Lung Leak Research Articles

Supercritical fluid-aerosolized vitamin E pretreatment decreases leak in isolated oxidant-perfused rat lungs

We hypothesized that direct pulmonary administration of supercritical fluid-aerosolized (SFA) vitamin E would decrease acute oxidative lung injury. We previously reported that rapid expansion of supercritical CO2 formed respirable particles of vitamin E and that administering SFA vitamin E to rats increased lung vitamin E levels and decreased neutrophil-mediated lung leak. In the present investigation, we found that pretreatment with SFA vitamin E protected isolated rat lungs against the oxidant-induced lung leak caused by perfusion with xanthine oxidase (XO) and purine, an enzyme system that generates superoxide union (O2-.) and hydrogen peroxide. SFA vitamin E droplets were 0.7-3 microns in diameter, and inhalation of the airborne droplets for 30 min deposited approximately 55 micrograms of vitamin E in rat lungs. Isolated rat lungs perfused with XO (0.02 U/ml) and purine (10 mM) gained more weight (1.75 +/- 0.12 g, n = 8), retained more Ficoll (11.5 +/- 1.2 mg/left lung, n = 7), and accumulated more Ficoll in their lung lavages (700 +/- 146 micrograms/ml, n = 8) than control lungs [0.25 +/- 0.06 g (n = 10), 6.2 +/- 1.2 mg/left lung (n = 9), and 141 +/- 31 micrograms/ml (n = 8), respectively, P < 0.05]. In contrast, isolated lungs from rats that were pretreated with SFA vitamin E had decreased (P < 0.05) weight gains (0.32 +/- 0.06 g, n = 7), Ficoll retentions (3.3 +/- 1.1 mg/left lung, n = 7), and lung lavage Ficoll concentrations (91 +/- 26 micrograms/ml, n = 6) after perfusion with XO and purine compared with isolated lungs from control rats perfused with XO and purine. This protective effect was not observed in rat lungs given sham treatments (CO2 alone or vitamin E acetate aerosolized with supercritical CO2). Our results suggest that direct pulmonary supplementation of vitamin E decreases susceptibility to vascular leakage caused by XO-derived oxidants.

Phagocytes and acute lung injury: dual roles for interleukin-1.

Interleukin-1 (IL-1) and neutrophils are increased in lungs of patients with the acute respiratory distress syndrome (ARDS). We found that rats given IL-1 intratracheally rapidly developed lung neutrophil accumulation and a neutrophil-dependent acute edematous lung leak. Lung leak was associated with increased lung lavage cytokine-induced chemoattractant (CINC) levels and increased oxidative stress that was manifested by increased exhaled H2O2 levels and increased lung oxidized glutathione levels. IL-1-induced lung leak was decreased by treatment with superoxide dismutase (SOD), dimethylsulfoxide (DMSO), supercritical fluid-aerosolized vitamin E, interleukin-1-receptor antagonist (IL-1ra), or liposome-associated PGE1 (Lip-PGE1). Importantly, Lip-PGE1 treatment also reduced ventilator dependence in a small clinical study of ARDS patients. Another series of investigations revealed that IL-1 pretreatment could prevent lung leak in rats given IL-1 intratracheally. These findings point to the possible dual effects of IL-1 with respect to the development of acute lung injury.

Modulating phosphatidic acid metabolism decreases oxidative injury in rat lungs.

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 40 nM), prevented oxidant-mediated capillary leak in isolated rat lungs given interleukin-8 (IL-8) intratracheally and perfused with human neutrophils. Lung leak was prevented by lung, but not neutrophil, lisofylline pretreatment. Furthermore, although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe (fMLP)-stimulated oxidant production in vitro. Lisofylline also prevented acute capillary leak in isolated rat lungs perfused only with the oxidant generator purine-xanthine oxidase but did not scavenge O2-(+) or H2O2 in vitro. Finally, lisofylline-mediated protection against lung leak in both models was associated with alterations in lung membrane free fatty acid acyl composition (as reflected by the decreased ratio [linoleate + oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent oxidant-induced capillary leak in isolated rat lungs and that protection appears to be mediated by blocking intrinsic lung linoleoyl phosphatidic acid metabolism. We speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane composition and renders this barrier less vulnerable to oxidative damage.

Synergism between endotoxin priming and exotoxin challenge in provoking severe vascular leakage in rabbit lungs.

