Exacerbation by granulocyte colony-stimulating factor of prior acute lung injury: implication of neutrophils

OBJECTIVE: Granulocyte colony-stimulating factor is widely prescribed to hasten recovery from cancer chemotherapy-induced neutropenia and has been reported to induce pulmonary toxicity. However, circumstances and mechanisms of this toxicity remain poorly known. DESIGN: To reproduce a routine situation in cancer patients receiving chemotherapy, we investigated the mechanisms underlying granulocyte colony-stimulating factor-induced exacerbation of alpha-naphthylthiourea-related pulmonary edema. SETTING: Laboratory research unit. SUBJECTS: Male specific-pathogen-free Sprague-Dawley rats. INTERVENTIONS: The effects of granulocyte colony-stimulating factor given alone or after alpha-naphthylthiourea used to induce acute lung injury were investigated. MEASUREMENTS AND MAIN RESULTS: Lung injury was assessed based on neutrophil sequestration (myeloperoxidase activity in lung tissue) and influx into alveolar spaces (bronchoalveolar lavage fluid cell quantification) and on edema formation (wet/dry lung weight ratio) and alveolar protein concentration into bronchoalveolar lavage fluid. Tumor necrosis factor-alpha and interleukin-1beta were measured in serum, lung homogenates, and isolated alveolar macrophage supernatants. In control rats, granulocyte colony-stimulating factor (25 microg/kg) significantly elevated circulating neutrophil counts without producing alveolar recruitment or pulmonary edema. alpha-Naphthylthiourea significantly increased the wet/dry lung weight ratio (4.68 +/- 0.04 vs. 4.38 +/- 0.07 in controls, p=.04) and induced alveolar protein leakage. Adding granulocyte colony-stimulating factor to alpha-naphthylthiourea exacerbated pulmonary edema, causing neutrophil sequestration in pulmonary vessels, significantly increasing lung myeloperoxidase activity (12.7 +/- 2.0 mOD/min/g vs. 1.1 +/- 0.4 mOD/min/g with alpha-naphthylthiourea alone; p<.0001), and increasing proinflammatory cytokine secretion. alpha-Naphthylthiourea-related pulmonary edema was not exacerbated by granulocyte colony-stimulating factor during cyclophosphamide-induced neutropenia or after lidocaine, which antagonizes neutrophil adhesion to endothelial cells. Tumor necrosis factor-alpha and interleukin-1beta concentrations in alveolar macrophage supernatants and lung homogenates were significantly higher with alpha-naphthylthiourea + granulocyte colony-stimulating factor than with either agent alone, and anti-tumor necrosis factor-alpha antibodies abolished granulocyte colony-stimulating factor-related exacerbation of alpha-naphthylthiourea-induced pulmonary edema. In rats with cyclophosphamide-induced neutropenia, tumor necrosis factor-alpha concentrations in alveolar macrophage supernatants and lung homogenates were significantly decreased compared with rats without neutropenia. CONCLUSION: Granulocyte colony-stimulating factor-related pulmonary toxicity may involve migration of neutrophils to vascular spaces, adhesion of neutrophils to previously injured endothelial cells, and potentiation of proinflammatory cytokine expression.     

Inhibition of nitric oxide synthesis by L-name exacerbates acute lung injury induced by hepatic ischemia-reperfusion.

Hepatic Kupffer cells and pulmonary alveolar macrophages together constitute a macrophage-axis involved in the regulation of regional and systemic inflammatory responses. Systemic inflammatory response syndrome induced by overproduced pro-inflammatory mediators is the major cause of adult respiratory distress syndrome. In the present study, we examined the anti-inflammatory role of nitric oxide (NO) in a rat model of acute lung injury induced by hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia by clamping the relevant branches of hepatic artery and portal vein, followed by a 4-h reperfusion achieved by removal of the vascular clamp. Four groups of animals were studied: sham control + saline; sham control + N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v., 10 min before reperfusion); HI/R + saline; HI/R + L-NAME. Results show that (1) administration of L-NAME to rats subjected to HI/R decreased plasma NO levels; however, the attenuation of NO increased plasma alanine aminotransferase (ALT) activity and superoxide generation in the ischemic lobes of liver, compared to HI/R alone. (2) Inhibition of NO synthesis with L-NAME in rats subjected to HI/R also enhanced systemic inflammatory response as assessed by the increase in the number of circulating leukocytes and levels of plasma tumor necrosis factor-alpha (TNFalpha) and interleukin 1-beta (IL-1beta). (3) The overwhelming systemic inflammatory response induced by administration of L-NAME in rats subjected to HI/R also augmented pulmonary vascular permeability and superoxide generation in the lung tissue. (4) Pulmonary alveolar macrophages isolated from rats subjected to HI/R + L-NAME produced higher levels of TNFalpha and IL-1beta in the supernatant of culture medium than that of rats subjected to HI/R alone. (5) There were no differences between the groups of sham + saline and sham + L-NAME in terms of plasma NO levels and ALT activity, circulating leukocytes, superoxide generation in the liver and lung, lavage protein levels, and TNFalpha and IL-1beta levels in plasma and bronchoalveolar lavage fluid. Our results suggest that inhibition of NO synthesis by L-NAME in rats subjected to HI/R not only augments ischemic liver injury, but also enhances the systemic inflammatory response and exacerbates remote lung injury. The increase in TNFalpha and IL-1beta production by alveolar macrophages may, in part, account for L-NAME-induced enhancement of acute lung injury.     

