RESUMEN
Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an active TLR-4 pathway. Proteome profiling array revealed that LPS combined with irradiation resulted in a strong phosphorylation of cAMP response element-binding protein (CREB). Inhibition of CREB binding protein (CBP) by the specific inhibitor ICG-001 not only abrogated the LPS-induced radioresistance but even led to an increase in radiosensitivity. The sensitization caused by ICG-001 could be attributed to a reduction of DNA double-strand break (DSB) repair. It is shown that in NSCLC cells LPS leads to a CREB dependent radioresistance which is, however, reversible through CBP inhibition by the specific inhibitor ICG-001. These findings indicate that the combined treatment with radiation and CBP inhibition may improve survival of NSCLC patients suffering from pulmonary infections.
RESUMEN
Pulmonary infections are frequent complications in lung cancer and may worsen its outcome and survival. Inflammatory mediators are suspected to promote tumor growth in non-small-cell lung cancer (NSCLC). Hence, bacterial pathogens may affect lung cancer growth by activation of inflammatory signalling. Against this background, we investigated the effect of purified lipoteichoic acids (LTA) of Staphylococcus aureus (S. aureus) on cellular proliferation and liberation of interleukin (IL)-8 in the NSCLC cell lines A549 and H226. A549 as well as H226 cells constitutively expressed TLR-2 mRNA. Even in low concentrations, LTA induced a prominent increase in cellular proliferation of A549 cells as quantified by automatic cell counting. In parallel, metabolic activity of A549 cells was enhanced. The increase in proliferation was accompanied by an increase in IL-8 mRNA expression and a dose- and time-dependent release of IL-8. Cellular proliferation as well as the release of IL-8 was dependent on specific ligation of TLR-2. Interestingly, targeting IL-8 by neutralizing antibodies completely abolished the LTA-induced proliferation of A549 cells. The pro-proliferative effect of LTA could also be reproduced in the squamous NSCLC cell line H226. In summary, LTA of S. aureus induced proliferation of NSCLC cell lines of adeno- and squamous cell carcinoma origin. Ligation of TLR-2 followed by auto- or paracrine signalling by endogenously synthesized IL-8 is centrally involved in LTA-induced tumor cell proliferation. Therefore, pulmonary infections may exert a direct pro-proliferative effect on lung cancer growth.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/patología , Proliferación Celular , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Neoplasias Pulmonares/patología , Ácidos Teicoicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Humanos , Técnicas In Vitro , Interleucina-8/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Staphylococcus aureus , Receptor Toll-Like 2/metabolismo , Células Tumorales CultivadasRESUMEN
The inflammatory tumor microenvironment plays a crucial role in tumor progression. In lung cancer, both bacterial infections and neutrophilia are associated with a poor prognosis. In this study, we characterized the effect of isolated human neutrophils on proliferation of the non-small cell lung cancer (NSCLC) cell line A549 and analyzed the impact of A549-neutrophil interactions on inflammatory mediator generation in naive and lipopolysaccharide (LPS)-exposed cell cultures. Co-incubation of A549 cells with neutrophils induced proliferation of resting and LPS-exposed A549 cells in a dose-dependent manner. In transwell-experiments, this effect was demonstrated to depend on direct cell-to-cell contact. This pro-proliferative effect of neutrophils on A549 cells could be attenuated by inhibition of neutrophil elastase activity, but not by oxygen radical neutralization. Correspondingly, neutrophil elastase secretion, but not respiratory burst, was specifically enhanced in co-cultures of A549 cells and neutrophils. Moreover, interference with COX-2 activity by indomethacin or the specific COX-2 inhibitor NS-398 also blunted the increased A549 proliferation in the presence of neutrophils. In parallel, a massive amplification of COX-2-dependent prostaglandin E2 synthesis was detected in A549-neutrophil co-cultures. These findings suggest that direct cell-cell interactions between neutrophils and tumor cells cause release of inflammatory mediators which, in turn, may enhance tumor growth in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Comunicación Celular/inmunología , Ciclooxigenasa 2/metabolismo , Neoplasias Pulmonares/inmunología , Neutrófilos/inmunología , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Dinoprostona/biosíntesis , Humanos , Elastasa de Leucocito/metabolismo , Lipopolisacáridos/farmacología , Neutrófilos/enzimología , Nitrobencenos/farmacología , Sulfonamidas/farmacología , Microambiente Tumoral/inmunologíaRESUMEN
Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Receptores ErbB/metabolismo , Lipopolisacáridos/inmunología , Neoplasias Pulmonares/patología , Proteínas de la Membrana/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/análisis , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Humanos , Indometacina/farmacología , Antígeno Ki-67/biosíntesis , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Nitrobencenos/farmacología , Sulfonamidas/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismoRESUMEN
