Nitric oxide-dependent inhibition of alveolar fluid clearance in hydrostatic lung edema

Formation of cardiogenic pulmonary edema in acute left heart failure is traditionally attributed to increased fluid filtration from pulmonary capillaries and subsequent alveolar flooding. Here, we demonstrate that hydrostatic edema formation at moderately elevated vascular pressures is predominantly caused by an inhibition of alveolar fluid reabsorption, which is mediated by endothelial-derived nitric oxide (NO). In isolated rat lungs, we quantified fluid fluxes into and out of the alveolar space and endothelial NO production by a two-compartmental double-indicator dilution technique and in situ fluorescence imaging, respectively. Elevation of hydrostatic pressure induced Ca(2+)-dependent endothelial NO production and caused a net fluid shift into the alveolar space, which was predominantly attributable to impaired fluid reabsorption. Inhibition of NO production or soluble guanylate cyclase reconstituted alveolar fluid reabsorption, whereas fluid clearance was blocked by exogenous NO donors or cGMP analogs. In isolated mouse lungs, hydrostatic edema formation was attenuated by NO synthase inhibition. Similarly, edema formation was decreased in isolated mouse lungs of endothelial NO synthase-deficient mice. Chronic heart failure results in endothelial dysfunction and preservation of alveolar fluid reabsorption. These findings identify impaired alveolar fluid clearance as an important mechanism in the pathogenesis of hydrostatic lung edema. This effect is mediated by endothelial-derived NO acting as an intercompartmental signaling molecule at the alveolo-capillary barrier.     

Alveolar pressure in fluid-filled occluded lung segments during permeability edema

In a model of increased hydrostatic pressure pulmonary edema Parker et al. (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 44: 267-276, 1978) demonstrated that alveolar pressure in occluded fluid-filled lung segments was determined primarily by interstitial fluid pressure. Alveolar pressure was subatmospheric at base line and rose with time as hydrostatic pressure was increased and pulmonary edema developed. To further test the hypothesis that fluid-filled alveolar pressure is determined by interstitial pressure we produced permeability pulmonary edema-constant hydrostatic pressure. After intravenous injection of oleic acid in dogs (0.01 mg/kg) the alveolar pressure rose from -6.85 +/- 0.8 to +4.60 +/- 2.28 Torr (P less than 0.001) after 1 h and +6.68 +/- 2.67 Torr (P less than 0.01) after 3 h. This rise in alveolar fluid pressure coincided with the onset of pulmonary edema. Our experiments demonstrate that during permeability pulmonary edema with constant capillary hydrostatic pressures, as with hemodynamic edema, alveolar pressure of fluid-filled segments seems to be determined by interstitial pressures.     

Beta-adrenergic agonist therapy accelerates the resolution of hydrostatic pulmonary edema in sheep and rats.

To determine whether beta-adrenergic agonist therapy increases alveolar liquid clearance during the resolution phase of hydrostatic pulmonary edema, we studied alveolar and lung liquid clearance in two animal models of hydrostatic pulmonary edema. Hydrostatic pulmonary edema was induced in sheep by acutely elevating left atrial pressure to 25 cmH(2)O and instilling 6 ml/kg body wt isotonic 5% albumin (prepared from bovine albumin) in normal saline into the distal air spaces of each lung. After 1 h, sheep were treated with a nebulized beta-agonist (salmeterol) or nebulized saline (controls), and left atrial pressure was then returned to normal. beta-Agonist therapy resulted in a 60% increase in alveolar liquid clearance over 3 h (P < 0.001). Because the rate of alveolar fluid clearance in rats is closer to human rates, we studied beta-agonist therapy in rats, with hydrostatic pulmonary edema induced by volume overload (40% body wt infusion of Ringer lactate). beta-Agonist therapy resulted in a significant decrease in excess lung water (P < 0.01) and significant improvement in arterial blood gases by 2 h (P < 0.03). These preclinical experimental studies support the need for controlled clinical trials to determine whether beta-adrenergic agonist therapy would be of value in accelerating the resolution of hydrostatic pulmonary edema in patients.     

