RESUMEN
Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) mRNA increased in response to LPS. Activation of nuclear factor-kappaB (NF-kappaB) presumably results in NF-kappaB binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-kappaB activation, cytokine message production, and TNF-alpha secretion. Maximum active NF-kappaB was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-alpha mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-alpha was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-kappaB activation and cytokine secretion in other cell types, did not inhibit NF-kappaB activation or TNF-alpha secretion. However, aspirin reduced the amount of both TNF-alpha and IL-6 mRNA present 30 min after LPS addition by half (P < 0.05).
Asunto(s)
Citocinas/biosíntesis , Endotoxinas/farmacología , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) activity and indexes of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1,726 +/- 117, 150 islet equivalent units) from Wistar-Furth rats were treated as 1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and 4) static culture plus LPS. TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures; yet the increase was more pronounced in the static culture group (P < 0.05). A decrease in insulin concentration was demonstrated in the LPS-stimulated HARV culture (P < 0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. Although nitrogenous compound analysis indicated a ubiquitous reliance on glutamine in all experimental groups, arginine was converted to ornithine at a twofold greater rate in the islets cultured in the HARV microgravity model system (P < 0.05). These studies demonstrate alterations in LPS-induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity-averaged cell culture methods or by three-dimensional cell assembly.
Asunto(s)
Aminoácidos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Simulación de Ingravidez , Animales , Técnicas de Cultivo de Célula/métodos , Separación Celular , Células Cultivadas , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Lactatos/metabolismo , Lipopolisacáridos/farmacología , Masculino , Compuestos de Nitrógeno/metabolismo , Pancreatectomía , Ratas , Ratas Endogámicas WFRESUMEN
Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-kappaB is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-kappaB may also be influenced by changes in O2 tension. It is thus proposed that acute changes in O2 tension surrounding AMs alter NF-kappaB activation and TNF secretion in these lung cells. AM-derived TNF secretion and NF-kappaB expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs (10(6)/ml) were incubated (37 degrees C at 5% CO2) for 2 h with LPS (Pseudomonas aeruginosa, 1 microgram/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay. Electrophoretic mobility shift and supershift assays were used to determine NF-kappaB activation and to identify NF-kappaB isoforms in AM extracts. In addition, mRNAs for selected AM proteins were determined with RNase protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF (P < 0.05) and enhanced expression of NF-kappaB (P < 0.05); the predominant isoforms were p65 and c-Rel. Increased mRNA bands for TNF-alpha, interleukin-1alpha, and interleukin-1beta were also observed in the hypoxic AMs. These results suggest that acute hypoxia in the lung may induce enhanced NF-kappaB activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.
Asunto(s)
Hipoxia/metabolismo , Macrófagos Alveolares/metabolismo , FN-kappa B/metabolismo , Enfermedad Aguda , Animales , Citocinas/genética , ADN/metabolismo , Masculino , FN-kappa B/genética , ARN Mensajero/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tumor necrosis factor (TNF) may be a major endogenous mediator of sepsis-induced acute organ injury. We proposed that treatment of septic pigs with the combined agents ibuprofen, a cyclooxygenase inhibitor, and histamine receptor antagonists, cimetidine (H2 antagonist) and diphenhydramine (H1 antagonist) would result in lower circulating levels of TNF and decreased parameters of sepsis-induced injury in these animals. To test this, plasma TNF activity, cardiac index, systemic and pulmonary arterial pressures, arterial PO2 and bronchoalveolar lavage protein content were monitored for 300 min in four groups of anesthetized pigs: saline-infused control pigs (n = 4); pigs infused for 60 min with Pseudomonas aeruginosa (5 x 10(8) organisms/mL, .3 mL/20 kg/min) (n = 5) and pigs infused for 60 min with P. aeruginosa plus ibuprofen (12.5 mg/kg) alone (n = 4) or ibuprofen plus cimetidine (150 mg) and diphenhydramine (30 mg/kg) at 0 and 120 min (CID, n = 4). Within 60 min, pigs infused with P. aeruginosa exhibited increased plasma TNF activity (>8-fold increase in ng/mL TNF; L929 cytolysis assay) and showed alterations in all hemodynamic and pulmonary parameters. Ibuprofen or CID administration in the septic pigs decreased peak TNF activity by 4.6 and 10.2 ng/mL, respectively, and CID treatment was correlated with better attenuation of certain sepsis-induced alterations. These results show that CID treatment attenuates sepsis-induced injury and that this is correlated with reduced plasma TNF activity in a porcine model of sepsis-induced acute organ injury.