Proinflammatory cytokines are centrally involved in tumor progression and survival in non-small cell lung cancer, and both the presence of infiltrating neutrophils and bacterial infection in the lung may indicate a poor prognosis. Against this background, we investigated the effect of the bacterial cell wall component lipopolysaccharide (LPS) on interleukin (IL)-6 and IL-8 synthesis in the non-small cell lung cancer line A549 and in A549-neutrophil cocultures. The LPS induced a dose-dependent and time-dependent release of IL-8 from A549 cells, whereas IL-6 could not be detected. Interestingly, in A549-neutrophil cocultures, IL-8 synthesis was massively amplified and IL-6 was also released, compared with the respective monocultures. The A549 cells were identified as the primary cellular source of these cytokines, as enhanced cytokine mRNA transcription was detected in this cell type, although not in neutrophils in the coculture system. Experiments done in transwells indicated that direct cell-cell contact was a prerequisite for the increased cytokine generation. Inhibition of tumor necrosis factor-alpha bioactivity by neutralizing antibodies and blocking cyclooxygenase-2 activity blunted the enhanced cytokine generation in the coculture system. Amplification of LPS-induced cytokine secretion could be reproduced when the small cell lung cancer cell line H69 was cocultured with neutrophils. When the Gram-positive cell wall component lipoteichoic acid was used instead of LPS, cytokine synthesis was also amplified in A549-neutrophil cocultures, to a similar extent to that observed with LPS. These data indicate that interaction between bacterial pathogens, neutrophils, and tumor cells might amplify the release of proinflammatory cytokines which may promote tumor growth in vivo.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Citocinas/biosíntesis , Inflamación/inmunología , Lipopolisacáridos/farmacología , Neoplasias Pulmonares/inmunología , Neutrófilos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Comunicación Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Técnicas de Cocultivo , Inhibidores de la Ciclooxigenasa 2/farmacología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inflamación/genética , Inflamación/fisiopatología , Mediadores de Inflamación/farmacología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Interleucina-8/efectos de los fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Neutrófilos/efectos de los fármacos , Neumonía Bacteriana/complicaciones , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/fisiopatología , ARN Mensajero/metabolismo , Ácidos Teicoicos/farmacología , Factores de Tiempo , Activación Transcripcional/efectos de los fármacosRESUMEN
OBJECTIVE: In sepsis, cardiac function is frequently depressed. Microcirculatory disturbances as evidenced in most organs may extend to the coronary circulation and may play a role in the occurrence of cardiac dysfunction. Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH), pore-forming exotoxins of clinically relevant bacteria, have recently been demonstrated to evoke cardiac dysfunction in isolated rat hearts by activating myocardial eicosanoid metabolism. alpha-Toxin activates synthesis of thromboxane (Tx) A2, ECH of cysteinyl-leukotrienes (Sibelius U, Grandel U, Buerke M, et al: Leukotriene-mediated coronary vasoconstriction and loss in myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused reat hearts. Crit Care Med 2003; 3:683-688, Sibelius U, Grandel U, Buerke M, et al: Staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts-Role of Tx formation. Circulation 2000; 101:78-85). We now investigated whether cardiac dysfunction in response to alpha-toxin and ECH is caused by disturbances of regional cardiac perfusion. DESIGN: A prospective, experimental study. SETTING: A research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Changes of regional perfusion were investigated by using colored microspheres in isolated rat hearts perfused with alpha-toxin or ECH either at constant coronary perfusion pressure or constant coronary flow rate. Significance of toxin-activated eicosanoid generation was evaluated by pharmacologic interventions. MEASUREMENTS AND MAIN RESULTS: By eliciting eicosanoid formation, both toxins caused an increase in coronary vascular resistance and a loss in contractile function. In ECH-perfused hearts, reduction of regional perfusion predominantly occurred in subendocardial sections in either perfusion mode (coronary perfusion pressure or coronary flow rate). When synthesis of cysteinyl-leukotrienes was blocked by the 5-lipooxygenase inhibitor MK-886, disturbances of regional perfusion and the associated cardiac dysfunction were largely prevented. Coronary perfusion of alpha-toxin caused a decrease of regional perfusion that was more pronounced in subepicardial layers. Inhibiting the release of TxA2 by blocking the cyclooxygenase with indomethacin attenuated the perfusion abnormalities and the cardiodepression in response to alpha-toxin. CONCLUSIONS: Bacterial exotoxins of clinically relevant bacteria may impair cardiac function by eliciting distinct coronary perfusion abnormalities via release of vasoactive eicosanoids.
