RESUMEN
Volatile anesthetics are known to attenuate inflammatory response and tissue damage markers in acute organ injury. It is unclear whether these beneficial effects of volatile anesthetics are mediated by the ether basic structure or by characteristics of their halogenations. We describe in an in vitro model of acute inflammation in pulmonary cells that halogenation (fluorinated carbon groups) is responsible for the immunomodulatory effects. The inflammatory response after coexposure to endotoxin and sevoflurane, diethyl-ether, or various water-soluble molecules carrying trifluorinated carbon (CF(3)) groups was evaluated in pulmonary epithelial and endothelial cells and in neutrophils. In epithelial and endothelial cells, expression of inflammatory mediators to LPS stimulation was dose-dependently decreased upon exposure to sevoflurane and other molecules with CF(3) groups. This was not observed for diethyl-ether or structure-similar nonfluorinated molecules. In neutrophils, chemotactic activity, as well as expression of surface CD11b and CD62L, was positively modified by molecules carrying CF(3) groups. Cytotoxicity could be excluded. These findings for the first time reveal in an in vitro model of acute inflammation that the immunomodulatory effects are not limited to volatile anesthetics but are associated with a much broader class of CF(3) group-containing molecules. The immunomodulatory effects could now be provided in a hydrophilic, injectable formulation for the treatment of patients suffering from acute organ injury, such as acute lung injury, in environments not suitable for volatile anesthetics.
Asunto(s)
Anestésicos/farmacología , Flúor/farmacología , Factores Inmunológicos/inmunología , Animales , Aorta/citología , Carbono/química , Caspasa 3/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Células Epiteliales/citología , Escherichia coli/metabolismo , Halógenos/química , Humanos , Inflamación , Lipopolisacáridos/metabolismo , Éteres Metílicos/farmacología , Microcirculación , Neutrófilos/citología , Ratas , SevofluranoRESUMEN
BACKGROUND: Acute renal failure is a frequent complication of sepsis. Hydroxyethyl starch (HES) is widely used in the treatment of such patients. However, the effect of HES on renal function during sepsis remains controversial. We established an in vitro model of tumor necrosis factor-alpha (TNF-alpha)-stimulated human proximal tubular epithelial (HK-2) cells to assess the possible effects of HES 130/0.42 and HES 200/0.5 on these activated cells. METHODS: HK-2 cells were stimulated with TNF-alpha in the presence or absence of HES 130/0.42 or 200/0.5. After 4, 10, and 18 h of incubation, monocyte chemoattractant protein-1 (MCP-1), a key chemoattractant for neutrophils and macrophages, was measured. In addition, viability and cytotoxicity assays were performed. RESULTS: MCP-1 expression was doubled upon TNF-alpha exposure. In the presence of 2% and 4% HES 200/0.5 in 98% (96%) medium over a stimulation time period of 10 h and 18 h, the MCP-1 concentration was decreased between 26% and 56% (P < 0.05). TNF-alpha stimulation resulted in a significant decrease of viability by 53%-63%, whereas viability decreased by only 32%-40% in coincubation with HES 130/0.42 (P < 0.005) and remained even less affected by TNF-alpha in the presence of HES 200/0.5 (P < 0.001). The TNF-alpha-induced cell death rate was attenuated in the presence of HES 200/0.5 (P < 0.05). CONCLUSIONS: This in vitro study shows that both HES products modulate cell injury upon inflammatory stimulation. The effect was more pronounced in the HES 200/0.5 group than for HES 130/0.42, suggesting a possible biological difference between the HES types.