Sesame oil protects against lead-plus-lipopolysaccharide-induced acute hepatic injury

Lead (Pb) increases lipopolysaccharide (LPS)-induced tumor necrosis factor alpha, which causes liver damage. In this study, we investigated the effect of sesame oil on Pb-plus-LPS (Pb + LPS)-induced acute liver damage in mice. Mice were given sesame oil (8 mL/kg orally) just after Pb acetate (10 mmol/kg i.p.) plus LPS (5 mg/kg i.p.). Aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-alpha, interleukin-1beta, nitric oxide, and inducible nitric oxide synthase levels were examined. Sesame oil significantly decreased serum aspartate aminotransferase and alanine aminotransferase levels in Pb + LPS-stimulated mice. Sesame oil reduced Pb + LPS-induced tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide production in serum and liver tissue. Furthermore, sesame oil decreased inducible nitric oxide synthase expression in leukocytes and liver tissue in Pb + LPS-treated mice. We hypothesize that the inhibition of proinflammatory cytokines and nitric oxide might be involved in sesame oil-associated protection against Pb + LPS-induced acute hepatic injury in mice.     

Activation of alveolar macrophages in acid-injured lung in rats: different effects of pentoxifylline on tumor necrosis factor-alpha and nitric oxide production

OBJECTIVE: To determine whether acid instillation augments tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages in rats, and to study the effects of treatment with pentoxifylline before acid instillation on the production of these inflammatory mediators. DESIGN: Controlled laboratory investigation on tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages of rats that had acid-induced lung injury. SETTING: University research laboratory. SUBJECT: Alveolar macrophages of rats. INTERVENTIONS: Alveolar macrophages were recovered by bronchoalveolar lavage at 4, 10, 16, 24, and 72 hrs after unilateral hydrochloric acid (pH, 1.0; volume, 0.1 mL) instillation into the lungs of rats. Alveolar macrophages then were cultured with or without lipopolysaccharide. One group of rats was pretreated with pentoxifylline before acid instillation. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages from both acid-instilled and contralateral lungs, which had recovered 24 hrs after acid instillation, produced significantly greater tumor necrosis factor-alpha and nitric oxide. Subsequent exposure to lipopolysaccharide, as a surrogate for bacterial infection, further promoted tumor necrosis factor-alpha and nitric oxide release. Alveolar macrophages from rats pretreated with pentoxifylline before acid instillation produced significantly less tumor necrosis factor-alpha and did not overproduce tumor necrosis factor-alpha when exposed to lipopolysaccharide. In contrast, pretreatment with pentoxifylline had no effect on nitric oxide production by alveolar macrophages. CONCLUSIONS: Acid instillation stimulates alveolar macrophages to produce tumor necrosis factor-alpha and nitric oxide. Pentoxifylline preserved innate production of tumor necrosis factor-alpha to lipopolysaccharide and did not inhibit the production of bactericidal nitric oxide. This may partly explain why pentoxifylline reduces acid aspiration-induced lung injury while maintaining the host's ability to combat bacterial infection after acid aspiration.     

Recombinant tumor necrosis factor/cachectin and interleukin 1 pretreatment decreases lung oxidized glutathione accumulation, lung injury, and mortality in rats exposed to hyperoxia

Single, preexposure, parenteral injection with both recombinant tumor necrosis factor/cachectin (TNF/C) and interleukin-1 (IL-1) prolonged the survival of rats (144 +/- 9 h) in continuous hyperoxia (greater than 99% O2 at 1 atm) when compared with rats injected with boiled TNF/C and boiled IL-1 (61 +/- 2 h), TNF/C alone (61 +/- 2 h), IL-1 alone (62 +/- 2 h), or saline (64 +/- 3 h). After exposure to hyperoxia for 52 h, pleural effusion volume, pulmonary artery pressure, total pulmonary resistance, and lung morphologic damage were decreased in those rats given TNF/C and IL-1 as compared with saline-injected rats. In parallel, ratios of reduced (GSH) to oxidized (GSSG) glutathione were greater (P less than 0.05) in lungs of TNF/C + IL-1-injected rats (91 +/- 20) than of saline-injected rats (30 +/- 4) that had been exposed to hyperoxia for 52 h. No differences were found in superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, or catalase activities in lungs of TNF/C + IL-1- or saline-treated, hyperoxia-exposed rats. Our results indicate that pretreatment with TNF/C and IL-1 favorably altered lung glutathione redox status, decreased lung injury, and enhanced survival of rats exposed to hyperoxia.     