Asunto(s)
Toxinas Bacterianas/farmacología , Circulación Coronaria/efectos de los fármacos , Eicosanoides/metabolismo , Proteínas de Escherichia coli/farmacología , Proteínas Hemolisinas/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Animales , Técnicas In Vitro , Masculino , Ratas , Ratas WistarRESUMEN
Bacterial toxins cause cardiac dysfunction and death through an inflammatory process, but the mechanism remains unclear. Simvastatin is recognized as having anti-inflammatory properties beyond its lipid-lowering effects. We examined Staphylococcus aureus alpha-toxin in isolated heart and in vivo models and tested simvastatin's effects in sepsis. Isolated Langendorff-perfused rat hearts were exposed to a recirculating perfusate containing alpha-toxin (0.5 microg mL(-1)). Compared with controls, there was a significant increase in coronary perfusion pressure and fall in myocardial performance. Significant increases in p53 expression and apoptosis (1.3 +/- 0.5 to 7.1 +/- 1.4 terminal deoxynucleaotidyl transferase nick end labeling-positive cells; P < 0.05) compared with controls were observed, but markers of necrosis were similar. In parallel experiments, anaesthetized rats receiving alpha-toxin (40 microg kg(-1), i.v.) had in vivo hemodynamic parameters and serum markers of necrosis monitored for 4 h before the hearts were analyzed for histological change, p53 expression, and apoptosis. Over 4 h, alpha-toxin exposure produced substantial hemodynamic effects. In addition, p53 expression (0.2 +/- 0.2 to 7.1 +/- 0.5 p53-positive myocytes; P < 0.05), TNF-alpha levels, the degree of apoptosis, and markers of necrosis were all significantly increased compared with control animals. Pretreatment with simvastatin protected against alpha-toxin-induced sepsis associated with reduced p53, TNF-alpha, apoptosis, and necrosis. We found significant changes in systemic hemodynamics, coronary perfusion pressure, myocardial function, and increased p53 expression with apoptosis due to bacterial exotoxin. In vivo changes were significantly inhibited by pretreatment with simvastatin. We provide novel evidence for the mechanisms by which septicemia causes myocardial depression and hint at a potential role for simvastatin as an inhibitor of apoptosis in sepsis.
Asunto(s)
Apoptosis/efectos de los fármacos , Cardiomiopatías/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Simvastatina/uso terapéutico , Animales , Cardiomiopatías/etiología , Cardiomiopatías/fisiopatología , Exotoxinas/toxicidad , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Miocardio/metabolismo , Miocardio/patología , Necrosis , Ratas , Sepsis/inducido químicamente , Sepsis/complicaciones , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils. SUBJECTS: Isolated human neutrophils from healthy volunteers. INTERVENTIONS: Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies. MEASUREMENTS: Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction. CONCLUSIONS: The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.