Asunto(s)
Derivados de Hidroxietil Almidón/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Sustitutos del Plasma/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Gelatina/farmacología , Humanos , Derivados de Hidroxietil Almidón/química , Inflamación , Soluciones Isotónicas/farmacología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , L-Lactato Deshidrogenasa/metabolismo , Microscopía Confocal , Lactato de Ringer , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Acute lung injury is a common complication in critically ill patients. Several studies suggest that volatile anesthetics have immunomodulating effects. The aim of the current study was to assess possible postconditioning with sevoflurane in an in vivo model of endotoxin-induced lung injury. METHODS: Rats were anesthetized, tracheotomized, and mechanically ventilated. Lipopolysaccharide (saline as control) was administered intratracheally. Upon injury after 2 h of propofol anesthesia, general anesthesia was continued with either sevoflurane or propofol for 4 h. Arterial blood gases were measured every 2 h. After 6 h of injury, bronchoalveolar lavage was performed and lungs were collected. Total cell count, albumin content, concentrations of the cytokines cytokine-induced neutrophil chemoattractant-1 and monocyte chemoattractant protein-1, and phospholipids were analyzed in bronchoalveolar lavage fluid. Expression of messenger RNA for the two cytokines and for surfactant protein B was determined in lung tissue. Histopathologic examination of the lung was performed. RESULTS: Significant improvement of the ratio of oxygen tension to inspired oxygen fraction was shown with sevoflurane (mean + or - SD: 243 + or - 94 mmHg [32.4 kPa]) compared with propofol (88 + or - 19 mmHg [11.7 kPa]). Total cell count representing effector cell recruitment as well as albumin content as a measure of lung permeability were significantly decreased in the sevoflurane-lipopolysaccharide group compared with the propofol-lipopolysaccharide group in bronchoalveolar lavage fluid. Expression of the cytokines protein in bronchoalveolar lavage fluid as well as messenger RNA in lung tissue was significantly lower in the sevoflurane-lipopolysaccharide group compared with the propofol-lipopolysaccharide group. CONCLUSIONS: Postconditioning with sevoflurane attenuates lung damage and preserves lung function in an in vivo model of acute lung injury.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Lipopolisacáridos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/prevención & control , Éteres Metílicos/toxicidad , Intercambio Gaseoso Pulmonar/fisiología , Animales , Presión Sanguínea/fisiología , Líquido del Lavado Bronquioalveolar/química , Recuento de Células , Células Cultivadas , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL1/genética , Quimiocinas/metabolismo , Células Endoteliales , Ensayo de Inmunoadsorción Enzimática , Hipercapnia/metabolismo , Enfermedades Pulmonares/patología , Masculino , Permeabilidad , Fosfolípidos/análisis , Propofol/farmacología , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Surfactantes Pulmonares/análisis , Surfactantes Pulmonares/aislamiento & purificación , ARN/biosíntesis , ARN/aislamiento & purificación , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SevofluranoRESUMEN
BACKGROUND: Endotoxin-induced lung injury is a useful experimental system for the characterization of immunopathologic mechanisms in acute lung injury. Although alveolar epithelial cells (AEC) are directly exposed to volatile anesthetics, there is limited information about the effect of anesthetics on these cells. In this study we investigated the effect of pretreatment with the inhaled anesthetic sevoflurane on lipopolysaccharide (LPS)-injured AEC. METHODS: AEC were incubated with 1.1 vol % sevoflurane for 0.5 h, followed by LPS stimulation for 5 h. Expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1beta (MIP-1beta), macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant-1 (CINC-1), and intercellular adhesion molecule-1 (ICAM-1) was analyzed. In addition, functional tests were performed through chemotaxis and adherence assays to underline the biological relevance of the findings. RESULTS: Exposure of AEC to sevoflurane resulted in a 50% downregulation of MCP-1 protein in the sevoflurane-LPS group when compared with non-sevoflurane- LPS cells (P < 0.05). MIP-1beta concentration in LPS-stimulated cells decreased by 32% with sevoflurane (P < 0.05), MIP-2 by 29% (P < 0.05), and CINC-1 by 20% (P < 0.05). ICAM-1 protein expression was attenuated by 36% (P < 0.05). This inhibition caused substantial changes in the inflammatory response of neutrophils. 33% less chemotactic activity was seen in sevoflurane-treated LPS cells (P < 0.001) as well as 47% decreased adhesion of neutrophils to AEC (P < 0.001). CONCLUSIONS: This study shows that sevoflurane alters the LPS-induced inflammatory response, not only with respect to the expression pattern of inflammatory mediators, but also regarding the biological consequences with less accumulation of effector cells such as neutrophils.