Hypothermia induces interleukin-10 and attenuates injury in the lungs of endotoxemic rats

We recently reported that hypothermia protects against intrapulmonary nitric oxide overproduction and nitric oxide-mediated lung injury in endotoxemic rats. Few studies have been performed to investigate whether hypothermia reduces inflammation by affecting favorable changes in chemokine and pro- and anti-inflammatory cytokine profiles. In this study, we tested the hypothesis that hypothermia decreases concentrations of growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1), interleukin (IL)-1beta, IL-6, and myeloperoxidase and increases concentration of IL-10 in the lungs endotoxemic rats. Twelve rats were anesthetized and randomized to treatment with either hypothermia (T = 18-24 degrees C; n = 6) or normothermia (T = 36-38 degrees C, n = 6). Endotoxin (15 mg/kg of Escherichia coli lipopolysaccharide) was administered intravascularly and lung tissue was harvested 150 min later. Three additional rats were sham instrumented and maintained as normothermic but not given endotoxin. Hematoxylin & eosin staining was performed for qualitative inspection of tissues. Quantitative analyses of lung homogenates were performed using enzyme-linked immunosorbent assays for IL-1beta, IL-6, IL-10, and GRO/CINC-1. Myeloperoxidase concentrations were determined using a colorimetric assay. Hypothermia attenuated the induction of intrapulmonary IL-1beta (P < 0.05), IL-6 (P < 0.05), GRO/CINC-1 (P < 0.05), and myeloperoxidase (P < 0.05) caused by endotoxin. Inspection of the lungs revealed that hypothermia similarly attenuated histological signs of injury, such as interstitial edema and neutrophil accumulation. Hypothermia increased the intrapulmonary concentration of IL-10 more than 3-fold over that measured in the normothermia (endotoxin-exposed) group (P < 0.05). Hypothermia inhibits neutrophil recruitment in the lungs of endotoxemic rats in part by decreasing proinflammatory cytokine expression. Additionally, hypothermia induces intrapulmonary IL-10 expression. Further studies are needed to investigate whether IL-10 mediates the anti-inflammatory effects of hypothermia.     

Gastric acid and particulate aspiration injury inhibits pulmonary bacterial clearance

OBJECTIVE: To establish a model of secondary bacterial pneumonia following gastric aspiration and to identify possible mechanisms involved in the suppressed antibacterial defenses following the initial pulmonary insult. DESIGN: A controlled, in vivo laboratory study. SETTING: Research laboratory of a health sciences university. SUBJECTS: Ninety-five Long-Evans rats. INTERVENTIONS: Animals were anesthetized for neck dissection and placement of a 14-gauge catheter in the trachea. Gastric aspirate (1.2 mL/kg of saline, pH 1.25, and 40 mg/mL sterile rat gastric particles) or an equal amount of normal saline (pH 5.3) was instilled intratracheally. One minute after this insult, animals received an intratracheal instillation of either 5.6 x 10 colony-forming units of Escherichia coli or an equal volume of normal saline. The animals remained in room air until kill at 4 hrs or 24 hrs after the intratracheal instillation. The lungs were homogenized for quantitative bacterial cultures. Bronchoalveolar lavage fluid was obtained for cell counts and measurements of albumin, tumor necrosis factor-alpha, interleukin-1 beta, cytokine-induced neutrophil chemoattractant-1, macrophage inflammatory protein-2, monocyte chemoattractant protein-1, and interleukin 10. MEASUREMENTS AND MAIN RESULTS: Animals that received gastric aspirate (followed by normal saline or E. coli) had increased injury as assessed by significant reductions in oxygenation and elevations in bronchoalveolar lavage albumin. At 24 hrs, animals that received gastric aspirate inoculation followed by E. coli had significantly higher pulmonary bacterial counts compared with animals that received E. coli alone. Gastric aspiration injury followed by bacterial inoculation also resulted in acute, but transient, increases in tumor necrosis factor-alpha, interleukin-1 beta, cytokine-induced neutrophil chemoattractant-1, and macrophage inflammatory protein-2 and more sustained elevations of monocyte chemoattractant protein-1 and interleukin-10. CONCLUSIONS: Lung injury increases and bacterial clearance decreases in this experimental model of E. coli pneumonia following gastric aspiration. Cytokine profiles suggest possible mechanisms for the impaired antibacterial host defense.     