Asunto(s)
Citocinas/biosíntesis , Lipopolisacáridos/inmunología , Neutrófilos/metabolismo , Sepsis/inmunología , Infecciones Estafilocócicas/inmunología , Ácidos Teicoicos/inmunología , Análisis de Varianza , Células Cultivadas , Humanos , Interleucina-10/inmunología , Interleucina-8/biosíntesis , Receptores de Lipopolisacáridos/inmunología , Neutrófilos/inmunología , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Receptores Toll-Like/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH) evoke cardiac dysfunction in isolated rat hearts by provoking myocardial synthesis of arachidonic acid-derived thromboxane A2 or the cysteinyl-leukotrienes, LTC4, LTD4, and LTE4, respectively. We investigated whether low doses of either toxin, which fail to induce cardiac depression by themselves, induce cardiac dysfunction when combined with free arachidonic acid. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Hearts were perfused with low doses of ECH or alpha-toxin in the absence or presence of arachidonic acid or the alternative eicosanoid precursor eicosapentaenoic acid (EPA). MEASUREMENTS AND MAIN RESULTS: Application of low-dose ECH with arachidonic acid increased coronary perfusion pressure, depressed left ventricular contractile function, provoked electrical instability, and induced a release of creatine kinase concomitant with the liberation of LTC4, LTD4, and LTE4 into the perfusate. All events were abolished when formation of cysteinyl-leukotrienes was blocked by the 5-lipoxygenase activity inhibitor MK-886, targeting 5-lipoxygenase activating protein. In the presence of arachidonic acid, low doses of alpha-toxin caused an increase in cerebral perfusion pressure and a decline of contractile performance, attributable to the release of thromboxane A2, as both events were mitigated by the cyclooxygenase-inhibitor indomethacin. High doses of ECH caused cardiac dysfunction even in the absence of arachidonic acid. However, in the presence of EPA, the cardiodepressant effect of ECH was blunted. Release of EPA-derived LTE5 at the expense of arachidonic acid-derived LTC4, LTD4, and LTE4 was noted in these hearts. CONCLUSIONS: The potency of the bacterial exotoxins ECH and alpha-toxin to cause coronary vasoconstriction and myocardial depression is dependent on the availability of free arachidonic acid and may be influenced by supplying omega-3 fatty acids as alternative lipid precursors.
Asunto(s)
Ácido Araquidónico/farmacología , Ácido Eicosapentaenoico/farmacología , Contracción Miocárdica/efectos de los fármacos , Animales , Toxinas Bacterianas , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Exotoxinas , Indometacina/farmacología , Leucotrienos/análisis , Masculino , Contracción Miocárdica/fisiología , Probabilidad , Ratas , Ratas Wistar , Valores de Referencia , Factores de Riesgo , Sensibilidad y Especificidad , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
BACKGROUND: Lipoteichoic acid (LTA) represents a major virulence factor in gram-positive sepsis. METHODS AND RESULTS: In the present study we perfused isolated rat hearts for 180 minutes with highly purified LTA from Staphylococcus aureus. A progressive decline of left ventricular contractile function paralleled by the expression of myocardial tumor necrosis factor-alpha (TNF-alpha) mRNA and protein as well as the release of TNF-alpha into the perfusate was observed in LTA-perfused hearts. Employment of an anti-TNF-alpha antibody completely prevented the loss in contractile function. When CD14, a prominent pathogen recognition receptor, was blocked by a specific antibody, induction of TNF-alpha mRNA and protein release as well as the associated cardiodepression was diminished in response to LTA. Synthesis of TNF-alpha protein was located to interstitial cells of LTA-challenged hearts as detected by immunohistochemistry. Besides progressive cardiodepression, coronary perfusion pressure (CPP) was moderately increased in LTA-perfused hearts. This was accompanied by the release of thromboxane A2 (TXA2) into the perfusate and the induction of cyclooxygenase (Cox)-2 mRNA and protein in the myocardium. Blocking of TXA2 by the nonspecific Cox inhibitor indomethacin, the thromboxane receptor antagonist daltroban, or the selective Cox-2 inhibitor NS-398 prevented the increase in CPP. CONCLUSIONS: LTA causes cardiac depression by activating myocardial TNF-alpha synthesis via CD14 and induces coronary vascular disturbances by activating Cox-2-dependent TXA2 synthesis. These phenomena may contribute to cardiac depression in gram-positive sepsis.