KGF‐2 targets alveolar epithelia and capillary endothelia to reduce high altitude pulmonary oedema in rats

High altitude pulmonary oedema (HAPE) severely affects non‐acclimatized individuals and is characterized by alveolar flooding with protein‐ rich oedema as a consequence of blood‐gas barrier disruption. Limited choice for prophylactic treatment warrants effective therapy against HAPE. Keratinocyte growth factor‐2 (KGF‐2) has shown efficiency in preventing alveolar epithelial cell DNA damages in vitro. In the current study, the effects of KGF‐2 intratracheal instillation on mortality, lung liquid balance and lung histology were evaluated in our previously developed rat model of HAPE. We found that pre‐treatment with KGF‐2 (5 mg/kg) significantly decreased mortality, improved oxygenation and reduced lung wet‐to‐dry weight ratio by preventing alveolar‐capillary barrier disruption demonstrated by histological examination and increasing alveolar fluid clearance up to 150%. In addition, KGF‐2 significantly inhibited decrease of transendothelial permeability after exposure to hypoxia, accompanied by a 10‐fold increase of Akt activity and inhibited apoptosis in human pulmonary microvascular endothelial cells, demonstrating attenuated endothelial apoptosis might contribute to reduction of endothelial permeability. These results showed the efficacy of KGF‐2 on inhibition of endothelial cell apoptosis, preservation of alveolar‐capillary barrier integrity and promotion of pulmonary oedema absorption in HAPE. Thus, KGF‐2 may represent a potential drug candidate for the prevention of HAPE.     

Alveolar epithelial fluid transport and the resolution of clinically severe hydrostatic pulmonary edema.

To characterize the rate and regulation of alveolar fluid clearance in the uninjured human lung, pulmonary edema fluid and plasma were sampled within the first 4 h after tracheal intubation in 65 mechanically ventilated patients with severe hydrostatic pulmonary edema. Alveolar fluid clearance was calculated from the change in pulmonary edema fluid protein concentration over time. Overall, 75% of patients had intact alveolar fluid clearance (>/=3%/h). Maximal alveolar fluid clearance (>/=14%/h) was present in 38% of patients, with a mean rate of 25 +/- 12%/h. Hemodynamic factors (including pulmonary arterial wedge pressure and left ventricular ejection fraction) and plasma epinephrine levels did not correlate with impaired or intact alveolar fluid clearance. Impaired alveolar fluid clearance was associated with a lower arterial pH and a higher Simplified Acute Physiology Score II. These factors may be markers of systemic hypoperfusion, which has been reported to impair alveolar fluid clearance by oxidant-mediated mechanisms. Finally, intact alveolar fluid clearance was associated with a greater improvement in oxygenation at 24 h along with a trend toward shorter duration of mechanical ventilation and an 18% lower hospital mortality. In summary, alveolar fluid clearance in humans may be rapid in the absence of alveolar epithelial injury. Catecholamine-independent factors are important in the regulation of alveolar fluid clearance in patients with severe hydrostatic pulmonary edema.     

Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury

We have previously reported that keratinocyte growth factor (KGF) attenuates alpha-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport. The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia. A 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P. aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance. Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 microCi of (131)I-albumin. In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals. In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF. KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation. In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P. aeruginosa pneumonia and LLC in early and late pneumonia. Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria.     

Hydraulic conductance of lung endothelial phenotypes and Starling safety factors against edema

Recent permeability studies comparing endothelial cell phenotypes derived from alveolar and extra-alveolar vessels have significant implications for interpreting the mechanisms of fluid homeostasis in the intact lung. These studies indicate that confluent monolayers of rat pulmonary microvascular endothelial cells had a hydraulic conductance (L(p)) that was only 5% and a transendothelial flux rate for 72-kDa dextran only 9% of values determined for rat pulmonary artery endothelial cell monolayers. On the basis of previous studies partitioning the filtration coefficients between alveolar and extra-alveolar vascular segments in rat lungs and previous studies of lymph albumin fluxes and permeability, the contribution of the alveolar capillary segment to total albumin flux in lymph was estimated to be less than 10%. In addition, the Starling safety factors against the edema calculated for the alveolar capillaries are quite different from those estimated for whole lung. Estimates of the edema safety factor due to increased filtration across the alveolar capillary wall based on the low L(p) indicate it is quantitatively the greatest safety factor, although it would be a minor safety factor for extra-alveolar vessels. Also, a markedly higher effective protein osmotic absorptive force for plasma proteins must occur in the capillaries relative to extra-alveolar vessels. The lower L(p) for alveolar capillaries also has implications for the sequence of hydrostatic edema formation, and it also may have a role in preventing exercise-induced alveolar flooding.     