Bronchoalveolar interleukin-1 beta: a marker of bacterial burden in mechanically ventilated patients with community-acquired pneumonia

OBJECTIVE: To assess the relationship between concentrations of bronchoalveolar cytokines and bacterial burden (quantitative bacterial count) in intubated patients with a presumptive diagnosis of community-acquired pneumonia. DESIGN: A cross-sectional and clinical investigation.SETTING Medical/surgical and respiratory intensive care unit of a tertiary 1,200-bed medical center. PATIENTS: According to the time course of community-acquired pneumonia at the time of study with bronchoalveolar lavage, 69 mechanically ventilated patients were divided into three subgroups: primary (n = 11), referral (n = 23), and treated (n = 35) community-acquired pneumonia. INTERVENTIONS: Bronchoalveolar lavage was performed in the most abnormal area on chest radiograph by fiberoptic bronchoscope. Bronchoalveolar lavage fluid was processed for quantitative bacterial culture. The concentrations of bronchoalveolar lavage cytokines (tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, interleukin-8, and interleukin-10) also were measured. MEASUREMENTS AND MAIN RESULTS: Thirty-two patients had a positive bacterial culture (bronchoalveolar lavage > or = 10 colony-forming units/mL)., and made up 76% of pathogens recovered at high concentrations. The concentrations of bronchoalveolar lavage interleukin-1 beta were 199.1 +/- 32.1 and 54.9 +/- 13.0 pg/mL (mean +/- se) in the patients with positive and negative bacterial culture, respectively (p < .001). Bronchoalveolar lavage interleukin- 1 beta was significantly higher in the patients with a high bacterial burden (p < .001), with mixed bacterial infection (p < .001), and with pneumonia (p < .001), compared with values in patients without these features. The relationship between bacterial load and concentrations of bronchoalveolar lavage interleukin-1 beta was very strong in the patients with primary and referral community-acquired pneumonia but was borderline in treated community-acquired pneumonia. CONCLUSIONS: The common pathogens were similar to the core pathogens of hospital-acquired pneumonia, probably due to antibiotic effects, delayed sampling, and superimposed nosocomial infection. Since the concentration of bronchoalveolar lavage interleukin-1 beta was correlated with bacterial burden in the alveoli, it may be a marker for progressive and ongoing inflammation in patients who have not responded to pneumonia therapy and who have persistence of bacteria in the lung.     

Resuscitation from experimental heatstroke by brain cooling therapy

We have used hypothermic retrograde jugular venous flush to cool the brain previously and to provide better resuscitation than peripheral cold saline infusion during heatstroke in the rat. The current study was performed to assess the effects of brain cooling further on production of reactive nitrogen species, reactive oxygen species, tumor necrosis factor-alpha, and interleukin-10 in both serum and brain during heatstroke. Rats, under general anaesthesia, were randomized into the following groups and given: (a) 36 degrees C or (b) 4 degrees C saline infusion in the external jugular vein immediately after onset of heatstroke. They were exposed to an ambient temperature of 43 degrees C for exactly 70 min to induce heatstroke. When the 36 degrees C saline-treated rats underwent heat stress, their survival time values were found to be 21-25 min. Immediately after the onset of heatstroke, resuscitation with an i.v. dose of 4 degrees C saline greatly improved survival (226-268 min). Compared with the normothermic controls, the 36 degrees C saline-treated heatstroke rats displayed higher levels of brain temperature, intracranial pressure, serum and hypothalamic nitric oxide metabolite, tumor necrosis factor-alpha and dihydroxybenzoic acid as well as hypothalamic inducible nitric oxide synthase immunoreactivity. In contrast, the values of mean arterial pressure, cerebral perfusion pressure, and hypothalamic levels of local blood flow, and partial pressure of oxygen were all significantly lower during heatstroke. The cerebrovascular dysfunction, the increased levels of nitric oxide metabolites, tumor necrosis factor-alpha, and dihydroxybenzoic acid in both the serum and the hypothalamus, and the increased levels of hypothalamic inducible nitric oxide synthase immunoreactivity occurred during heatstroke were significantly suppressed by brain cooling. Although the serum and hypothalamic interleukin-10 maintained at a negligible level before stress, they were significantly elevated by brain cooling during heatstroke. These findings suggest that brain cooling may resuscitate persons who had heatstroke by decreasing overproduction of reactive nitrogen species, tumor necrosis factor-alpha, reactive oxygen species and cerebrovascular dysfunction, but increasing production of interleukin-10.     