Asunto(s)
Endotoxinas/farmacología , Epitelio/efectos de los fármacos , Éteres Metílicos/farmacología , Alveolos Pulmonares/efectos de los fármacos , Anestésicos/farmacología , Anestésicos por Inhalación/farmacología , Animales , Adhesión Celular , Quimiocina CCL2/metabolismo , Quimiocina CCL4 , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Pulmón/citología , Proteínas Inflamatorias de Macrófagos/metabolismo , Monocinas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ratas , SevofluranoRESUMEN
BACKGROUND: Endotoxin causes acute lung injury, which can lead to acute respiratory distress syndrome. Because local anesthetics are known to attenuate inflammatory reactions, ropivacaine was tested for its possible antiinflammatory effect in lipopolysaccharide-induced lung injury in rat alveolar epithelial cells (AECs) and rat pulmonary artery endothelial cells (RPAECs) in vitro and in vivo. METHODS: AECs and RPAECs were stimulated for 4 h with lipopolysaccharide or lipopolysaccharide and 1 mum ropivacaine. Messenger RNA (mRNA) for intercellular adhesion molecule 1 was assessed. Isolated neutrophils were incubated with stimulated target cells to quantify adhesion and neutrophil-induced cytotoxicity in AECs and RPAECs. In vivo, lipopolysaccharide was instilled intratracheally with or without 1 mm intratracheally or intravenously administered ropivacaine. Bronchoalveolar lavage was performed 5 h later to determine neutrophil and albumin content, as well as concentrations of inflammatory mediators. RESULTS: In AECs and RPAECs, ropivacaine attenuated lipopolysaccharide-induced up-regulation of mRNA for intercellular adhesion molecule 1 by 41% and 24%, respectively (P < 0.05). In the presence of ropivacaine, increased neutrophil adhesion was down-regulated by 58% and 44% (P < 0.005), whereas cytotoxicity in AECs and RPAECs was diminished by 28% and 33%, respectively (P < 0.05). Enhanced neutrophil count in lipopolysaccharide lungs was reduced by 56% in the presence of intratracheally instilled ropivacaine (81% with intravenous ropivacaine; P < 0.005). Albumin was decreased by 46% with intratracheal ropivacaine (38% with intravenous ropivacaine; P < 0.05), and inflammatory mediators were decreased by 48-59% (69-81% with intravenous ropivacaine; P < 0.01). CONCLUSIONS: Ropivacaine intervention substantially attenuated the inflammatory response in acute lung injury and thus may carry an interesting potential for antiinflammatory treatment.
Asunto(s)
Amidas/farmacología , Anestésicos Locales/farmacología , Antiinflamatorios , Factores Inmunológicos , Neumonía/prevención & control , Adulto , Animales , Líquido del Lavado Bronquioalveolar/citología , Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotoxinas , Células Epiteliales/efectos de los fármacos , Humanos , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular/genética , Lipopolisacáridos , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neumonía/inducido químicamente , Neumonía/patología , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Ribosómico 18S/genética , Ratas , Ratas Wistar , Ropivacaína , Albúmina Sérica/metabolismoRESUMEN
BACKGROUND: Early stages of diabetic nephropathy are characterized by alterations of glomerular filtration, increased tubular sodium and water reabsorption, and systemic volume expansion, which may be a major cause for the development of hypertension. As a significant fraction of renal salt and water transport is mediated by the proximal tubular Na+/H+ exchanger NHE3, we investigated its regulation in rats with STZ-induced diabetes mellitus. METHODS: Male Sprague-Dawley rats were injected +/- streptozotocin (STZ, 60 mg/kg), and sacrificed after 2, 7 or 14 days. Renal cortical BBM vesicles were prepared to measure Na+/H+ exchange (NHE) activity and NHE3 protein abundance. Cortical NHE3 mRNA was extracted to perform Northern blot analysis. Pharmacological inhibitors were used in vivo and in vitro in order to identify isoform specificity conferring changes in NHE activity mediated by the diabetic milieu. RESULTS: Compared to control rats, STZ rats were clearly hyperglycemic at all time points studied. NHE activity was significantly increased by 40 and 37% in diabetic rats after 7 and 14 days, respectively, but not after 2 days. The increase in Na+/H+ exchange activity was not inhibited by HOE-642 (3 microM). Administration of exogenous insulin to diabetic rats resulted in lower blood sugars, but not NHE activity. Moreover, serum glucose concentration did not correlate with NHE activity in any subgroup nor in all animals analyzed together. However, in STZ rats supplemented with exogenous insulin NHE activity was positively correlated with serum insulin concentrations (r = 0.86, p < 0.01). In vivo, the increase in NHE activity induced by STZ could be completely inhibited when rats were fed 6 ppm of HOE-642 with the diet over 14 days. The changes in Na+/H+ exchange activity were not paralleled by changes in NHE3 protein or mRNA abundance in diabetic rats at any of the time points investigated. CONCLUSIONS: These results suggest that proximal tubular Na/H exchange activity is modified in the early stage of diabetes mellitus.