Lipopolysaccharides (LPS) of gram-negative bacteria prime rabbit lungs for enhanced thromboxane-mediated vasoconstriction upon subsequent challenge with the exotoxin Escherichia coli hemolysin (HlyA) (Walmrath et al. J. Exp. Med. 1994;180:1437-1443). We investigated the impact of endotoxin priming and subsequent HlyA challenge on lung vascular permeability while maintaining constancy of capillary pressure. Rabbit lungs were perfused in a pressure-controlled mode in the presence of the thromboxane receptor antagonist BM 13.505, with continuous monitoring of flow. Perfusion for 180 min with 10 ng/ml LPS did not provoke vasoconstriction or alteration of capillary filtration coefficient (Kfc) values. HlyA (0.021 hemolytic units/ml) induced thromboxane release and a transient decrease in perfusion flow in the absence of significant changes in Kfc. Similar results were obtained when LPS and HlyA were coapplied simultaneously. However, when the HlyA challenge was undertaken after 180 min of LPS priming, a manifold increase in Kfc values was noted, with concomitant severe lung edema formation, although capillary pressure remained unchanged. Thus, endotoxin primes the lung vasculature to respond with a severe increase in vascular permeability to a subsequent low-dose application of HlyA. Such synergism between endotoxin priming and exotoxin challenge in provoking lung vascular leakage may contribute to the pathogenesis of respiratory failure in sepsis and severe lung infection.

TNF mediates lung leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally.

Interleukin-1 (IL-1) is increased in lung lavages obtained from patients with acute respiratory distress syndrome, and administering IL-1 intratracheally to rats causes an acute, neutrophil-dependent, oxidative lung leak. We found that rats given IL-1 intratracheally had increased lung lavage fluid tumor necrosis factor (TNF) levels, and that rats treated with TNF binding protein (TNFbp) intravenously did not develop the increased lung leak that occurs after administration of IL-1 intratracheally. In contrast, rats given IL-1 intratracheally and TNFbp intravenously had the same elevations in lung lavage neutrophil accumulation and lung lavage cytokine-induced neutrophil chemoattractant levels as rats given IL-1 intratracheally. Our results show that TNFbp decreases neutrophil-mediated lung leak, but not lung neutrophil accumulation, after administration of IL-1 intratracheally in rats.

Mepacrine decreases lung leak in rats given interleukin-1 intratracheally.

We hypothesized that phospholipase A2 (PLA2) metabolites contribute to the acute, neutrophil-dependent, edematous lung leak that develops after administration of interleukin-1 (IL-1) intratracheally to rats and tested this premise by using mepacrine to inhibit PLA2 activity in vivo. We found that lung PLA2 activity, lung lavage phospholipid content, lung leak index, lung weight gain, and lung lavage protein concentrations were increased in rats given IL-1 intratracheally compared with sham-treated control rats. By comparison, lungs of mecaprine and IL-1-treated rats had decreased PLA2 activity, lavage phospholipid content, leak, weight gain, and lavage protein increases compared with rats given IL-1 intratracheally. Mepacrine treatment also decreased lung neutrophil accumulation, but not lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, in rats given IL-1 intratracheally. In parallel experiments, mepacrine treatment reduced the adhesion of human neutrophils to IL-1-treated human umbilical vein endothelial cells in vitro. Our results indicate that PLA2 activity participates in the lung neutrophil retention and pulmonary vascular leak that develops in rats given IL-1 intratracheally.

Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally.

Interleukin-1 (IL-1) is increased in lung lavages from patients with the acute respiratory distress syndrome, and administering IL-1 intratracheally causes neutrophil accumulation and a neutrophil-dependent oxidative leak in lungs of rats. In the present study, we found that rats pretreated intraperitoneally with lisofylline [(R)-1-(5-hydroxyhexyl)-3, 7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species, did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1. However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbers of lung lavage neutrophils as rats given IL-1 intratracheally. Lungs of rats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment of LSF. Our results reveal that LSF decreases lung leak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

The Effect of Heat Shock Response on the Tumor Necrosis Factor-alpha-induced Acute Lung Injury in Rats

