthine oxidase before hemorrhage, but not before endotoxemia, decreased p65-CBP interactions while increasing those between CREB and CBP. These alterations in CREB-CBP and p65-CBP interactions were functionally significant because xanthine oxidase inhibition before hemorrhage resulted in increased expression of the CREB-dependent gene c-Fos and decreased expression of macrophage inflammatory protein-2, a NF-kappaB-dependent gene. The present results show that the coactivator CBP has an important role in modulating transcription in vivo under clinically relevant pathophysiological conditions.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Endotoxemia/fisiopatologia , Hemorragia/fisiopatologia , Pulmão/fisiopatologia , NF-kappa B/fisiologia , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Animais , Proteína de Ligação a CREB , Quimiocina CXCL2 , Quimiocinas/genética , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Fator de Transcrição RelA , Transcrição Gênica/fisiologia , Xantina Oxidase/antagonistas & inibidoresRESUMO
Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotoxemia/enzimologia , Hemorragia/enzimologia , Pulmão/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ativação de Neutrófilo , Antagonistas Adrenérgicos alfa/administração & dosagem , Antagonistas Adrenérgicos beta/administração & dosagem , Ração Animal , Animais , Endotoxemia/metabolismo , Endotoxemia/patologia , Ativação Enzimática , Hemorragia/metabolismo , Hemorragia/patologia , Injeções Intraperitoneais , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Fentolamina/administração & dosagem , Propranolol/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tungstênio/administração & dosagem , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/metabolismoRESUMO
Acute lung injury is characterized by accumulation of neutrophils in the lungs, accompanied by the development of interstitial edema and an intense inflammatory response. To assess the role of neutrophils as early immune effectors in hemorrhage- or endotoxemia-induced lung injury, mice were made neutropenic with cyclophosphamide or anti-neutrophil antibodies. Endotoxemia- or hemorrhage-induced lung edema was significantly reduced in neutropenic animals. Activation of the transcriptional regulatory factor nuclear factor-kappaB after hemorrhage or endotoxemia was diminished in the lungs of neutropenic mice compared with nonneutropenic controls. Hemorrhage or endotoxemia was followed by increases in pulmonary mRNA and protein levels for interleukin-1beta (IL-1beta), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). Endotoxin-induced increases in proinflammatory cytokine expression were greater than those found after hemorrhage. The amounts of mRNA or protein for IL-1beta, MIP-2, and TNF-alpha were significantly lower after hemorrhage in the lungs of neutropenic versus nonneutropenic mice. Neutropenia was associated with significant reductions in IL-1beta and MIP-2 but not in TNF-alpha expression in the lungs after endotoxemia. These experiments show that neutrophils play a central role in initiating acute inflammatory responses and causing injury in the lungs after hemorrhage or endotoxemia.
Assuntos
Endotoxemia/imunologia , Hemoptise/imunologia , Neutrófilos/imunologia , Síndrome do Desconforto Respiratório/imunologia , Animais , Quimiocina CXCL2 , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas/metabolismo , Endotoxemia/induzido quimicamente , Expressão Gênica/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-1/metabolismo , Lipopolissacarídeos , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neutropenia/imunologia , Neutrófilos/enzimologia , Peroxidase/análise , RNA Mensageiro/análise , Síndrome do Desconforto Respiratório/induzido quimicamente , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: Acute tubular necrosis (ATN) and the ensuing renal failure induced by ischemia and reperfusion injury (I/R) remain a major cause of morbidity and mortality among patients in the intensive care unit. Although it is well established that exogenous tumor necrosis factor-alpha (TNF) induces renal injury, it remains unknown whether ischemia and/or reperfusion activates the signaling mechanisms required for renal TNF production. We hypothesized that ischemia and/or reperfusion would activate the oxidant sensitive TNF transcription factor, nuclear factor kappa B (NFkappaB), and thereby lead to renal TNF production. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, after which various periods of renal ischemia, with or without reperfusion, were induced in rats. At different time intervals, kidneys were harvested and NFkappaB activation (electrophoretic mobility shift assay), TNF mRNA content (RT-PCR), and TNF bioactivity (WEHI-164 cell clone cytotoxicity assay) were determined. RESULTS: Results indicate that 15 minutes of ischemia alone activates NFkappaB, whereas peak activation occurred at 30 minutes of ischemia alone. NFkappaB remained activated through 60 minutes reperfusion. Thirty minutes of ischemia is required to induce renal TNF mRNA production; however, renal TNF protein expression and bioactivity peaked following 1 hour of ischemia and 2 hours reperfusion. CONCLUSIONS: These results are the initial demonstration that renal ischemia, with or without reperfusion, activates the TNF transcription factor NFkappaB and increases TNF bioactivity in the kidney.