Beneficial effects of nitric oxide inhalation on pulmonary bacterial clearance

OBJECTIVE: The beneficial effects of nitric oxide inhalation on oxygenation during acute respiratory distress syndrome are well described. In contrast, the effects of nitric oxide on pulmonary inflammatory response are much less known in vivo. The objectives of this study were to evaluate the effects of nitric oxide inhalation on bacterial clearance during bacterial pneumonia and on alveolar neutrophil functions. DESIGN: Controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Severe pneumonia was induced by alveolar instillation of live Pseudomonas aeruginosa (1.5 x 10(8) colony-forming units/kg) in rats. After instillation, rats were exposed to oxygen alone (FIO(2) 100%) or to oxygen (FIO(2) approximately 100%) plus nitric oxide (10 ppm) during 24 hrs. MEASUREMENTS AND MAIN RESULTS: Oxygen plus nitric oxide inhalation compared with oxygen alone increased recruitment of alveolar neutrophils (32.5 +/- 4.6 x 10(6) cells/mL vs. 23.4 +/- 1.9 x 10(6) cells/mL, p <.05) and improved bacterial clearance in the bronchoalveolar lavage fluid (8.1 +/- 4.2 x 10(2) vs. 1.6 +/- 1.0 x 10(5) colony-forming units/mL, p <.05) and in the pulmonary parenchyma (1.7 +/- 1.14 x 10(7) vs. 3.4 +/- 1.5 x 10(8) colony-forming units/mL, p <.05). However, neither protein concentration in the bronchoalveolar lavage fluid nor mortality rates were modified by nitric oxide inhalation. The ex vivo alveolar neutrophil functions were similar regardless of whether rats previously inhaled nitric oxide. In vitro experiments demonstrated that nitric oxide donor had a direct bactericidal effect against P. aeruginosa and did not modify alveolar neutrophil functions. CONCLUSIONS: These results suggest a beneficial effect of nitric oxide inhalation on bacterial clearance of P. aeruginosa attributable to both a direct bactericidal effect and an influx of alveolar neutrophils with preserved functions.     

Can the interleukin-6 response to endotoxin be predicted? Studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines

OBJECTIVES: To evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 gene, gender, the monocyte density of the endotoxin receptor CD14, or the inflammatory cytokines tumor necrosis factor-alpha or interleukin-1beta influence the interleukin-6 response of whole blood to endotoxin. DESIGN: Analysis of interleukin-6 release from endotoxin-stimulated human whole blood. SETTING: Medical research laboratory. PATIENTS: Healthy human blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The interleukin-6 -174 G/C and the tumor necrosis factor -308 G/A promoter polymorphisms were determined by real-time polymerase chain reaction assay by using specific fluorescence labeled hybridization probes. Monocyte CD14 expression was assessed by flow cytometry. After incubation of whole blood with endotoxin, plasma concentrations of interleukin-6, tumor necrosis factor-alpha, and interleukin-1beta were measured by means of chemiluminescence. The interleukin-6 concentrations were lower (p = .005) in individuals who were CG heterozygotes compared with individuals homozygous for the C or the G. The difference between C and G homozygotes was not significant (p = .67). The interleukin-6 response was enhanced in men compared with women (p = .015). There was no correlation between interleukin-6 concentrations and monocyte CD14 density. Interleukin-6 concentrations correlated with the concentrations of tumor necrosis factor-alpha (r = .59, p = .01) and interleukin-1beta (r = .47, p = .01). There was no linkage between the tumor necrosis factor -308 and the interleukin-6 -174 polymorphisms. CONCLUSIONS: The interleukin-6 response to endotoxin was influenced by gender and correlated with the concentrations of more proximal cytokine tumor necrosis factor-alpha and interleukin-1beta. The interleukin-6 -174 G/C promoter polymorphism can only partly predict the interleukin-6 response of human whole blood to endotoxin stimulation, and the results were different from previous reporter gene assays that reported higher interleukin-6 concentrations for the G allele. Tumor necrosis factor -308 G homozygotes produce the lowest tumor necrosis factor concentrations. The number of tumor necrosis factor -308 G homozygotes was not higher among interleukin-6 -174 heterozygotes, and thus this cannot account for their significantly smaller interleukin-6 production.     

Detrimental effects of nitric oxide inhibition on hepatic encephalopathy in rats with thioacetamide-induced fulminant hepatic failure

Hepatic encephalopathy is a complex neuropsychiatric syndrome seen secondary to acute liver failure, chronic parenchymal liver disease, or portal-systemic anastomosis. Vasodilatation induced by nitric oxide (NO) may be involved in the development of hepatic coma. However, there are no comprehensive data concerning the effects of NO inhibition on the severity of hepatic encephalopathy. Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg kg-1 day-1) for 3 days. Rats were divided into two groups to receive either NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 day-1 via intragastric gavage) or normal saline (N/S) from 2 days prior to TAA administration for 5 days. Severity of encephalopathy was assessed by counts of motor activity and neurobehaviour test scores. Plasma levels of endotoxin, tumour necrosis factor-alpha and nitrate/nitrite were determined by the chromogenic Limulus assay, enzyme-linked immunosorbent assay and colorimetric assay, respectively. Compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving L-NAME (59% vs. 18%, P < 0.01). Inhibition of NO had detrimental effects on the counts of motor activities (P < 0.05) and neurobehaviour score (P < 0.01). Rats treated with L-NAME had significantly higher plasma levels of endotoxin (26.7 +/- 3.8 pg mL-1) and tumour necrosis factor-alpha (29.4 +/- 6.5 pg mL-1) compared with rats treated with N/S (13.2 +/- 2.7 pg mL-1 and 11.2 +/- 2.6 pg mL-1, respectively, P < 0.01). Plasma levels of endotoxin and tumour necrosis factor-alpha, but not of nitrate/nitrite, were significantly correlated with the severity of hepatic encephalopathy (P < 0.05). Chronic L-NAME administration had detrimental effects on the severity of encephalopathy in TAA-treated rats, suggesting a protective role of NO in the development of fulminant hepatic failure.     