Asunto(s)
Insuficiencia Cardíaca/microbiología , Corazón/microbiología , Lipopolisacáridos , Staphylococcus aureus/fisiología , Ácidos Teicoicos , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Insuficiencia Cardíaca/inducido químicamente , Técnicas In Vitro , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/farmacología , Receptores de Lipopolisacáridos/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/inmunología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina G/inmunología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Ácidos Teicoicos/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/efectos de los fármacosRESUMEN
OBJECTIVE: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis. We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified hemolysin for 60 min. MEASUREMENTS AND MAIN RESULTS: Low concentrations of the toxin in the perfusate (0.1-0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase). Moreover, 0.2 hemolytic units/mL hemolysin evoked ventricular fibrillation within 10 mins of toxin application. These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate. The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase. In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective. In addition, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886. CONCLUSIONS: Low doses of hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature. Bacterial exotoxins such as hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.
Asunto(s)
Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/complicaciones , Escherichia coli , Exotoxinas/efectos adversos , Insuficiencia Cardíaca/microbiología , Proteínas Hemolisinas/efectos adversos , Leucotrienos/fisiología , Contracción Miocárdica , Choque Séptico/microbiología , Vasoconstricción , Fibrilación Ventricular/microbiología , Animales , Circulación Coronaria , Relación Dosis-Respuesta a Droga , Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/fisiopatología , Técnicas In Vitro , Indoles/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Estudios Prospectivos , Ratas , Choque Séptico/inmunología , Choque Séptico/fisiopatología , Disfunción Ventricular Izquierda/inmunología , Disfunción Ventricular Izquierda/microbiología , Disfunción Ventricular Izquierda/fisiopatología , Fibrilación Ventricular/inmunología , Fibrilación Ventricular/fisiopatología , Presión VentricularRESUMEN
The virulence of pathogenic bacteria is critically dependent on their ability to produce toxins that attack eukaryotic target cells. Microbial toxins are either structural components of the bacterial cell wall (endotoxins) or actively secreted proteins (exotoxins). Sepsis and septic shock, which represent major causes of mortality in modern intensive care medicine, are caused by an inadequate inflammatory and immunological host response to bacterial infection. Emerging evidence suggests that the systemic spread of microbial toxins, rather than bacteremia itself, is the crucial event in the pathogenesis of this dramatic dysregulation. The endothelium, with its diversity of physiological functions is a main target of bacterial toxins. The resulting endothelial dysfunction is believed to contribute to the underlying pathomechanisms and the collapse of homeostasis of organ function. In vitro, bacterial toxins induce subtle alterations of endothelial cell function rather than massive cell damage. Furthermore, bacterial toxins targeting endothelial cells severely alter the behavior of extravascular cells and circulating leukocytes via excessive formation of vasoactive mediators and overexpression of adhesion molecules. Research on the effects of microbial toxins on vascular endothelium has broadened our general understanding of microbial strategies to induce organ damage, even in the absence of viable bacteria. Combining antitoxin strategies with antibiotic therapy may prove to be of benefit to patients suffering from bacterial sepsis in the future.