Assuntos
Isquemia/imunologia , Rim/irrigação sanguínea , Rim/imunologia , NF-kappa B/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Masculino , NF-kappa B/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Fator de Necrose Tumoral alfa/genéticaRESUMO
Activation of the nuclear regulatory factor NF-kappaB occurs in the lungs of patients with the acute respiratory distress syndrome (ARDS) and may contribute to the increased expression of immunoregulatory cytokines and other proinflammatory mediators in this setting. Because of the important role that NF-kappaB activation appears to play in the development of acute lung injury, we examined cytoplasmic and nuclear NF-kapppaB counterregulatory mechanisms, involving IkappaB proteins, in alveolar macrophages obtained from 7 control patients without lung injury and 11 patients with established ARDS. Cytoplasmic levels of the NF-kappaB subunits p50, p65, and c-Rel were significantly decreased in alveolar macrophages from patients with ARDS, consistent with enhanced migration of liberated NF-kappaB dimers from the cytoplasm to the nucleus. Cytoplasmic and nuclear levels of IkappaBalpha were not significantly altered in alveolar macrophages from patients with established ARDS, compared with controls. In contrast, nuclear levels of Bcl-3 were significantly decreased in patients with ARDS compared with controls (P = 0.02). No IkappaBgamma, IkappaBbeta, or p105 proteins were detected in the cytoplasm of alveolar macrophages from control patients or patients with ARDS. The presence of activated NF-kappaB in alveolar macrophages from patients with established ARDS implies the presence of an ongoing stimulus for NF-kappaB activation. In this setting, appropriate counterregulatory mechanisms to normalize nuclear levels of NF-kappaB and to suppress NF-kappaB-mediated transcription, such as increased cytoplasmic and nuclear IkappaBalpha levels or decreased Bcl-3 levels, appeared to be induced. Nevertheless, even though counterregulatory mechanisms to NF-kappaB activation are activated in lung macrophages of patients with ARDS, NF-kappaB remains activated. These results suggest that fundamental abnormalities in transcriptional mechanisms involving NF-kappaB and important in the inflammatory response occur in the lungs of patients with ARDS.
Assuntos
Proteínas I-kappa B , Macrófagos Alveolares/metabolismo , NF-kappa B/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Proteína 3 do Linfoma de Células B , Lavagem Broncoalveolar , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Macrófagos Alveolares/imunologia , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa , Subunidade p50 de NF-kappa B , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Síndrome do Desconforto Respiratório/imunologia , Índice de Gravidade de Doença , Fator de Transcrição RelA , Fatores de Transcrição/metabolismoRESUMO
Acute inflammatory lung injury occurs frequently in the setting of severe infection or blood loss. Accumulation of activated neutrophils in the lungs and increased pulmonary proinflammatory cytokine levels are major characteristics of acute lung injury. In the present experiments, we examined mechanisms leading to neutrophil accumulation and activation in the lungs after endotoxemia or hemorrhage. Levels of IL-1 beta, TNF-alpha, and macrophage inflammatory protein-2 mRNA were increased in lung neutrophils from endotoxemic or hemorrhaged mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic, hemorrhaged, or control mice. The transcriptional regulatory factors NF-kappa B and cAMP response element binding protein were activated in lung but not blood neutrophils after hemorrhage or endotoxemia. Xanthine oxidase inhibition, achieved by feeding allopurinol or tungsten-containing diets, did not affect neutrophil trafficking to the lungs after hemorrhage or endotoxemia. Xanthine oxidase inhibition did prevent hemorrhage- but not endotoxemia-induced increases in proinflammatory cytokine expression among lung neutrophils. Hemorrhage- or endotoxemia-associated activation of NF-kappa B in lung neutrophils was not affected by inhibition of xanthine oxidase. cAMP response element binding protein activation was increased after hemorrhage, but not endotoxemia, in mice fed xanthine oxidase-inhibiting diets. Our results indicate that xanthine oxidase modulates cAMP response element binding protein activation and proinflammatory cytokine expression in lung neutrophils after hemorrhage, but not endotoxemia. These findings suggest that the mechanisms leading to acute inflammatory lung injury after hemorrhage differ from those associated with endotoxemia.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Endotoxemia/metabolismo , Hemorragia/metabolismo , Pulmão/metabolismo , NF-kappa B/fisiologia , Ativação de Neutrófilo , Espécies Reativas de Oxigênio/fisiologia , Animais , Movimento Celular , Quimiocina CXCL2 , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotoxemia/enzimologia , Endotoxemia/patologia , Ativação Enzimática , Hemorragia/enzimologia , Hemorragia/patologia , Interleucina-1/biossíntese , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monocinas/biossíntese , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Peroxidase/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Xantina Oxidase/antagonistas & inibidoresRESUMO