Asunto(s)
Bacteriemia/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Bacteriemia/metabolismo , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Cimetidina/farmacología , Difenhidramina/farmacología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Hipertensión Pulmonar/tratamiento farmacológico , Ibuprofeno/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Porcinos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
BACKGROUND: Evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in heart diseases such as atherosclerosis. We used porcine coronary arteries and smooth muscle cells cultured from these vessels to study the regulation of production of TNF-alpha. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-kappaB, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappaB. MATERIAL AND METHODS: LPS was used to stimulate TNF-alpha production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-alpha release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-kappaB activation was determined in whole cell extracts by electrophoretic mobility shift assay. RESULTS: In segments of coronary arteries, LPS stimulated TNF-alpha release which increased with time to a maximum at 6 h (485 +/- 19 units/g tissue) and remained elevated at this level for 24 h. In contrast, the level of TNF-alpha measured at 24 h in medium from coronary tissue not exposed to LPS was 11.1 +/- 4.1 units/g tissue. In the presence of LPS, both FSK and 8-Br-cAMP significantly reduced TNF-alpha release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-alpha was 126 +/- 24 and 71.6 +/- 22 units/g tissue, respectively (P < 0.05 vs LPS alone). Tissue levels of cAMP were significantly elevated in the presence of FSK. Similar results were obtained with smooth muscle cells cultured from the coronary arteries; i.e., LPS stimulated TNF-alpha release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-alpha stimulated by LPS was associated with activation of NF-kappaB. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappaB by LPS. CONCLUSIONS: Porcine coronary arteries produce TNF-alpha from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-kappaB. However, activation of NF-kappaB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-alpha gene. These results support the view that coronary vessels can be a source of TNF-alpha possibly involved in heart disease.
Asunto(s)
Vasos Coronarios/fisiología , AMP Cíclico/metabolismo , Músculo Liso Vascular/fisiología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Secuencia de Bases , Colforsina/farmacología , Vasos Coronarios/efectos de los fármacos , Cardiopatías/etiología , Técnicas In Vitro , Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/genética , Sondas de Oligonucleótidos/genética , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
OBJECTIVES: We recently reported that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from both human vascular tissue and cultured smooth muscle cells. In the current study, we tested the hypothesis that increased intracellular cyclic adenosine 3',5'-monophosphate (cAMP) could inhibit TNF-alpha release. DESIGN: Prospective, repeated-measures analysis. SETTING: Academic research laboratory. SUBJECTS: Segments of internal mammary artery and saphenous vein from patients undergoing coronary artery bypass surgery. MEASUREMENTS AND MAIN RESULTS: Segments of saphenous vein and internal mammary artery and confluent smooth muscle cells cultured from these vessels were incubated in the presence of 20 micrograms/mL bacterial lipopolysaccharide, alone or with the addition of forskolin or 8-Br-cAMP. At 0, 1, 3, 6, 18, and 24 hrs, the incubation medium was removed from vessel segments or cells and was analyzed for biologically active TNF-alpha, using the L929 cell cytotoxicity assay. cAMP was extracted from tissue and cells with 0.1 N HCl and was analyzed by radioimmunoassay. Bacterial lipopolysaccharide stimulated the release of TNF-alpha from internal mammary smooth muscle cells at all time points. For example, at 6 hrs, TNF-alpha concentration in the medium from lipopolysaccharide-stimulated cells was 20 +/- 1.6 U/mg of cell protein, compared with 0.9 +/- 0.5 U/mg of cell protein in control cell medium (p < .05). Forskolin-inhibited bacterial lipopolysaccharide stimulated TNF-alpha release. In the presence of lipopolysaccharide and forskolin, TNF-alpha release at 6 hrs was 8.6 +/- 1.5 U/mg of cell protein (p < .05 vs. in the presence of bacterial lipopolysaccharide alone). Bacterial lipopolysaccharide, alone, had no effect on intracellular cAMP. Forskolin increased intracellular cAMP levels to 74.0 +/- 12 pmol/mg of cell protein at 6 hrs from a control level of 7.7 +/- 0.4 pmol/mg (p < .05). The 8-Br-cAMP, an agent that mimics the action of intracellular cAMP, also inhibited TNF-alpha release stimulated by lipopolysaccharide. Similar inhibition by forskolin and 8-Br-cAMP on TNF-alpha release was obtained with smooth muscle cells from saphenous vein. Finally, in tissue segments from either internal mammary artery or saphenous vein, both forskolin and 8-Br-cAMP inhibited lipopolysaccharide-stimulated TNF-alpha release. CONCLUSIONS: These results are consistent with the conclusion that vascular tissue, particularly the smooth muscle cell, is a source of TNF-alpha. Further, bacterial lipopolysaccharide-stimulated tumor TNF-alpha release can be inhibited by increased intracellular cAMP.