The proMMP-2 activation rate in patients with chronic viral liver disease

BACKGROUND: Crush syndrome has been described as extensive muscle damage, leading to acute renal failure. The aim of this study was to evaluate the possible role of nitric oxide, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in crush syndrome. PATIENTS AND METHODS: A total of 17 patients suffering from crush syndrome, 7 patients without crush syndrome and 10 healthy controls were enrolled in the study. Plasma nitrate, TNF-alpha, IL-1 beta levels and biochemical parameters were measured. RESULTS: All patients with crush syndrome demonstrated acute renal failure. Plasma nitrate levels were elevated significantly in the crush syndrome patients compared with patients without crush syndrome (33.5 +/- 20.1 vs. 15.3 +/- 5 micromol/l, p=0.014). There was no significant difference in TNF-alpha and IL-1 beta levels between control and patient groups. CONCLUSION: Increased plasma nitrate levels in the crush syndrome may be related either to the elevated production of NO or the diminished excretion of nitrate or both.     

Nitric oxide donors alter cardiomyocyte cytokine secretion and cardiac function

OBJECTS: The mechanisms by which nitric oxide produces beneficial/detrimental effects on physiologic function are unclear. In this study, we hypothesized that nitric oxide promotes cyclic guanosine monophosphate (cGMP) formation, which, in turn, promotes cardiomyocyte secretion of inflammatory cytokines as well as accumulation of intracellular Na+/Ca2+; these factors contribute to altered cardiac contractile function. DESIGN: Laboratory study. SETTING: Medical Center. SUBJECTS: Adult Sprague Dawley rats weighing 325-350 g. INTERVENTIONS: Cardiomyocytes were prepared by collagenase perfusion of rat hearts; cells were plated (5 x 10(4) cells/microtiter well) and challenged with either vehicle or nitric oxide donor (S-nitroso-N-acetyl-penicillamine [SNAP] or PAPA NONOATE, 3-[2-Hydroxy-2-nitroso-1-propythdrazinol]-1-propanamine], NOC-15 [PAPA-NO], 0.3 or 1.0 mM of each nitric oxide donor) in the presence/absence of methylene blue (10 microM/L to inhibit cGMP). After 3 hrs, supernatants were collected to measure nitrite/nitrate (nitric oxide), cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6), and cGMP levels; cells were then loaded with a fluorescent indicator (Fura-2AM or sodium-binding benzofurzan isophthalate) to measure myocyte Ca2+ or Na+, respectively. Parallel experiments included the addition of nitric oxide donor (0.3 or 1.0 mM SNAP or PAPA-NO) to perfused hearts in presence or absence of the methylene blue to examine cGMP-mediated effects on myocardial contraction-relaxation, while other experiments determined a) potential lipopolysaccharide contamination of myocyte preparations; and b) whether a cGMP analogue recapitulated the effects of nitric oxide donors on cytokine secretion. MEASUREMENTS AND MAIN RESULTS: Nitric oxide donors produced a dose-dependent increase in cGMP levels in myocyte supernatants as well as an increase in myocyte cytokine secretion, increased myocyte loading of Na+/Ca2+, and produced myocardial contractile dysfunction. Addition of the cGMP analog, 8-bromo-cGMP, recapitulated the effects of nitric oxide donors on myocyte cytokine secretion. Nitric oxide donor-related effects were ablated by pretreatment of myocytes or isolated hearts with methylene blue. Treatment of myocytes with recombinant bactericidal/permeability-increasing protein to scavenge lipopolysaccharide confirmed that cytokine responses to nitric oxide donors were not related to lipopolysaccharide contamination of myocyte preparations. CONCLUSIONS: We suggest that nitric oxide synthesis in injury and disease promotes cGMP formation, which, in turn, modulates cardiac contraction/relaxation by a) altering cardiomyocyte secretion of inflammatory cytokines and b) altering myocyte handling of Na+/Ca2+.     