Hemorrhage and endotoxemia are associated with neutrophil accumulation in the lungs and the development of acute inflammatory lung injury. Because alterations in the rate of apoptosis may affect the number and function of neutrophils in the lungs, we determined the percentage of neutrophils undergoing apoptosis in the lungs of control, hemorrhaged, or endotoxemic mice. In control mice, 18.5 +/- 1.2% of pulmonary neutrophils were apoptotic. The proportion of apoptotic neutrophils in the lungs was significantly decreased 1 h after hemorrhage (6.5 +/- 1.6%, P < 0.01 compared to control) or endotoxemia (7.0 +/- 0.9%, P < 0.01 compared to control). Between 1 and 24 h after endotoxemia or hemorrhage, the proportion of apoptotic neutrophils in the lung remained significantly depressed compared to that in control, unmanipulated mice. By 48 h, the proportion of apoptotic neutrophils returned to baseline levels in the lungs of hemorrhaged (21.4 +/- 1.4%) or endotoxemic (16.4 +/- 1. 6%) mice. Lung neutrophil IL-1beta mRNA was significantly increased from that of control mice [i.e., 0.12 +/- 0.06 relative absorbance units (RAU)] 1 h after hemorrhage (5.19 +/- 0.068 RAU, P < 0.05 compared to control) or endotoxemia (8.90 +/- 1.53 RAU, P < 0.01 compared to control). In IL-1beta-deficient mice, there was no significant difference in lung neutrophil apoptosis or neutrophil entry into the lung after hemorrhage or endotoxemia compared to wild-type mice. Our results show that apoptosis among lung neutrophils is decreased for more than 24 h after hemorrhage or endotoxemia. Although IL-1beta expression is increased in lung neutrophils under these conditions, IL-1beta is not responsible for either the influx of neutrophils into the lung or the reduction of apoptosis in neutrophil populations after hemorrhage or endotoxemia.
Assuntos
Apoptose/imunologia , Endotoxemia/imunologia , Hemorragia/imunologia , Interleucina-1/imunologia , Neutrófilos/imunologia , Animais , Movimento Celular , Interleucina-1/biossíntese , Interleucina-1/genética , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/citologiaRESUMO
Endotoxemia produces elevations in catecholamine levels in the pulmonary and systemic circulation as well as rapid increases in neutrophil number and proinflammatory cytokine expression in the lungs. In the present experiments, we examined the effects of endogenous and exogenous adrenergic stimulation on endotoxin-induced lung neutrophil accumulation and activation. Levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and macrophage inflammatory protein (MIP)-2 mRNAs were increased in lung neutrophils from endotoxemic mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic or control mice. Treatment with the beta-adrenergic antagonist propranolol before endotoxin administration did not affect trafficking of neutrophils to the lungs or the expression of IL-1beta, TNF-alpha, or MIP-2 by lung neutrophils. Administration of the alpha-adrenergic antagonist phentolamine before endotoxemia did not alter lung neutrophil accumulation as measured by myeloperoxidase (MPO) levels but did result in significant increases in IL-1beta, TNF-alpha, and MIP-2 mRNA expression by lung neutrophils compared with endotoxemia alone. Administration of the alpha1-adrenergic agonist phenylephrine before endotoxin did not affect trafficking of neutrophils to the lungs but was associated with significantly increased expression of TNF-alpha and MIP-2 mRNAs by lung neutrophils compared with that found after endotoxin alone. In contrast, treatment with the alpha2-adrenergic agonist UK-14304 prevented endotoxin-induced increases in lung MPO and lung neutrophil cytokine mRNA levels. The suppressive effects of UK-14304 on endotoxin-induced increases in lung MPO were not affected by administration of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester. These data demonstrate that the initial accumulation and activation of neutrophils in the lungs after endotoxemia can be significantly diminished by alpha2-adrenergic stimulation. Therapy with alpha2-adrenergic agents may have a role in modulating inflammatory pulmonary processes associated with sepsis-induced acute lung injury.