Asunto(s)
AMP Cíclico/farmacología , Músculo Liso Vascular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Colforsina/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Lipopolisacáridos/farmacología , Ratones , Músculo Liso Vascular/metabolismo , Estudios ProspectivosRESUMEN
Tumor necrosis factor (TNF) is involved in the pathogenesis of acute sepsis-induced organ injury and has been implicated as a mediator of metabolic alterations observed during sepsis. Pancreatic islet cell function may be significantly compromised during sepsis or endotoxemia, and sepsis also increases plasma levels of epinephrine, a modifier of islet insulin secretion. We proposed that islets exposed to bacterial lipopolysaccharide (LPS) produce TNF and that epinephrine attenuates islet secretory activity. We monitored the effects of LPS and epinephrine on TNF and insulin activity of isolated Wistar-Furth rat islets (pancreas digested with collagenase, islets isolated using Ficoll gradients; n = 4 islet populations, each with 632 +/- 11 islets/2.5 ml culture medium). Islets were incubated (37 degrees C, 5% CO2) 3 days. LPS (Escherichia coli, 1 microgram/ml) and epinephrine (14 micrograms/ml) were added to the islets, and incubations were continued for 1-4 h. Glucose (Beckman Glucose Analyzer), insulin (radioimmunoassay), and TNF (L929 cytotoxicity assay) were measured in the islet medium samples at 1- to 4-h time points. In the conditioned medium, glucose decreased (P < 0.05), insulin increased (P < 0.05), and exposure to LPS did not alter these levels [P = not significant (NS)] but did increase TNF activity by 400% (P < 0.05). Epinephrine reduced insulin by 38-43% (P < 0.05) and TNF by 20-25% (P < 0.05) but had no effect on glucose levels (P = NS). We conclude that insulin is secreted from isolated islets and that exposure to LPS acutely increases islet-derived TNF activity, whereas epinephrine modifies TNF and insulin secretion of rat pancreatic islets.
Asunto(s)
Islotes Pancreáticos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Medios de Cultivo/metabolismo , Epinefrina/farmacología , Glucosa/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Lipopolisacáridos/farmacología , Masculino , Ratas , Ratas Endogámicas WFRESUMEN
OBJECTIVES: Based on our previous study that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from human vascular tissue and smooth muscle cells, we tested the hypothesis that release of TNF could be inhibited by pretreatment with glucocorticoids. DESIGN: Prospective, repeated-measures analysis of concentration-response relationships. SETTING: Academic anesthesiology research laboratory. SUBJECTS: Segments of internal mammary artery and saphenous vein were obtained during coronary artery bypass surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Confluent human smooth muscle cells, cultured from saphenous vein and internal mammary artery, were exposed to 20 micrograms/mL of bacterial lipopolysaccharide following pretreatment for 18 hrs with either 0.1, 1.0, or 10.0 microM of dexamethasone. At 1, 3, 6, 18, and 24 hrs, the culture medium was removed and analyzed for biologically active TNF-alpha using the L929 cell cytotoxicity assay. Smooth muscle cells exposed to bacterial lipopolysaccharide but not treated with dexamethasone served as controls. In control internal mammary cells, bacterial lipopolysaccharide stimulated TNF-alpha release in a time-dependent manner to a peak of 36 +/- 2.3 U/mg of cell protein at 6 hrs, compared with 0.7 +/- 0.3 U/mg of cell protein in cells not exposed to lipopolysaccharide. Dexamethasone inhibited bacterial lipopolysaccharide-stimulated release at all time points in a concentration-dependent manner. For instance, at 6 hrs, TNF-alpha was 12 +/- 2.2, 6.9 +/- 1.7, and 2.3 +/- 0.9 U/mg of cell protein for cells pretreated with 0.1, 1.0, and 10.0 microM of dexamethasone, respectively (p < .05 vs. control). In separate experiments, segments of internal mammary artery and saphenous vein were obtained from five patients who received 1 g of methylprednisolone intravenously during induction of anesthesia, and from seven patients who did not receive methylprednisolone. Bacterial lipopolysaccharide induced release of TNF-alpha from vascular tissues of untreated patients in a time-dependent manner (e.g., 733 +/- 44 U/g of tissue at 6 hrs in saphenous vein). In contrast, in patients treated with methylprednisolone, bacterial lipopolysaccharide did not stimulate release from vascular tissues incubated for up to 24 hrs. CONCLUSIONS: These results indicate that human vascular tissue, particularly the smooth muscle cell, may be a source of TNF-alpha and that glucocorticoids inhibit release stimulated by bacterial lipopolysaccharide.