Background : Heat-treated cells are known to be protected from lysis by TNF, which is considered to play a central role in the pathogenesis of sepsis-induced acute lung injury. The objective of the study was to investigate the effect of heat shock response by heat-pretreatment on the acute lung injury of the rats induced by intratracheally administered TNF-, Methods : We intratracheally instilled either saline or TNF (R&D, 500ng) with and without heat pretreatment in Sprague-Dawley rats weighing 250~350 g. The heated rats were raised their rectal temperature to and was maintained thereafter for 13 minutes at 18 h before intratracheal administration of saline or TNF. After 5 h of intratracheal treatment, lung leak, lung myeloperoxidase activity (MPO) and heat shock proteins were measured in rats. Lung leak index was defined as counts per minute of in the right lung divided by counts per minutes of in 1.0 ml of blood. All data are expressed as means SE. Results : There is no difference in acute lung leak index ( vs ) among the rats given saline intratracheally with and without heat pretreatment, but MPO activity showed a decreased tendency in heat-pretreated rats () compared with heat-unpretreated rats () (P=0.064). Rats administered TNF intratracheally with heat-pretreatment had decreased lung leak index () and lung MPO activity () compared with those of heat-unpretreated and TNF-administered rats (, ) (P

Nitric oxide decreases lung injury after intestinal ischemia.

After injury to a primary organ, mediators are released into the circulation and may initiate inflammation of remote organs. We hypothesized that the local production of nitric oxide (NO) may act to limit the spread of inflammation to secondarily targeted organs. In anesthetized rats, 30 min of intestinal ischemia followed by 2 h of reperfusion (I/R) did not increase lung albumin leak. However, after treatment with NG-nitro-L-arginine methyl ester (L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO. The site of action of NO appeared to be the lung and not the gut because 1) after treatment with L-NAME, local delivery of NO to the lung by inhalation abolished the increase in intestinal I/R-induced lung leak; 2) L-NAME had no effect on epithelial permeability (51Cr-labeled EDTA clearance) of reperfused small bowel; and 3) after treatment with L-NAME, local delivery of NO to the gut by luminal perfusion did not improve epithelial permeability of reperfused intestines. Furthermore, L-NAME increased, and inhaled NO decreased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lung leak in rats subjected to intestinal I/R. We conclude that endogenous lung NO limits secondary lung injury after intestinal I/R by decreasing pulmonary neutrophil retention.

Tumor necrosis factor induced acute lung leak in rats: less than with interleukin-1.

Although local tumor necrosis factor-alpha (TNF) release by alveolar macrophages has been postulated to contribute to the development of acute respiratory distress syndrome (ARDS), the effects of instilling TNF intratracheally on the development of acute lung leak are not known. Our goal was to determine the effect of intratracheally administered TNF on the development of acute lung leak in rats. We found that rats given TNF (500 ng) 5 hours previously intratracheally had increased (p < 0.05) lung lavage cytokine induced neutrophil chemoattractant (CINC) concentrations, lung lavage neutrophils, lung myeloperoxidase (MPO) activity, and lung leak compared to saline-treated control rats. However, all of the responses following TNF instillation were much lower than the responses to interleukin-1 alpha (IL-1) instillation. For example, instilling 50 ng of IL-1 caused 6.4 times the increases in lung lavage CINC concentrations, 15.5 times the increase in lung lavage neutrophils, 3.6 times the increase in lung MPO activity and 3.8 times the increase in lung leak caused by giving 500 ng of TNF intratracheally. Co-treatment with TNF-binding protein decreased both lung MPO and lung leak increases in rats given TNF intratracheally. These observations suggest that locally elevated levels of TNF may induce lung neutrophil recruitment and acute lung leak but that IL-1 is a much more potent agent than TNF in causing lung neutrophil accumulation and lung leak.

Keratinocyte growth factor attenuates lung leak induced by alpha-naphthylthiourea in rats

To investigate the effect of pretreatment with keratinocyte growth factor on acute permeability pulmonary edema. Prospective, randomized, controlled animal study. University research laboratory. Specific pathogen-free Sprague-Dawley rats. Acute permeability pulmonary edema was induced with an injection of alpha-naphthylthiourea, and lung leak was assessed in an isolated perfused lung model over 180 mins. Leak was confirmed with wet/dry lung weight ratios, and the alveolar fluid protein concentration was measured after bronchoalveolar lavage. The effect of pretreatment with keratinocyte growth factor (injected intratracheally 48 hrs before the experiment) on alpha-naphthylthiourea-induced pulmonary edema was assessed (keratinocyte growth factor/alpha-naphthylthiourea group). Control groups (Control and keratinocyte growth factor/Control) were also studied. Histopathology was performed for each of the four groups. The alpha-naphthylthiourea produced an acute permeability pulmonary edema detected by lung leak over the 180-min ex vivo period of monitoring the isolated perfused lung (leak = 8+/-mL; wet/dry weight ratio 14.7+/-2; lavage protein 3.1+/-1 mg/mL). Pretreatment with keratinocyte growth factor significantly attenuated these parameters (leak = 2.3+/-0.4 mL; wet/dry weight ratio 7.1 +/- 0.5; lavage protein 0.28 +/-0.03 mg/mL), which were not significantly different from the control group and the keratinocyte growth factor/control group. Histopathology showed abundant type II pneumocyte hyperplasia in the lungs of animals pretreated with keratinocyte growth factor, and marked pulmonary edema in animals pretreated with alpha-naphthylthiourea. Less edema was apparent in the keratinocyte growth factor/alpha-naphthylthiourea group. All data are expressed as mean +/- SEM. Pretreatment with keratinocyte growth factor significantly attenuates pulmonary edema induced by alpha-naphthylthiourea. The mechanisms of this protection are likely related to type II pneumocyte hyperplasia, but remain to be specifically elucidated.