Asunto(s)
Toxinas Bacterianas/inmunología , Endotelio Vascular/inmunología , Sepsis/inmunología , Animales , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/microbiología , Endotelio Vascular/fisiopatología , Endotoxinas/inmunología , Endotoxinas/metabolismo , Exotoxinas/inmunología , Exotoxinas/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Modelos Biológicos , Insuficiencia Multiorgánica/inmunología , Insuficiencia Multiorgánica/microbiología , Insuficiencia Multiorgánica/patología , Peptidoglicano/metabolismo , Ácidos Teicoicos/metabolismoRESUMEN
OBJECTIVE: Exposure of neutrophils to low doses of bacterial lipopolysaccharides enhances their readiness to respond with inflammatory mediator generation including oxygen radical formation to a subsequently applied inflammatory stimulus ("priming"). In the present study, we investigated the role of lipid mediator synthesis and the impact of the anti-inflammatory cytokine interleukin-10 on the lipopolysaccharide-dependent priming of human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated neutrophils from healthy volunteers. INTERVENTIONS: Incubation of isolated neutrophils with endotoxin. MEASUREMENTS AND MAIN RESULTS: Evidence for two distinct priming mechanisms was obtained. The first was strictly serum component dependent, proceeded via CD14, and was not inhibited by even high concentrations of interleukin-10. The second priming mechanism was serum component independent but nevertheless proceeded via CD14. It was linked with neutrophil synthesis of the platelet activating factor and resulted in the appearance of leukotrienes, in particular leukotriene B4, as far as exogenous arachidonic acid was provided. The employment of a platelet-activating factor receptor antagonist (WEB 2086) blocked leukotriene synthesis, and both WEB 2086 and a 5-lipoxygenase inhibitor (MK-886) suppressed the respiratory burst linked with this second priming pathway. This sequence of priming events was inhibited by interleukin-10, when this cytokine was coadministered with the priming agent lipopolysaccharide, whereas late interleukin-10 admixture was ineffective. CONCLUSIONS: We conclude that two mechanisms of lipopolysaccharide priming of human neutrophil respiratory burst can be differentiated. One displays serum component dependence, is independent of neutrophil lipid mediator generation, and is not affected by interleukin-10. The other is serum independent although being operated via CD14, employs autocrine loops of platelet-activating factor and leukotriene B4 synthesis, and is sensitive to the inhibitory capacity of interleukin-10. These features may be relevant when the goal is to pharmacologically modify neutrophil functions in septic events.
Asunto(s)
Interleucina-10/farmacología , Leucotrienos/biosíntesis , Lipopolisacáridos/farmacología , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/biosíntesis , Estallido Respiratorio/efectos de los fármacos , Ácido Araquidónico/farmacología , Comunicación Autocrina , Azepinas/farmacología , Escherichia coli , Humanos , Técnicas In Vitro , Indoles/farmacología , Antagonistas de Leucotrieno/farmacología , Leucotrieno B4/biosíntesis , Receptores de Lipopolisacáridos/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/antagonistas & inhibidores , Estudios Prospectivos , Superóxidos/metabolismo , Triazoles/farmacologíaRESUMEN
BACKGROUND: Simvastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, has been shown to lower serum cholesterol levels in clinical use. Moreover, statins exert beneficial effects in vascular diseases by inhibition of leukocyte rolling, adherence, and transmigration. The aim of this study was to determine if pretreatment with simvastatin attenuates Staphylococcus aureus alpha-toxin-induced increase in leukocyte-endothelial interactions during exotoxemia. METHODS AND RESULTS: The effects of simvastatin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation. Simvastatin (50 or 100 microg/kg) was administered 18 hours before the study. Activation of microcirculation was induced by bolus administration of 40 microg/kg S aureus alpha-toxin. Exotoxemia resulted in a significant and time-dependent increase in leukocyte rolling, adherence, and transmigration of leukocytes as well as P-selectin expression on the intestinal vascular endothelium. Pretreatment with simvastatin significantly inhibited exotoxin-induced leukocyte rolling from 71+/-10 to 14+/-4.7 cells/min (P<0.01) and adherence from 14+/-3.5 to 0.4+/-0.2 cells (P<0.01). In addition, simvastatin pretreatment significantly inhibited transmigration of leukocytes from 10.5+/-1.2 to 4.2+/-0.9 (P<0.05) cells. Immunohistochemical detection of endothelial cell adhesion molecule P-selectin showed a 50% decrease in endothelial cell surface expression after simvastatin treatment. Furthermore, simvastatin treatment resulted in enhanced expression of endothelial cell NO synthase III in the intestinal microcirculation. CONCLUSIONS: These results demonstrate that simvastatin interferes with exotoxin-induced leukocyte-endothelial cell interactions, which may be relevant in various infectious diseases. Statin treatment may offer a new therapeutic strategy for these clinical conditions.