Asunto(s)
Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Lipopolisacáridos , Arterias Mamarias/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Vena Safena/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVES: In septic shock, the principal source of increased plasma concentrations of tumor necrosis factor alpha (TNF) is considered to be the macrophage. Release from the macrophage is stimulated by bacterial lipopolysaccharide (endotoxin). We tested the hypothesis that vascular tissue also responds to endotoxin by releasing TNF. DESIGN: Prospective repeated measures analysis of timed-release curves. SETTING: Anesthesia research laboratory in an academic medical center. SUBJECTS: With Institutional Review Board approval and patient consent, segments of internal mammary artery and saphenous vein were obtained during coronary artery bypass surgery. INTERVENTIONS: None MEASUREMENTS AND MAIN RESULTS: Segments of saphenous veins were incubated for 24 hrs in the presence or absence of bacterial lipopolysaccharide. At 0.5, 1, 3, 6, and 24 hrs, medium was assayed for TNF. In other experiments, smooth muscle cells were cultured from saphenous veins, incubated with our without bacterial lipopolysaccharide, and a time-course of TNF release determined. Bacterial lipopolysaccharide (20 micrograms/mL) significantly stimulated release of TNF from venous tissue in a time-dependent manner. At 0.5 hrs, TNF was undetectable in untreated tissue and was 48 +/- 8 U/g wet tissue weight in the presence of bacterial lipopolysaccharide. At 3 hrs, TNF was 43 +/- 27 U/g wet tissue weight in untreated and 388 +/- 185 U/g wet tissue weight in treated (p < .01 vs. control) tissue. Segments of internal mammary artery responded in a similar manner. In smooth muscle cells cultured from saphenous vein and internal mammary artery, bacterial lipopolysaccharide triggered the release of TNF. At 3 hrs, the release of TNF in control cells was 0.2 +/- 0.15 U/mg cell protein and 17 +/- 2 U/mg in the presence of 20 micrograms/mL of bacterial lipopolysaccharide (p < .01 vs. control). CONCLUSIONS: Human blood vessels, both artery and vein, produce TNF potentially from a smooth muscle cell source in response to bacterial lipopolysaccharide.
Asunto(s)
Escherichia coli , Lipopolisacáridos/farmacología , Músculo Liso Vascular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Análisis de Varianza , Células Cultivadas , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Arterias Mamarias , Músculo Liso Vascular/efectos de los fármacos , Vena Safena , Choque Séptico/sangre , Choque Séptico/inmunología , Factores de TiempoRESUMEN
This study examined the kinetics of IL-6 release into the systemic circulation in a porcine model of bacterial sepsis induced by infusion of live Pseudomonas aeruginosa. Three groups of animals were studied. Group I (n = 12) animals received a 1 hr infusion of live P. aeruginosa. Group II (n = 6) animals received monoclonal antibody to tumor necrosis factor-alpha (TNF-alpha) (15 mg/kg) prior to induction of sepsis. Group III (n = 7) animals received sterile saline only. TNF-alpha and interleukin-6 (IL-6) levels rose sharply, in group I following pseudomonas infusion. Following a peak at 120 min after the bacterial infusion (4.8 +/- 0.7 U/ml at 120 min vs 0.4 +/- 0.2 U/ml at 0 min), TNF-alpha levels subsequently declined prior to the end of the experiment. In contrast, IL-6 levels rose sharply, subsequent to TNF-alpha, peaked at 180 min, and remained significantly elevated throughout the study period (5.3 +/- 0.9 ng/ml vs 0.05 +/- 0.01 ng/ml, 0 min). In animals pretreated with monoclonal antibody to TNF-alpha, no increase in TNF-alpha activity was detected at any time during the period of study. IL-6 levels in antibody-treated animals, although greatly attenuated, still rose significantly above baseline (2.02 +/- 0.8 ng/ml at 180 min vs 0.05 +/- 0.01 ng/ml at 0 min) and above levels in control animals. We conclude that although TNF-alpha plays an important role in synthesis and release of IL-6, there is a TNF-alpha-independent pathway for release of IL-6 in sepsis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Bacteriemia/veterinaria , Interleucina-6/sangre , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/terapia , Factor de Necrosis Tumoral alfa/inmunología , Animales , Bacteriemia/sangre , Bacteriemia/tratamiento farmacológico , Presión Sanguínea , Frecuencia Cardíaca , Infusiones Intravenosas , Porcinos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Septic shock is characterized by surges of tumor necrosis factor-alpha (TNF-alpha) along with myocardial dysfunction and systemic hypotension. TNF-alpha promotes the release of immunoreactive endothelin (ET). Because TNF-alpha is elevated in septic shock, we hypothesized that elevated levels of endothelin can contribute to cardiac dysfunction and hypotension. We infused live Pseudomonas aeruginosa into anesthetized, hemodynamically monitored young swine and measured ET and TNF-alpha. Septic swine developed systemic arterial hypotension and had significantly elevated TNF-alpha (4.15 +/- .41 U/ml at 1 h versus .40 +/- .13 U/ml at time zero) compared to control animals. ET levels were significantly elevated at 4 h (52.38 +/- 12.88 pg/ml vs. 10.45 +/- 1.82 pg/ml at time zero) and correlated negatively with the decline in cardiac output. We then passively immunized swine using anti TNF-alpha prior to the induction of sepsis to examine if TNF played a central role in the release ET. The anti TNF-alpha effectively removed circulating TNF-alpha bioactivity in septic animals. Anti-TNF-alpha-treated animals did not develop significant systemic arterial hypotension and had significant attenuation in endothelin (19.01 +/- 4.18 pg/ml at 4 h compared to 52.38 +/- 12.88 pg/ml in septic animals at 4 h) which correlated with preservation of cardiac output. TNF-alpha may cause cardiac dysfunction in sepsis syndrome through increased release of ET.