EFFECTS OF INHALED NITRIC OXIDE (NO) IN PREMATURE LAMBS WITH MODERATE HYALINE MEMBRANE DISEASE (HMD). † 1467

Gas exchange improvment by low dose NO inhalation without increase of vascular permeability was reported by Kinsella et al. in experimental severe HMD (Pediatr Res 1995; 38: 337A). Our hypothesis was that inhaled NO could be deleterious in less severe respiratory failure by increasing pulmonary blood flow and/or altering vascular permeability. The effects of inhaled NO on gas exchange and lung vascular permeability in moderate HMD were studied. Eighteen moderatly premature lambs (130 days gestation, term=147 days) were randomly assigned to treatment with 20 ppm inhaled NO from the onset of ventilation(HO) or control. After porcine surfactant administration (Curosurf® 100 mg/kg), ventilator was set to maintain PaCO2 values between 40 and 50 mmHg and SpO2 values between 90 and 95%. After 4 hours,125 I-labeled albumine and 51Cr-tagged red blood cells were injected. One hour later, gamma-counting was performed on lung tissue and blood. Wet and dry weights of blood and lung were measured. Pulmonary edema was assessed by measurement of extravascular lung water content (Qwl) normalized for bloodless dry lung weight. Vascular permeability was evaluated by the Lung Leak Index (LLl) measurement (Am Rev Respir Dis. 1993; 148: 1194-203). Oxygenation (OI = Mean Airway Pressure*FiO2/PaO2) and ventilatory index (VI = Mean Airway Pressure*Respiratory Rate) were calculated at 1 and 5 hours. OI was significantly lower after 1 hour of ventilation in the NO vs. control group. However, OI and ventilatory requirement were not significantly different after 5 hours of ventilation. In spite of similar extravascular lung water content in both groups, vascular protein leak was increased in NO group. In conclusion, no benefit of short term NO inhalation (5 hours) could be demontrated for gas exchange in premature lambs with moderate HMD. Moreoever increased lung vascular protein leak in the NO group suggests an alteration of vascular permeability. Benefits of long term NO inhalation should be demonstrated before its clinical use in moderate HMD.Table

Pro-oxidant Iron Is Present in Human Pulmonary Epithelial Lining Fluid: Implications for Oxidative Stress in the Lung

Bronchoalveolar lavage (BAL) provides a sample of lung surface lining fluid. Several pulmonary diseases appear to involve tissue damage caused by reactive oxygen species generated by inhaled oxidants or by phagocytic cells activated within the lung. We show that BAL from normal healthy controls contain chelatable redox active iron that is pro-oxidant towards free radical reactions. Adult patients with the acute respiratory distress syndrome (ARDS) are known to be under severe oxidative stress and to have high mortality rates. Patients who survived ARDS were found to have chelatable redox active iron in their lung fluid, whereas nonsurvivors had no such iron present. Severe lung leak in nonsurvivors was paradoxically associated with increased levels of transferrin and iron-binding antioxidant activity. We suggest that the presence of pro-oxidant iron in normal lung fluid is an important factor which makes the lung vulnerable to oxidative stress.