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Simvastatina/farmacología , Toxemia/prevención & control , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Técnicas de Cultivo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Hemodinámica , Proteínas Hemolisinas , Inmunohistoquímica , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Masculino , Venas Mesentéricas/anatomía & histología , Venas Mesentéricas/efectos de los fármacos , Venas Mesentéricas/fisiopatología , Microscopía por Video , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo III , Selectina-P/análisis , Selectina-P/inmunología , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patología , Infecciones Estafilocócicas/prevención & control , Toxemia/inmunología , Toxemia/patología , Vénulas/efectos de los fármacos , Vénulas/fisiopatologíaRESUMEN
Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c-ANCA)], a leukocyte serine protease, are highly specific for Wegener's Granulomatosis (WG). A pathogenetic role for c-ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti-PR3 antibodies (anti-PR3) and c-ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti-PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1beta and delayed release of IL-6, IL-8, and thromboxane A2 (TxA2). The secretory response was reproduced by c-ANCA but not by human and murine control IgG and anti-CD14 antibodies. Because F(ab)2 fragments of anti-PR3 were ineffective, coligation of Fc gamma receptors (FcgammaR) was apparently mandatory for monocyte activation. Using soluble receptors for TNF-alpha and IL-1beta and a Tx receptor antagonist, we noted that the "early" cytokines functioned as inducers of TxA2, which then activated IL-8 release. In contrast, IL-6 formation was an independent event. We concluded that anti-PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF-alpha, IL-1beta, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Citocinas/sangre , Granulomatosis con Poliangitis/inmunología , Monocitos/inmunología , Activación Neutrófila/inmunología , Inhibidores de Proteasas/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/farmacología , Anticuerpos Monoclonales/farmacología , Técnicas de Cultivo de Célula/métodos , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Cinética , Activación Neutrófila/efectos de los fármacos , Prostaglandinas/fisiologíaRESUMEN
Among the anti-neutrophil cytoplasmic Abs (ANCA), those targeting proteinase 3 (PR3) have a high sensitivity and specificity for Wegener's granulomatosis (WG). A pathogenetic role for these autoantibodies has been proposed due to their capacity of activating neutrophils in vitro. Recently, PR3 was also detected in human renal tubular epithelial cells (TEC). In the present study, the effect of murine monoclonal anti-PR3 Abs (anti-PR3) and purified c-ANCA targeting PR3 from WG serum on isolated human renal tubular cell signaling and inflammatory mediator release was characterized. Priming of TEC with TNF-alpha resulted in surface expression of PR3, as quantified in immunofluorescence studies and by flow cytometry. Moreover, PR3 was immunoprecipitated on surface-labeled TEC. Primed TEC responded to anti-PR3 with a dose- and time-dependent activation of phosphoinositide hydrolysis, resulting in a remarkable accumulation of inositolphosphates. Control IgG was entirely ineffective, whereas PR3-ANCA reproduced the phosphoinositide response. The signaling response was accompanied by a pronounced release of superoxidanion into the cell supernatant. Moreover, large amounts of PGE(2) and, to a lesser extent, of thromboxane B(2), the stable metabolite of TxA(2), were secreted from anti-PR3-stimulated TEC. In parallel, a rise in intracellular cAMP levels was observed, which was blocked by the cyclooxygenase inhibitor indomethacin. We conclude that anti-PR3 Abs directly target renal TECs, thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway. Associated metabolic events such as the release of reactive oxygen species and lipid mediators may directly contribute to the development of renal lesions and loss of kidney function in WG.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Mediadores de Inflamación/metabolismo , Túbulos Renales/inmunología , Túbulos Renales/metabolismo , Serina Endopeptidasas/inmunología , Transducción de Señal/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Reacciones Antígeno-Anticuerpo , Ácido Araquidónico/metabolismo , Autoantígenos/biosíntesis , Autoantígenos/inmunología , Membrana Celular/enzimología , Membrana Celular/inmunología , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Células Epiteliales/enzimología , Humanos , Hidrólisis , Sueros Inmunes/metabolismo , Sueros Inmunes/farmacología , Líquido Intracelular/metabolismo , Túbulos Renales/enzimología , Mieloblastina , Fosfatidilinositoles/metabolismo , Serina Endopeptidasas/biosíntesis , Superóxidos/metabolismo , Tromboxano A2/metabolismoRESUMEN
The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.