Asunto(s)
Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Túbulos Renales Proximales/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Estreptozocina , Enfermedad Aguda , Animales , Enfermedad Crónica , Retroalimentación , Masculino , Ratas , Ratas Sprague-Dawley , Intercambiador 3 de Sodio-HidrógenoRESUMEN
BACKGROUND: Acute lung injury caused by gastric aspiration is a frequent occurrence in unconscious patients. Acute respiratory distress syndrome in association with gastric aspiration carries a mortality of up to 30% and accounts for up to 20% of deaths associated with anesthesia. Although the clinical condition is well known, knowledge about the exact inflammatory mechanisms is still incomplete. This study was performed to define the role of alveolar macrophages in this inflammatory response. In addition, potentially modifying effects of intratracheally applied nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate were investigated. METHODS: Rat alveolar macrophages were depleted by intratracheal administration of clodronate liposomes, and lung injury was evaluated 6 h after instillation of 0.1N hydrochloric acid. In a second set of experiments, pyrrolidine dithiocarbamate was intratracheally instilled 3 h after hydrochloric acid application, and injury parameters were determined. RESULTS: Depletion of alveolar macrophages resulted in decreased production of inflammatory mediators in acid aspiration (23-80% reduction of messenger RNA or protein of inflammatory mediators; P < 0.05) and consequently also in diminished neutrophil recruitment (36% fewer neutrophils; P < 0.01). Treatment with pyrrolidine dithiocarbamate was highly effective in decreasing neutrophil recruitment (66%; P < 0.01) and vascular permeability (80%; P < 0.001). CONCLUSIONS: These data suggest that alveolar macrophages play an essential role in the inflammatory response of acid-induced lung injury. For the first time, attenuation of acid-induced lung injury with an inhibitor, applied after the onset of injury, is shown.
Asunto(s)
Macrófagos Alveolares/fisiología , FN-kappa B/antagonistas & inhibidores , Neumonía por Aspiración/tratamiento farmacológico , Pirrolidinas/uso terapéutico , Tiocarbamatos/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar/química , Permeabilidad Capilar , Quimiocina CCL2/fisiología , Modelos Animales de Enfermedad , Mediadores de Inflamación/fisiología , Masculino , Neumonía por Aspiración/etiología , Ratas , Ratas Wistar , Síndrome de Dificultad Respiratoria/etiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury. METHODS: Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed. RESULTS: A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation. CONCLUSION: These data suggest that alveolar macrophages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.
Asunto(s)
Quimiocina CCL2/inmunología , Activación de Macrófagos/inmunología , Activación Neutrófila/inmunología , Neumonía/inmunología , Neumonía/patología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , Animales , Comunicación Celular/inmunología , Endotoxinas , Lipopolisacáridos , Macrófagos/inmunología , Masculino , Neutrófilos/inmunología , Ratas , Ratas WistarRESUMEN
Albumin filtered by the glomerulus is reabsorbed in the proximal tubule. We have shown previously that proteinuria stimulates the proximal tubular Na(+)/H(+) exchanger 3 (NHE3) in rats. Activation of NHE3 may be a pathophysiologically important factor in the development of renal salt and water retention observed in the nephrotic syndrome. For examining whether albumin is a specific inducer of proximal tubular Na(+)/H(+) exchange and to determine the molecular mechanisms by which it regulates Na(+)/H(+) exchange, the effect of albumin on NHE3 in opossum kidney cells was studied. Albumin activated Na(+)/H(+) exchange in a time- and dose-dependent manner up to 100% in 48 h. In the early phase of stimulation (2 to 12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, increased NHE3 activity was accompanied by increase in cell surface NHE3 protein. The increase in surface NHE3 was associated with increased bidirectional trafficking of NHE3 protein between intracellular compartments and the cell surface. At 48 h, total cell NHE3 protein abundance and mRNA were increased as well. Whereas NHE3 translation was increased, NHE3 protein half-life remained unchanged. The effects of albumin on NHE3 protein abundance were modified by hydrocortisone in a complicated pattern. These results indicate that albumin directly regulates proximal tubular NHE3 at multiple levels.
Asunto(s)
Albúminas/fisiología , Intercambiadores de Sodio-Hidrógeno , Animales , Células Cultivadas , Ratas , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na(+)/H(+) exchanger 3 (NHE3), a principal Na(+) transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na(+)/H(+) exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2-12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.