The study for the roles of intratracheally administered histamine in the neutrophil-mediated acute lung injury in rats:

Background : Neutrophils are considered to play critical roles in the development of acute respiratory distress syndrome. Histamine, which is distributed abundantly in lung tissue, increases the rolling of neutrophills via increase of P-selectin expression on the surface of endothelial cells and is known to have some interrelationships with IL-1, IL-8 and TNF-. We studied to investigate the effect of the histamine on the acute lung injury of the rats induced by intratracheal insufflation of TNF- which has less potency to cause lung injury compared to IL-1 in rats. Methods : We intratracheally instilled saline or TNF(R&D, 500ng), IL-1(R&D, 50ng)or histamine of varius dose(1.1, 11 and ) with and without TNF separately in Sprague-Dawley rats weighing 270-370 grams. We also intratracheally treated IL-1(50ng) along with histamine(). In cases, there were synergistic effects induced by histamine on the parameters of TNF-induced acute lung injury, antihistamines(Sigma, mepyramine as a receptor blockade and ranitidine as a receptor blockade, 10 mg/kg in each)were co-administered intravenously to the rats treated TNF along with histamine() intratraeheally. Then after 5 h we measured lung lavage neutrophil numbers, lavage cytokine-induced neutrophil chemoattractants(CINC), lung myeloperoxidase activity(MPO) and lung leak. We also intratracheally insufflated TNF with/without histamine(), then after 24 h measured lung leak in rats. Statistical analyses were done by Kruskal-Wallis nonparametric ANOVA test with Dunn's multiple comparison test or by Mann-Whitney U test. Results : We found that rats given TNF, histamine alone(11 and ), and TNF with histamine(l.1, 11, and ) intratracheally had increased (p had increased MPO activity (P=0.0251) compared with TNF-treated rats. TNF and TNF with histamine(1.1, 11, and ) intratracheally had all increased (p had increased (p=0.0367) lavage neutrophil numbers compared with TNF treated rats. But there were no additive effect of histamine with TNF compared with TNF alone in acute lung leak on 5 h and 24 h in rats. Treatment of rats with the and antagonists resulted in inhibitions of lavage neutrophil accumulations and lavage CINC activity elevations elicited by co-treated histamine in TNF-induced acute lung injury intratracheally in rats. We also found that rats given IL-1 along with histamine intratracheally did not have increase in lung leak compared with IL-1 treated rats. Conclusion : Histamine administered intratracheally did not have synergistic effects on TNF-induced acute lung leak inspite of additive effects on increase in MPO activity and lavage neutrophil numbers in rats. These observations suggest that instilling histamine intratracheally would not play synergistic roles in neutrophil-mediated acute lung injury in rats.

Confocal- and electron-microscopic localization of FITC-albumin in H2O2-induced pulmonary edema.

Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) was used to detect sites of protein leakage in rat lungs perfused for 15-60 min with H2O2 (75, 150, and 300 microM). Leaky vessels were localized by confocal laser microscopy. Endothelial routes of protein leakage were identified by electron microscopy after photo-conversion of FITC-BSA to an osmiophilic diaminobenzidine product. Transport of FITC-BSA into the alveolar interstitium was assessed by immunogold labeling and anti-FITC antibodies. We detected leakage of FITC-BSA through transendothelial gaps in the pulmonary arterial endothelium after 30 min of perfusion with 300 microM H2O2 and after 60 min of perfusion with 150 microM H2O2. Junctional permeability and distribution of ZO-1 protein in the arterial endothelium were unchanged. Microvascular permeability to FITC-BSA was not increased in lungs perfused with H2O2. In lungs perfused with 300 microM H2O2, progressive extravasation of albumin was associated with significant increases in water content and perfusing pressure. We conclude that the pulmonary arterial endothelium is the primary target of circulating H2O2.

Gut Ischemia-Reperfusion Produces Lung Injury via a Mechanism Which Involves Xanthine Oxodase and Phospholipase A2.

The gut and the neutrophil (PMN) have been emphasized to play mechanistic roles in the development of adult respiratory distress syndrome and multiple organ failure. Reactive oxygen metabolites and various lipid mediators have been implicated as key players in these pathophysiology. We have previously shown that gut ischemia/reperfusion (I/R) provokes neutrophil-mediated lung injury. We hypothesized in this study that gut I/R produces lung injury via a mechanism which involves xanthine oxidase (XO) and phospholipase A2 (PLA2). Methods: Sprague-Dawley rats were divided into five groups. 1) Normal rats, 2) I/R group: Rats were subjected to 45min of superior mesenteric artery (SMA) occlusion, 3) Sham-laparotomy group (Lap): Rats underwent the same procedures except SMA occlusion, 4) XO-inactivated I/R group (Tung+I/R): Rats were fed tungsten enriched, molybdenum depleted diet for three weeks to inactivate XO before SMA clamp, 5) PLA2-inactivated I/R group (Quin+I/R): Rats were pretreated with a PLA2 inhibitor, quinacrine (10mg/kg, IV), prior to the induction of gut Ischemia. Six hr after reperfusion, circulating PMN priming was determined by the difference in superoxide generation with and without the activating stimulus, N-formyl-Met-Leu-Phe (fMLP). Pulmonary PMN sequestration was quantitated by myeloperoxidase (MPO) activity, while lung endothelial permeability was assessed by 125I albumin lung/blood ratio. Results: Gut I/R markedly enhanced PMN superoxide production in the presence of fMLP, increased pulmonary MPO activities, and provoked 125I albumin lung leak. Although the Tung+I/R and Quin+I/R groups did not alter I/R-induced pulmonary MPO activities, these groups attenuated gut I/R-induced circulating PMN priming and abrogated gut I/R-induced lung injury. Conclusion: These data suggest that both XO and PLA2 activation are proximal steps in the pathogenesis of lung injury following splanchnic hypoperfusion.