Asunto(s)
Endotelinas/sangre , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Choque Séptico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Endotelinas/agonistas , Hemodinámica/efectos de los fármacos , Inmunización , Infusiones Intravenosas , Choque Séptico/microbiología , Porcinos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
A number of key mediators are implicated in the pathophysiology of sepsis. In previous studies of a septic porcine model, ibuprofen pretreatment prevented the early but not the late rise in pulmonary vascular resistance index (PVRI) and the early but not the late fall in arterial PO2 (PaO2), whereas monoclonal antibody to tumor necrosis factor alpha (anti-TNF alpha) prevented the late but not the early rise in PVRI and the late but not the early fall in PaO2. This study examined the impact of pretreatment with combined ibuprofen and anti-TNF-alpha on the course of sepsis and acute lung injury (ALI) in pigs. Three groups were studied for 5 hours. Groups I (n = 9) and II (n = 5) received a 1-hour infusion of Pseudomonas aeruginosa. Group II received ibuprofen (12.5 mg/kg) and anti-TNF-alpha (5 mg/kg) before P. aeruginosa, and a further bolus of ibuprofen at 120 minutes. Group III (n = 11) received sterile saline. Group I demonstrated a significant (p < 0.05) rise in plasma TNF-alpha that was abolished in group II. The SVRI in group II did not change significantly from baseline through the study and the SVRI rose sharply in group I following onset of the infusion of P. aeruginosa, as did PVRI. There was no significant change in PVRI from baseline in group II, except for the final 60 minutes; PVRI in group II was significantly less than in group I throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Gasto Cardíaco/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/fisiopatología , Ibuprofeno/farmacología , Neutrófilos/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Resistencia Vascular/efectos de los fármacos , Animales , Antígenos CD18 , Gasto Cardíaco/fisiología , Dimercaprol , Quimioterapia Combinada , Infecciones por Bacterias Gramnegativas/sangre , Recuento de Leucocitos , Oxígeno/metabolismo , Presión Parcial , Premedicación , Pseudomonas aeruginosa , Circulación Pulmonar , Superóxidos/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/metabolismo , Resistencia Vascular/fisiologíaRESUMEN
Tumor necrosis factor (TNF alpha), both by direct action and by trafficking cells of the immune system, is implicated in cardiopulmonary derangements and PMN-mediated microvascular injury associated with gram-negative sepsis. We examined the effects of pretreatment with a monoclonal antibody to TNF alpha on PMN function, hemodynamic derangements, and alveolar capillary membrane damage in a septic porcine model. Anti-TNF alpha profoundly improved hemodynamic consequences in this model. Reduction in PMN CD11/18 receptor expression, lung myeloperoxidase activity, and attenuation of peripheral neutropenia (all P < 0.05) indicate that pretreatment significantly reduced lung sequestration of PMNs seen in septic controls. In contrast, PMN oxygen radical (O2-) generation was not significantly different from unprotected septic animals. Despite the presence of circulating PMNs primed for O2- burst, alveolar capillary membrane damage, assessed by bronchoalveolar lavage protein content and arterial PO2 was markedly attenuated in the treatment group (P < 0.05). We conclude that anti-TNF alpha suppresses systemic hemodynamic actions of TNF alpha. Further, it prevents upregulation of PMN adhesion receptors inhibiting PMN/endothelial cell interaction. This prevents formation of a "microenvironment," protected from circulating oxidant scavengers, into which sepsis-activated PMNs release their toxic products. Pretreatment with anti-TNF alpha monoclonal antibody thus affords global protection in porcine Gram-negative sepsis.