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Toxinas Bacterianas/toxicidad , Exotoxinas/toxicidad , Corazón/efectos de los fármacos , Proteínas Hemolisinas/toxicidad , Molécula 1 de Adhesión Intercelular/genética , Leucotrienos/metabolismo , Neutrófilos/fisiología , Animales , Ácido Araquidónico/farmacología , Corazón/fisiopatología , Humanos , Hidrazonas , Técnicas In Vitro , Miocardio/patología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Sondas de Oligonucleótidos , Perfusión , Ratas , TiofenosRESUMEN
The effect of Staphylococcus aureus alpha toxin (alpha-toxin) on selectin-mediated neutrophil adhesion was investigated in polymorphonuclear leukocyte- (PMN) induced vasocontraction and endothelial dysfunction. Adherence of human PMNs to rat aortic endothelium increased significantly following stimulation of the endothelium with alpha-toxin (0.1, 0.5, and 1 microg/mL). This effect could be significantly attenuated by monoclonal antibodies directed against P-selectin or fucoidin, a carbohydrate known to block selectins. Unstimulated human PMNs (10(6)cells/mL) were added to organ chambers containing rat aortic rings stimulated with alpha-toxin (0.5 microg/mL). PMNs elicited a significant vasocontraction in alpha-toxin-stimulated, but not in control aortic, rings (142+/-12 mg versus 12+/-4 mg, P < 0.05). This PMN-induced vasocontraction was virtually blunted by pretreatment with MAb directed against P-selectin or fucoidin (P < 0.05). Endothelial function as assessed by endothelium-dependent vasorelaxation to acetylcholine was substantially inhibited after induction of PMN-induced vasocontraction in alpha-toxin-stimulated aortic rings. This endothelial dysfunction was reduced by P-selectin MAb or fucoidin. In contrast, endothelium-independent relaxation to sodium nitrite was not altered by PMN incubation, indicating that vascular smooth muscle function was unaffected. Thus, PMN-endothelial interaction following S. aureus a-toxin activation of the vascular endothelium is at least, in part, mediated by selectins. As a consequence, PMN-induced vasocontraction and endothelial dysfunction occur. Such mechanisms may be involved in microcirculation abnormalities encountered in sepsis or septic shock due to S. aureus infection.
Asunto(s)
Toxinas Bacterianas/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Proteínas Hemolisinas/farmacología , Neutrófilos/metabolismo , Vasoconstricción/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/fisiopatología , Azepinas/farmacología , Adhesión Celular/efectos de los fármacos , Fibrinolíticos/farmacología , Humanos , Técnicas In Vitro , Neutrófilos/citología , Ratas , Trombina/farmacología , Triazoles/farmacologíaRESUMEN
Staphylococcus aureus alpha-toxin is a pore-forming bacterial exotoxin that has been implicated as a significant virulence factor in human staphylococcal diseases. In primary cultures of rat pneumocyte type II cells and the human A549 alveolar epithelial cell line, purified alpha-toxin provoked rapid-onset phosphatidylinositol (PtdIns) hydrolysis as well as liberation of nitric oxide and the prostanoids PGE(2), PGI(2), and thromboxane A(2). In addition, sustained upregulation of proinflammatory interleukin (IL)-8 mRNA expression and protein secretion occurred. "Priming" with low-dose IL-1beta markedly enhanced the IL-8 response to alpha-toxin, which was then accompanied by IL-6 appearance. The cytokine response was blocked by the intracellular Ca(2+)-chelating reagent 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, the protein kinase C inhibitor bis-indolyl maleimide I, as well as two independent inhibitors of nuclear factor-kappaB activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We conclude that alveolar epithelial cells are highly reactive target cells of staphylococcal alpha-toxin. alpha-Toxin pore-associated transmembrane Ca(2+) flux and PtdIns hydrolysis-related signaling with downstream activation of protein kinase C and nuclear translocation of nuclear factor-kappaB are suggested to represent important underlying mechanisms. Such reactivity of the alveolar epithelial cells may be relevant for pathogenic sequelae in staphylococcal lung disease.