Assuntos
Catecolaminas/farmacologia , Catecolaminas/fisiologia , Lipopolissacarídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Endotoxinas/farmacologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The cytokine interleukin (IL) 18 (formerly interferon gamma-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL-18-stimulated HIV-1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 microM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-kappaB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-kappaB.
Assuntos
HIV-1/efeitos dos fármacos , Interleucina-18/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Replicação Viral/efeitos dos fármacos , Sequência de Bases , Transporte Biológico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , HIV-1/fisiologia , Humanos , Interleucina-6/biossíntese , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Sondas de Oligonucleotídeos , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The heart is a tumor necrosis factor (TNFalpha) producing organ. Locally (v systemically)-produced TNFalpha likely contributes to myocardial dysfunction via direct suppression of myocardial contractile function, the induction of myocardial apoptosis, and the genesis of cardiac hypertrophy. Although recent studies have demonstrated increased myocardial TNFalpha following endotoxemia, it remains unknown whether shock, in the absence of sepsis, activates myocardial nuclear factor kappa B (NFkappaB, a TNFalpha transcription factor) and/or increases TNFalpha in the heart. To study this, rats were hemorrhaged and resuscitated, after which hearts were harvested and analysed for evidence of NFkappaB activation (electrophoretic mobility shift assay) and assayed for TNFalpha levels. Hemorrhage and resuscitation activated NFkappaB and resulted in a dramatic increase in myocardial TNFalpha. This study constitutes the initial demonstration that hemorrhagic shock activates the signaling mechanisms which culminate in increased myocardial TNFalpha. Indeed, this may have important clinical implications, since hemorrhage is a frequent complication of both iatrogenic and accidental trauma, as well as a potent instigator of multiple organ failure.
Assuntos
Hemorragia/metabolismo , Miocárdio/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Coração , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The nuclear regulatory factor (NF)-kappa B is activated in the lungs of patients with acute respiratory distress syndrome (ARDS). In experimental models of acute lung injury, activation of NF-kappa B contributes to the increased expression of immunoregulatory cytokines and other proinflammatory mediators in the lungs. Because of the important role that NF-kappa B activation appears to play in the development of acute lung injury, we examined cytoplasmic and nuclear NF-kappa B counterregulatory mechanisms in lung mononuclear cells, using a murine model in which inflammatory lung injury develops after blood loss. Sustained activation of NF-kappa B was present in lung mononuclear cells over the 4-h period after blood loss. The activation of NF-kappa B after hemorrhage was accompanied by alterations in levels of the NF-kappa B regulatory proteins I kappa B alpha and Bcl-3. Cytoplasmic and nuclear I kappa B alpha were increased and nuclear Bcl-3 was decreased during the first hour after blood loss, but, by 4 h posthemorrhage, cytoplasmic and nuclear I kappa B alpha levels were decreased and nuclear levels of Bcl-3 were increased. Inhibition of xanthine oxidase activity in otherwise unmanipulated unhemorrhaged mice resulted in increased levels of I kappa B alpha and decreased amounts of Bcl-3 in nuclear extracts from lung mononuclear cells. No changes in the levels of nuclear I kappa B alpha or Bcl-3 occurred after hemorrhage when xanthine oxidase activity was inhibited. These results demonstrate that blood loss, at least partly through xanthine oxidase-dependent mechanisms, produces alterations in the levels of both I kappa B alpha and Bcl-3 in lung mononuclear cell populations. The effects of hemorrhage on proteins that regulate activation of NF-kappa B may contribute to the frequent development of inflammatory lung injury in this setting.