Liposome-encapsulated hemoglobin does not exacerbate endotoxin-induced lung injury

OBJECTIVE: To test the hypothesis that liposome encapsulated hemoglobin (LEH), an experimental oxygen-carrying fluid, exacerbates endotoxin-induced lung injury in the rat. DESIGN: Prospective, randomized animal study. SETTING: University animal laboratory. METHODS: Anesthetized Sprague-Dawley rats (n = 8-13) were infused with LEH (10% of estimated total blood volume) or vehicle (0.9% NaCl). Thirty minutes later, Escherichia coli endotoxin (3.6 mg/kg, i.v.) or vehicle (0.9% NaCl) was administered, and skeletal muscle oxygen tension as well as lung injury were assessed at 2, 4, and 8 hrs. Oxygen tension was measured using a miniaturized thin film oxygen sensor placed in the rectus abdominis muscle, and lung injury was evaluated by determining lung weights, lung myeloperoxidase activity, lung tissue tumor necrosis factor-alpha level, and protein concentration in bronchoalveolar lavage fluid. RESULTS: The intravenous bolus injection of E. coli endotoxin elevated lung water content (33% +/- 5%; p < .01 vs. sham controls), myeloperoxidase activity (56% +/- 6%; p < .01), and tumor necrosis factor-alpha production (1320 +/- 154 pg/g lung tissue; p < .05 vs. undetected levels in sham controls), as well as induced protein accumulation in bronchoalveolar lavage fluid (258% +/- 38%; p < .01) and skeletal muscle hypoxia (52 +/- 8 mm Hg; p < .05). Pretreatment with LEH, which when infused alone did not induce lung injury, had no effect on these responses. CONCLUSION: In this specific model of endotoxin-induced lung injury, LEH does not exacerbate microvascular leakage and leukosequestration, the hallmarks of adult respiratory distress syndrome.     

Nitric oxide attenuates platelet-activating factor induced nasal airway plasma extravasation in healthy subjects

BACKGROUND: Paranasal sinuses and the nose are important sources of nitric oxide (NO) in humans but the relevance of NO production to the control of nasal airway plasma exudation and its response to inflammatory mediators such as platelet-activating factor (PAF) in healthy subjects is not well known. DESIGN: In this study we aimed to evaluate the effect of the nitric oxide synthase (NOS) inhibitor NG L-arginine methyl ester (L-NAME) on nasal airway plasma extravasation at baseline and after an acute challenge with PAF that induces most symptoms of rhinitis. Eleven healthy subjects were enrolled in the study. Plasma extravasation in the nasal airway was assessed by measuring the albumin content of nasal lavage. RESULTS: PAF challenge caused a significant increase in concentrations of albumin in the nasal lavage fluid (from 0.59 +/- 0.13 mg dL(-1) to 2.46 +/- 0.45 mg dL(-1)) after placebo. Pretreatment with L-NAME significantly prevented the increase of albumin in the nasal lavage fluid induced by PAF as compared to placebo (from 0.53 +/- 0.11 mg dL(-1) to 1.70 +/- 0.28 mg dL(-1); P < 0.005). CONCLUSION: Topical administration of a NO inhibitor is able to attenuate the nasal airway plasma extravasation induced by PAF, suggesting that NO release in vivo is involved in the nasal response to PAF.     

Changes in glucocorticoid and mineralocorticoid receptors of liver and kidney cytosols after pathologic stress and its regulation in rats

OBJECTIVE: As effectors, glucocorticoid and mineralocorticoid receptors play an important role in pathologic stress. This study was designed to observe the changes in glucocorticoid receptor of liver cytosols and mineralocorticoid receptor of kidney cytosols after pathologic stress in rats. DESIGN: Controlled laboratory study. SETTING: Medical university. SUBJECTS: Male Wistar rats (weight range, 180-200 g). INTERVENTIONS: Rats received a low-degree or heavy-degree immersion scald that covered 10% or 35% total body surface area and were randomly divided to receive either tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, KPV peptide (Ac-D-Lys-L-Pro-D-Val), or saline (control). The binding capacity and the apparent dissociation constant of the steroid-binding sites of normal, low-degree, and heavy-degree scalded rats were measured by radioligand-binding assay, with [3H]dexamethasone and aldosterone as the ligand, respectively. MEASUREMENTS AND MAIN RESULTS: The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after heavy-degree scald (208.45 +/- 30.78 fmol/mg of protein) was lower than that of the control group (306.71 +/- 27.96 fmol/mg of protein; p < .01). The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after low-degree scald (296.64 +/- 16.06 fmol/mg of protein) was not significantly different compared with the control group (p > .05). There were two types of mineralocorticoid receptor in kidney cytosols in rats, and their binding capacity and apparent dissociation constant were not identical. The binding capacity of mineralocorticoid receptor in rats 12 hrs after heavy-degree scald (binding capacity 1, 22.40 +/- 5.40 fmol/mg of protein; binding capacity 2, 196.30 +/- 32.50 fmol/mg of protein) was lower than that of the control group (binding capacity 1, 41.60 +/- 7.20 fmol/mg of protein; binding capacity 2, 317.60 +/- 70.00 fmol/mg of protein; p < .01). The binding capacity of mineralocorticoid receptor in kidney cytosols in rats 12 hrs after low-degree scald (binding capacity 1, 41.40 +/- 5.00 fmol/mg of protein; binding capacity 2, 314.80 +/- 45.70 fmol/mg of protein) was not significantly different compared with the control group (p > .05). The injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide (Ac-D-Lys-L-Pro-D-Val) might prevent a reduction in the binding capacity of glucocorticoid receptor in hepatic cytosols and mineralocorticoid receptor in kidney cytosols in rats with heavy-degree scald in vivo. CONCLUSIONS: These studies suggest that the glucocorticoid receptor of hepatic cytosols and the mineralocorticoid receptor of renal cytosols decreased in rats with heavy-degree immersion scald and that the injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide might increase the level of glucocorticoid receptor and mineralocorticoid receptor in vivo.     