N-acetylcysteine pretreatment attenuates paraquat-induced lung leak in rats.

We investigated the effects of N-acetylcysteine (NAC) pretreatment on paraquat-induced lung inflammation and leak. We found that administering a single intravenous dose (60 mg/kg) of paraquat rapidly (2 h) increased lung leak, lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, and lung myeloperoxidase (MPO) activity in rats. Rats pretreated with NAC (150 mg/kg, intraperitoneally) had increased lung tissue glutathione (GSH + GSSG) levels compared to saline-pretreated rats. In addition, rats pretreated with NAC and then given paraquat 2.5 h later had decreased lung leak compared to saline-pretreated rats given paraquat. In contrast, NAC pretreated rats given paraquat had the same lung lavage CINC levels and lung tissue MPO activity as saline-pretreated rats given paraquat. Our results indicate that paraquat causes an oxidative injury which may be decreased by the GSH-increasing or other properties of NAC.

Cytokine-induced neutrophil chemoattractant is necessary for interleukin-1-induced lung leak in rats.

Because interleukin-1 (IL-1) is not a potent chemoattractant intrinsically, our goal was to determine whether cytokine-induced neutrophil chemoattractant (CINC) mediates neutrophil accumulation and leak that occur in lungs of rats given IL-1 intratracheally. We found that rats given IL-1 intratracheally had increased lung lavage CINC concentrations, lung myeloperoxidase (MPO) activity, lung lavage neutrophil numbers, and lung leak compared with saline-treated control rats. In parallel, rats given increasing doses of anti-CINC antibody along with IL-1 intratracheally had progressively decreased lung lavage CINC concentrations, lung lavage neutrophil numbers, and lung leak, but the same lung MPO activity, as did rats given only IL-1 intratracheally. In addition, lavage CINC concentrations correlated with lavage neutrophil numbers in rats. Because CINC is a chemoattractant in vitro and because anti-CINC antibody decreased lung leak induced by IL-1, CINC was then given alone intratracheally. Rats given only CINC intratracheally had the same lung MPO activity, lung lavage neutrophil numbers, and lung leak as did saline-treated control rats. These observations suggest that instilling IL-1 intratracheally induces lung leak via a CINC-dependent pathway but that administering CINC alone in concentrations exceeding the levels measured in lung lavages from IL-1-treated rats is not sufficient to cause lung neutrophil accumulation and lung leak.

CD11b Blockade Prevents Lung Injury despite Neutrophil Priming after Gut Ischemia/Reperfusion

Gut ischemia/reperfusion (I/R) provokes lung injury via a mechanism that involves neutrophils [polymorphonuclear neutrophils (PMNs)]. CD11b/CD18 (alpha mB2) is the integrin receptor on PMNs critical for adhesion-dependent oxidative burst. The purpose of this study was to investigate the mechanistic role of CD11b in the process of gut I/R-induced lung injury. Sprague-Dawley rats underwent 45 minutes of superior mesenteric artery (SMA) occlusion with and without CD11b monoclonal antibody treatment (IB6) (1 mg/kg, i.v.), before SMA clamping. At 2-hour reperfusion, PMN presence in tissue was quantitated by myeloperoxidase activity and circulating PMN priming determined by the difference in superoxide production with and without N-formyl-methionyl-leucyl-phenylalanine, whereas lung leak was assessed by 125I-albumin lung/blood ratio. In sum, CD11b blockade prevented gut I/R-induced lung leak, but did not attenuate gut I/R-induced PMN priming or tissue PMN accumulation. In conclusion, gut I/R promotes PMN priming and PMN adhesion in both local and distant beds via receptors other than CD11b, but this B2 integrin receptor is critical for PMN-mediated endothelial injury.