Assuntos
Hemorragia/fisiopatologia , Proteínas I-kappa B , Pulmão/metabolismo , NF-kappa B/biossíntese , Alopurinol/farmacologia , Animais , Proteína 3 do Linfoma de Células B , Sequência de Bases , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Genes de Imunoglobulinas , Hemorragia/patologia , Homeostase , Inflamação , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Proteínas Proto-Oncogênicas/biossíntese , Fatores de TranscriçãoRESUMO
The expression of proinflammatory and immunoregulatory cytokines rapidly increases in the lungs after hemorrhage, and such alterations contribute to the frequent development of acute inflammatory lung injury in this setting. Blood loss also produces elevations in catecholamine concentrations in the pulmonary and systemic circulation. In the present experiments, we used alpha- and beta-adrenergic receptor blockade to examine in vivo interactions between hemorrhage-induced adrenergic stimulation and pulmonary cytokine expression. Treatment of mice with the alpha-adrenergic receptor antagonist phentolamine prevented not only the elevation in mRNA levels of IL-1beta, TNF-alpha, and TGF-beta1, the increase in IL-1beta protein, but also the activation of nuclear factor (NF)-KB and cyclic AMP response element binding protein, which occurred in lung cells of untreated animals during the first hour after hemorrhage. In contrast, treatment before hemorrhage with the beta-adrenergic receptor antagonist propranolol was associated with increases in mRNA levels for IL-1beta, TNF-alpha, and TGF-beta1, which were greater than those present in untreated hemorrhaged mice, and did not prevent hemorrhage-associated increases in lung IL-1beta protein. Treatment with propranolol prevented hemorrhage-induced phosphorylation of cyclic AMP response element binding protein, but increased hemorrhage-associated activation of NF-KB. These results demonstrate that hemorrhage initially increases pulmonary cytokine expression through alpha- but not beta-adrenergic stimulation, and suggest that such alpha-adrenergic-mediated effects occur through activation of the transcriptional regulatory factor NF-kappaB.
Assuntos
Catecolaminas/fisiologia , Citocinas/biossíntese , Hemorragia/imunologia , Pulmão/metabolismo , NF-kappa B/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas/genética , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fentolamina/farmacologia , Fosforilação , RNA Mensageiro/análise , Receptores Adrenérgicos/fisiologiaRESUMO
Increased expression of proinflammatory cytokines appears to be an important factor contributing to the development of acute lung injury. In murine models, mRNA levels of proinflammatory and immunoregulatory cytokines, including IL-1alpha, IL-1beta, TGF-beta1, and TNF-alpha, are increased in intraparenchymal lung mononuclear cells 1 h after hemorrhage. Binding elements for the nuclear transcriptional regulatory factors, nuclear factor kappaB (NF-kappaB), CCAAT/enhancer binding protein beta (C/EBPbeta), serum protein 1 (Sp1), activator protein 1 (AP-1), and the cyclic AMP response-element binding protein (CREB) are present in the promoter regions of numerous cytokine genes, including those whose expression is increased after blood loss. To investigate early transcriptional mechanisms which may be involved in regulating pulmonary cytokine expression after hemorrhage, we examined in vivo activation of these five nuclear transcriptional factors among intraparenchymal lung mononuclear cells obtained in the immediate post-hemorrhage period. Activation of NF-kappaB and CREB, but not C/EBPbeta, Sp1, or AP-1, was present in lung mononuclear cells isolated from mice 15 min after hemorrhage. Inhibition of xanthine oxidase by prior feeding with either an allopurinol-supplemented or a tungsten-enriched diet prevented hemorrhage-induced activation of CREB, but not NF-kappaB. These results demonstrate that hemorrhage leads to rapid in vivo activation in the lung of CREB through a xanthine oxidase-dependent mechanism and of NF-kappaB through other pathways, and suggest that the activation of these transcriptional factors may have an important role in regulating pulmonary cytokine expression and the development of acute lung injury after blood loss.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hemorragia/metabolismo , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/patologia , NF-kappa B/metabolismo , Animais , Hemorragia/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/metabolismoRESUMO