Cytokines down-regulate alpha1-adrenergic receptor expression during endotoxemia

OBJECTIVE: The reduced pressure response to norepinephrine in septic patients has directed our interest to the regulation of alpha1-adrenergic receptors in vitro and in vivo during conditions mimicking acute sepsis. DESIGN: Prospective animal trial followed by a controlled cell culture study. SETTING: Laboratory of the Department of Anesthesiology. SUBJECTS: Male Sprague-Dawley rats weighing 200 to 250 g and a mesangial cell line. INTERVENTIONS: Experimental endotoxemia was induced in rats with lipopolysaccharide, and blood pressure dose-response studies with norepinephrine were performed. Alpha1-receptor gene expression was determined in various organs by a specific RNase protection assay, and tissue concentrations of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha were measured. Rat renal mesangial cells were incubated with these cytokines or with nitric oxide donors to investigate the regulation of alpha1-adrenergic receptors during severe inflammation on a cellular level. MEASUREMENTS AND MAIN RESULTS: The pressor effect of norepinephrine was markedly diminished during endotoxemia. The animals showed down-regulated mRNA levels of alpha1A-, alpha1B- and alpha1D-receptors in all organs investigated, and the tissue concentrations of interleukin-1beta and tumor necrosis factor-alpha were highly increased during experimental endotoxemia. Incubation of cultured rat renal mesangial cells with the cytokines resulted in diminished alpha -receptor gene expression and [3H]prazosin binding capacity, whereas incubation of the cells with nitric oxide donors did not affect alpha1B-receptor expression. In line, blocking of cytokine-induced nitric oxide synthesis by coincubation of mesangial cells with N(G)-nitro-L-arginine methyl ester did not influence cytokine-induced down-regulation of alpha1B-receptors. CONCLUSIONS: Our data show that endotoxemia causes a systemic down-regulation of alpha1-receptors on the level of gene expression and suggest that this effect is likely mediated by proinflammatory cytokines in a synergistic but nitric oxide-independent fashion. We propose that this down-regulation of alpha1-adrenergic receptors contributes to the attenuated blood pressure response to norepinephrine and, therefore, to septic circulatory failure in patients.     

Effect of the nitric oxide inhibitor, L-N(G)-monomethylarginine, on accumulation of interleukin-6 and interleukin-8, and nuclear factor-kappaB activity in a human endothelial cell line.

OBJECTIVE: To determine the effect of the nitric oxide synthase inhibitor, L-N(G)-monomethylarginine, on interleukin-6 and interleukin-8 accumulation, and nuclear factor-kappaB expression in an endothelial cell model of sepsis. DESIGN: Controlled cell culture experiments examining the immunomodulatory effects of nitric oxide synthase inhibition. SUBJECTS: A human endothelial cell line (EA.hy926). MEASUREMENTS AND RESULTS: Cells were incubated with tumor necrosis factor-alpha and interleukin (IL)-1beta in the presence of L-N(G)-monomethylarginine (L-NMMA). IL-6 and IL-8 were measured in culture supernatants using enzyme immunoassay. Nuclear factor-kappaB was measured using electrophoretic mobility shift assay and was quantified using phosphorimaging. IL-6 accumulation was decreased (p < .05) and IL-8 accumulation increased (p < .01) with L-NMMA. Increased nuclear factor-kappaB expression in stimulated cells was unaltered on exposure to L-NMMA. Cell viability was unaffected. CONCLUSIONS: Excessive production of nitric oxide has been implicated in septic shock, and the use of nitric oxide synthase inhibitors has been suggested. The immunoregulatory actions of nitric oxide synthase inhibitors affects the profile of cytokine release. This effect is not mediated through modulation of nuclear factor-kappaB. These findings have implications for the use of nitric oxide synthase inhibiting agents in septic shock.