Asunto(s)
Antígenos CD/fisiología , Neutrófilos/inmunología , Oxígeno/sangre , Síndrome de Dificultad Respiratoria/fisiopatología , Sepsis/fisiopatología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Líquido del Lavado Bronquioalveolar/química , Antígenos CD11 , Antígenos CD18 , Radicales Libres , Neutrófilos/química , Neutrófilos/citología , Porcinos , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Tumor necrosis factor-alpha (TNF), an inflammatory cytokine released by macrophages, may be a mediator of lung injury during septicemia. We previously reported that the cyclooxygenase inhibitor ibuprofen and histamine receptor antagonists cimetidine (H2 antagonist) and diphenhydramine (H1 antagonist) attenuate lung injury and reduce circulating TNF surges during porcine sepsis. Since pulmonary alveolar macrophages (PAM) may participate in early sepsis by producing TNF, we hypothesized that the TNF activity of PAM is reduced by ibuprofen, cimetidine, and diphenhydramine. To test this, we examined changes in PAM-derived TNF bioactivity and cell viability of freshly isolated porcine PAM during exposure to bacterial endotoxin (LPS), ibuprofen, cimetidine, and diphenhydramine. The TNF activity (% L929 cytotoxicity of PAM conditioned medium) was elevated in LPS-stimulated PAM cultures (15 to 25% increase at 1 to 6 h and 40 to 43% increase at 6 to 48 h, compared with non-LPS-stimulated cultures), and ibuprofen (150 micrograms/ml) added with LPS decreased the TNF activity for 24 h (20 to 28% reduction at 1 to 24 h). Ibuprofen added 1 h after LPS was less effective in reducing the PAM-derived TNF activity (20 to 22% reduction at 2 to 6 h). Cimetidine (112 micrograms/ml) reduced the TNF activity of LPS-stimulated PAM cultures during the first 4 h of LPS exposure (15 to 24% decrease at 1 to 4 h). Diphenhydramine (150 micrograms/ml) attenuated the PAM-derived TNF activity but also decreased viability of PAM, indicating a toxic effect of this agent on PAM.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cimetidina/farmacología , Difenhidramina/farmacología , Ibuprofeno/farmacología , Macrófagos Alveolares/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Histamina/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Ratones , PorcinosRESUMEN
Pulmonary alveolar macrophages (PAM) function as phagocytes of inhaled particulate matter and microorganisms at the air-tissue interface of lung alveoli. Changes in cellular ATP concentrations ([ATP]) and phagocytic function during acute hypoxia may be important in conditions associated with low alveolar O2. We proposed that acute hypoxia would decrease phagocytosis and reduce [ATP] in freshly isolated PAM. Phagocytic function (fluorescent microscopic technique determining percent phagocytosis in live cells) was monitored by recording uptake and retention of glutaraldehyde-fixed red blood cells (GRBC) in isolated rabbit PAM during acute incubations in air (20% O2) or hypoxia (1.7% O2). Macrophage [ATP] were determined spectrophotometrically. Acute hypoxia for 30 to 150 min decreased phagocytic function 30 to 56% in PAM without significantly affecting cell adherence and viability. Pre-exposure of PAM to hypoxia before addition of GRBC resulted in an even greater reduction in phagocytosis (97% decrease by 30 min), and recovery of phagocytic function occurred 60 to 90 min after returning PAM to air. The cellular retention of phagocytosed GRBC (percentage of PAM with GRBC and number of GRBC/PAM) was reduced 30% by 1 h of hypoxia. Compared with [ATP] of PAM in air, [ATP] of PAM exposed to hypoxia were reduced 55 and 35% at 30 and 60 min, respectively. Compared with [ATP] of cells with GRBC in air at 0 and 30 min, PAM with GRBC in hypoxia for 30 min had, respectively, 61 and 40% lower [ATP]. By 60 min with GRBC, PAM [ATP] in air and hypoxia were similar but were 50% lower than [ATP] at time 0.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina Trifosfato/metabolismo , Hipoxia de la Célula/inmunología , Macrófagos Alveolares/inmunología , Fagocitosis , Animales , Adhesión Celular , Supervivencia Celular , Glutaral , Microscopía Fluorescente , ConejosRESUMEN