Hyperoxia-associated production of reactive oxygen species leads to neutrophil infiltration into the lungs and increased pulmonary proinflammatory cytokine expression. However, the initial events induced by hyperoxia, and leading to acute inflammatory lung injury, remain incompletely characterized. To explore this issue, we examined nuclear transcriptional regulatory factor (NF-kappaB and NF-IL-6) activation and cytokine expression in the lungs following 12 to 48 h of hyperoxia exposure. No increases in cytokine (IL-1beta, IL-6, IL-10, TGF-beta, TNF-alpha, IFN-gamma) expression nor in NF-kappaB activation were found after 12 h of hyperoxia. Following 24 h of hyperoxia, NF-kappaB activation and increased levels of TNF-alpha mRNA were present in pulmonary lymphocytes. By 48 h of hyperoxia, amounts of IFN-gamma and TNF-alpha protein as well as mRNA were increased in the lungs, and NF-kappaB continued to show activation, even though no histologic abnormalities were present. These results show that hyperoxia activates NF-kappaB in the lungs before any increase in proinflammatory cytokine protein occurs, and suggest that NF-kappaB activation may represent an initial event in the proinflammatory sequence induced by hyperoxia.
Assuntos
Regulação da Expressão Gênica , Hiperóxia/genética , Interferon gama/biossíntese , Pulmão/imunologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Líquido da Lavagem Broncoalveolar , Hiperóxia/imunologia , Interferon gama/genética , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa/genéticaRESUMO
The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.
Assuntos
DNA Mitocondrial/genética , Deleção de Genes , Adulto , Linhagem Celular , Células Clonais , Feminino , Humanos , Modelos Genéticos , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: The expression of proinflammatory cytokines is rapidly increased in experimental models of the acute respiratory distress syndrome (ARDS), in patients at risk for ARDS, and in patients with established ARDS. Because multiple cytokines are present in bronchoalveolar lavage fluid, a common, proximal activation mechanism may operate in these settings. The proinflammatory cytokines whose expression is increased in the lungs of patients with ARDS have binding sequences in their enhancer/promoter regions for transcriptional regulatory proteins, such as nuclear factor-kappa B (NF-kappa B), nuclear factor-IL6 (NF-IL6), cyclic adenosine monophosphate responsive element binding protein, serum protein-1, and activating protein-1. To test the hypothesis that activation of one or more of these nuclear transcriptional regulatory factors might provide a common mechanism for the simultaneous expression of multiple cytokine genes in the setting of ARDS, we measured activation of these factors in alveolar macrophages from patients with ARDS and from controls. DESIGN: Prospective, clinical study. SETTING: Medical and surgical intensive care units at a university hospital and a county hospital. PATIENTS: Twelve patients, six with established ARDS and six control patients without lung injury. INTERVENTIONS: Patients with ARDS and controls underwent fiberoptic bronchoscopy and bronchoalveolar lavage. Alveolar macrophages were isolated from lavage fluid and the nuclear proteins were extracted. Activation of transcriptional factors NF-kappa B, NF-IL6, cyclic adenosine monophosphate responsive element binding protein, activating protein-1, and serum protein-1 was determined using an electrophoretic mobility shift assay, followed by densitometry of the autoradiographed gels. MEASUREMENTS AND MAIN RESULTS: There were no significant differences in gender, age, tobacco smoking, Acute Physiology and Chronic Health Evaluation II score, quantity of lavage fluid, or number of alveolar macrophages in lavage specimens in the patient groups. Acute Lung Injury score and the Pao2/Fio2 ratio differed significantly between controls and ARDS patients: 0.46 +/- 0.17 vs. 2.74 +/- 0.14 (p < .0001) and 310 +/- 45 torr (41.3 +/- 6.0 kPa) vs. 150 +/- 11 torr (21.3 +/- 1.5 kPa) (p < .006), respectively. The mean Fio2 of the control patients was not significantly different from the mean Fio2 of ARDS patients: 0.47 +/- 0.11 vs. 0.55 +/- 0.6 (p = .53). Patients with ARDS had significantly (p < .02) increased activation of NF-kappa B in alveolar macrophages compared with patients without the syndrome. There was no evidence of increased activation of the transcriptional factors activating protein-1, serum protein-1, NF-IL6, or cyclic adenosine monophosphate responsive element binding protein in alveolar macrophages from ARDS vs. control patients. CONCLUSIONS: These experiments demonstrated increased in vivo activation of the nuclear transcriptional regulatory factor NF-kappa B (but not NF-IL6, cyclic adenosine monophosphate responsive element binding protein, activating protein-1, or serum protein-1) in alveolar macrophages from patients with ARDS. Because binding sequences for NF-kappa B are present in the enhancer/promoter sequences of multiple proinflammatory cytokines, activation of NF-kappa B may contribute to the increased expression of multiple cytokines in the lung in the setting of established ARDS.