Tumor necrosis factor (TNF) is implicated in the pathophysiology of gram-negative sepsis. This study examined physiologic and biochemical effects of pretreatment with an anti-TNF alpha monoclonal antibody immediately before the onset of sepsis. Three groups of anesthetized ventilated pigs were studied for 300 minutes. Groups 1 (n = 12) and 2 (n = 6) received a 1-hour infusion of live Pseudomonas aeruginosa. Group 2 was pretreated with anti-TNF alpha monoclonal antibody (15 mg/kg). Group 3 (n = 8) received intravenous sterile saline. Group 1 exhibited a significant rise in plasma TNF activity, which was abolished in group 2. Cardiac index was reduced in both groups 1 and 2 in the first hour but recovered in group 2 (3.3 +/- 0.4 l/min per square meter at 300 minutes in group 2 vs 1.3 +/- 0.2 L/min per square meter in group 1). Metabolic acidosis was attenuated (arterial pH, 7.39 +/- 0.01 in group 2 vs 7.16 +/- 0.03 at 300 minutes in group 1). Increased extravascular lung water was also attenuated (5.9 +/- 0.7 in group 2 vs 13.2 +/- 1.5 mL/kg at 300 minutes in group 1). However, pulmonary hypertension and hypoxemia, which are known cyclooxygenase effects, were not affected. In the early phase of the study, plasma thromboxane B2 levels were elevated in both groups 1 and 2. We conclude that anti-TNF alpha monoclonal antibody offered significant protection against the effects of sepsis, but that other mediators may be responsible for the early changes seen in this model.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Bacteriemia/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/inmunología , Animales , Bacteriemia/complicaciones , Bacteriemia/fisiopatología , Agua Pulmonar Extravascular/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Pulmón/efectos de los fármacos , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/fisiopatología , Choque Séptico/etiología , Choque Séptico/prevención & control , Porcinos , Tromboxano B2/sangre , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Plasma tumor necrosis factor (TNF) activity, cardiac index, extravascular lung water, systemic and pulmonary arterial pressures, pulmonary vascular resistance index, and arterial PO2 were monitored for 300 min in four groups of anesthetized pigs: saline-infused animals (n = 5), saline-infused animals given ibuprofen (12.5 mg/kg iv) at 0 and 120 min (n = 4), animals infused for 60 min with live Pseudomonas aeruginosa (Ps, 5 x 10(8) organisms/ml at 0.3 ml.20 kg-1.min-1, n = 6), and animals infused for 60 min with Ps plus ibuprofen administered at 0 and 120 min (n = 4). Infusion of Ps induced significant elevations (greater than 4-fold increase in units/ml of TNF by 60 min, P less than 0.05) in plasma TNF activity (L929 cytolysis assay) and alterations (P less than 0.05) in all hemodynamic and pulmonary parameters within 30-60 min. Ibuprofen administration in sepsis significantly decreased peak TNF activity by 2 units/ml and attenuated many of the physiological alterations due to sepsis. These results show that ibuprofen attenuates sepsis-induced injury and that alterations of acute septic insult are correlated with reduced plasma TNF activity in septic animals given ibuprofen.
Asunto(s)
Ibuprofeno/farmacología , Enfermedades Pulmonares/sangre , Infecciones por Pseudomonas/sangre , Factor de Necrosis Tumoral alfa/análisis , Enfermedad Aguda , Animales , Análisis de los Gases de la Sangre , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Agua Pulmonar Extravascular/efectos de los fármacos , Enfermedades Pulmonares/fisiopatología , Infecciones por Pseudomonas/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Porcinos , Resistencia Vascular/efectos de los fármacosRESUMEN
Ibuprofen pretreatment attenuates the enhanced neutrophil (PMN) respiratory burst and reduces increased plasma tumor necrosis factor (TNF) activity in porcine sepsis-induced acute lung injury (ALI). These septic responses have been linked to increased alveolar-capillary membrane (ACM) permeability. This study was designed to establish whether delayed ibuprofen treatment would have the same effect and to examine the relationship between PMN oxidant generation and TNF. Three groups of anesthetized, ventilated pigs (15-25 kg) were used. Group Ps received Pseudomonas aeruginosa (5 x 10(8) CFU/mL at 0.3 mL/20 kg/min) for one hour IV; The control group (Con) received 0.9% NaCl. Group D-Ibu received ibuprofen 12.5 mg/kg as a delayed bolus at 30 minutes and again at 120 minutes after Ps. Protein (BAL-P, microgram/mL) in harvested bronchoalveolar lavage fluid and extravascular lung water (EVLW, mL/kg) were used to estimate the integrity of the ACM. Superoxide anion (O2-) generation (ferricytochrome c reduction) from circulating PMNs and plasma TNF activity (L929 fibroblast bioassay) were measured. The EVLW increased significantly (p less than 0.05), as did BAL-P (p less than 0.01), in the P. aeruginosa-treated animals at 300 minutes. These increases were abolished in Group D-Ibu: EVLW, 6.6 +/- 1.0 baseline vs. 14.6 +/- 2.6 Ps 300 vs. 6.8 +/- 0.9 D-Ibu 300; BAL-P, 175 +/- 28 baseline vs. 984 +/- 186 Ps 300 vs. 284 +/- 42.8 D-Ibu 300. Both enhanced PMN oxidant activity and increased plasma TNF activity were significantly attenuated by delayed ibuprofen treatment. These data support the efficacy of the nonsteroidal anti-inflammatory drug, ibuprofen, when used after the onset of a septic stimulus.
Asunto(s)
Inhibidores de la Ciclooxigenasa , Enfermedades Pulmonares/sangre , Neutrófilos/metabolismo , Infecciones por Pseudomonas/complicaciones , Factor de Necrosis Tumoral alfa/análisis , Animales , Líquido del Lavado Bronquioalveolar , Permeabilidad Capilar , Agua Pulmonar Extravascular/metabolismo , Hemodinámica , Ibuprofeno/farmacología , Recuento de Leucocitos , Pulmón/irrigación sanguínea , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/fisiopatología , Neutrófilos/efectos de los fármacos , Alveolos Pulmonares/fisiopatología , Superóxidos/metabolismo , Porcinos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The microcirculation contains mononuclear phagocytes, with features characteristic of macrophages, adhered to luminal capillary surfaces by intercellular adhesion plaques. These pulmonary intravascular macrophages may play an important role in regulating lung vascular tone and capillary permeability, and may modulate capillary endothelial cell growth and replication by the secretion of soluble mediators (i.e., arachidonate metabolites, cytokines). This study describes a technique which utilizes in situ lung perfusion to remove intravascular macrophages in large numbers from the microcirculation of porcine lung (n = 26). This technique yielded 3.8 +/- 0.5 x 10(8) (mean +/- SEM) mononuclear cells which were highly phagocytic toward particulate carbon (phagocytic index, 80 +/- 6%). Harvested mononuclear phagocytes reestablished intercellular adhesion plaques when placed on small vessel porcine pulmonary artery endothelial cell monolayers and exhibited histochemical characteristics typical of monocyte/macrophage lineage cells. Mononuclear cells obtained from lung microcirculation displayed size heterogeneity varying from 10.4 to 16.5 microns in diameter. Both large and small cell populations phagocytosed particulate carbon. Morphometric studies performed on collagenase-treated lung demonstrated that in situ perfusion removed significant numbers of intravascular macrophages in lung capillaries. The technique described permits the rapid removal of anchored mononuclear phagocytes from lung capillaries with minimal postmortem delay.
Asunto(s)
Separación Celular , Pulmón/irrigación sanguínea , Macrófagos , Animales , Recuento de Células , Separación Celular/métodos , Histocitoquímica , Pulmón/citología , Pulmón/efectos de los fármacos , Macrófagos/química , Macrófagos/fisiología , Macrófagos/ultraestructura , Colagenasa Microbiana/farmacología , Microcirculación , Perfusión , Fagocitosis , PorcinosRESUMEN
Tumor necrosis factor (TNF) may be involved in the pathogenesis of acute lung injury (ALI) associated with septicemia. Therefore, we measured plasma TNF activity during sepsis and development of lung injury in a porcine model of ALI. Plasma samples were obtained from anesthetized saline-infused control pigs (n = 10) and those infused for 1 h with live Pseudomonas aeruginosa (10(8) organisms/ml, 0.3 ml/20 kg/min) (n = 16). TNF activity was measured in plasma using the L929 fibroblast cytolytic assay. L929 cytotoxicity caused by TNF-alpha or TNF-beta was determined in plasma by measuring the cytotoxicity neutralized by TNF antisera. No significant TNF activity was detected in control pig plasma. In septic pigs, TNF activity appeared in plasma 15 min after onset of septicemia and remained elevated throughout the experiment (6.1 +/- 10.2% to 80.0 +/- 5.0%, 15 and 300 min, respectively). The appearance of pulmonary arterial hypertension, increased lung water, decreased lung compliance, and deteriorating gas exchange was correlated with the rise in plasma TNF activity, which reached a peak at 90 to 120 min in septic pigs. Our results provide evidence that both TNF subtypes are present in plasma during septicemia. Anti-TNF-alpha and anti-TNF-beta neutralized TNF activity in whole septic plasma at 15, 30, and 45 min after onset of septicemia, and the antibodies blocked TNF activity in serially diluted septic plasma at all time points up to 210 min of sepsis. TNF activity in septic plasma at 210 to 300 min was not neutralized entirely by TNF antisera.(ABSTRACT TRUNCATED AT 250 WORDS)