Assuntos
Macrófagos Alveolares/metabolismo , NF-kappa B/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Adulto , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , NF-kappa B/genética , Estudos Prospectivos , Síndrome do Desconforto Respiratório/genética , Índice de Gravidade de Doença , Ativação TranscricionalRESUMO
Hemorrhage rapidly increases the expression of proinflammatory and immunoregulatory cytokines in the lungs. Binding elements for the nuclear transcriptional regulatory factors (NF)-kappa B and NF-IL6 (C/EBP beta) are present in the promoter regions of multiple cytokine genes, including those whose expression is increased after blood loss. In the present experiments, we found increased activation in vivo of NF-kappa B in lung mononuclear cells, but not in splenocytes, taken from mice 1 h after hemorrhage. In contrast, hemorrhage did not activate NF-IL6 in lung cells or splenocytes. Inhibition of xanthine oxidase by prior feeding of a tungsten-enriched diet prevented hemorrhage-induced activation in lung cells of NF-kappa B. Incubating splenocytes in vitro with xanthine oxidase activated NF-kappa B but not NF-IL6. Xanthine oxidase-induced activation of NF-kappa B was inhibited by manganese superoxide dismutase, but not by catalase. These results suggest that xanthine oxidase-mediated superoxide anion-dependent activation of NF-kappa B occurs in vivo and in vitro. This mechanism may contribute to increased lung cytokine responses after hemorrhage.
Assuntos
Hemorragia/fisiopatologia , Pulmão/metabolismo , NF-kappa B/fisiologia , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Catalase/farmacologia , Proteínas de Ligação a DNA/fisiologia , Hemorragia/patologia , Zíper de Leucina , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Sondas de Oligonucleotídeos/genética , Baço/metabolismo , Baço/patologia , Superóxido Dismutase/farmacologia , Xantina Oxidase/antagonistas & inibidoresRESUMO
Hemorrhage rapidly increases plasma xanthine oxidase levels as well as the expression of proinflammatory and immunoregulatory cytokines in the lungs. To determine the role of circulating xanthine oxidase (XO), as well as other plasma factors, in affecting pulmonary cytokine expression, we conducted studies in which plasma from hemorrhaged mice was transferred into unhemorrhaged recipient mice. Administration of posthemorrhage plasma to recipient mice increased the levels of mRNA for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta 1 (TGF-beta 1) in lung mononuclear cells. No enhancement of mRNA levels for these cytokines was found in the lungs of mice given allopurinol-treated posthemorrhage plasma or fed a tungsten-enriched, XO-depleting diet prior to transfer of posthemorrhage plasma. Among the nuclear transcriptional regulatory factors examined, only the cyclic AMP response-element binding protein (CREB) was activated in nuclear extracts from lung mononuclear cells of mice that were given posthemorrhage plasma. No activation of nuclear factor-kappa B (NF-kappa B), nuclear factor interleukin 6 (NF-IL6), activating protein-1 (AP-1), or serum protein-1 (SP-1) was found. These results suggest that the mechanism for hemorrhage-induced increases in pulmonary cytokine expression is by activation of the enhancer CREB through a tissue XO-dependent pathway initiated by plasma-borne mediators.