Keratinocyte growth factor augments pulmonary innate immunity through epithelium-driven, GM-CSF-dependent paracrine activation of alveolar macrophages

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of insults. In this study, we tested the hypothesis that KGF augments pulmonary host defense. We found that a single dose of intrapulmonary KGF enhanced the clearance of Escherichia coli or Pseudomonas aeruginosa instilled into the lungs 24 h later. KGF augmented the recruitment, phagocytic activity, and oxidant responses of alveolar macrophages, including lipopolysaccharide-stimulated nitric oxide release and zymosan-induced superoxide production. Less robust alveolar macrophage recruitment and activation was observed in mice treated with intraperitoneal KGF. KGF treatment was associated with increased levels of MIP1γ, LIX, VCAM, IGFBP-6, and GM-CSF in the bronchoalveolar lavage fluid. Of these, only GM-CSF recapitulated in vitro the macrophage activation phenotype seen in the KGF-treated animals. The KGF-stimulated increase in GM-CSF levels in lung tissue and alveolar lining fluid arose from the epithelium, peaked within 1 h, and was associated with STAT5 phosphorylation in alveolar macrophages, consistent with epithelium-driven paracrine activation of macrophage signaling through the KGF receptor/GM-CSF/GM-CSF receptor/JAK-STAT axis. Enhanced bacterial clearance did not occur in response to KGF administration in GM-CSF(-/-) mice, or in mice treated with a neutralizing antibody to GM-CSF. We conclude that KGF enhances alveolar host defense through GM-CSF-stimulated macrophage activation. KGF administration may constitute a promising therapeutic strategy to augment innate immune defenses in refractory pulmonary infections.     

High surface tension pulmonary edema induced by detergent aerosol

The effect of the detergent dioctyl sodium sulfosuccinate on pulmonary extravascular water volume (PEWV) was studied in adult anesthetized mongrel dogs. The detergent was dissolved as a 1% solution in a vehicle of equal volumes of 95% ethanol and normal saline and administered by ultrasonic nebulizer attached to the inspiratory tubing of a piston ventilator. Two hours following detergent aerosol PEWV measured gravimetrically was increased compared with either animals receiving no aerosol or those receiving an aerosol of vehicle alone. Loss of surfactant activity and increased alveolar surface tension were demonstrated by Wilhelmy balance studies of minced lung extracts, by a fall in static compliance, and by evidence of atelectasis and instability noted by gross observation and by in vivo microscopy. No significant changes in colloid oncotic pressure or pulmonary microvascular hydrostatic pressure were observed. These data suggest that pulmonary edema can be induced by increased alveolar surface tension and support the concept that one of the major roles of pulmonary surfactant is to prevent pulmonary edema.     

Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema

Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of 2.50-47.62 pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.2 pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of 20.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(2). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of 2.2-9.3 pg/mug) was similar to that of controls at baseline (2.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.     

Keratinocyte growth factor‐2 intratracheal instillation significantly attenuates ventilator‐induced lung injury in rats

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor‐2 (KGF‐2), a potent epithelial cell mitogen, may have a role in preventing ventilator‐induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF‐2 in VILI in rats. Forty healthy adult male Sprague‐Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high‐volume zero positive end‐expiratory pressure) and HVZP+KGF‐2 were given intratracheally equal PBS and 5 mg/kg KGF‐2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF‐2 were administered in the same manner in Groups Control and KGF‐2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor‐α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre‐treatment with KGF‐2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF‐2 might be considered a promising prevention for human VILI or other acute lung injury diseases.     

Keratinocyte growth factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 lung epithelial cells

Acute respiratory distress syndrome (ARDS) is a syndrome characterized by the rapid influx of protein-rich edema fluid into the air spaces. The magnitude of alveolar epithelial cell injury is a key determinant of disease severity and an important predictor of patient outcome. The alveolar epithelium is positioned at the interface of the host response in the initiation, progression, and recovery phase of the disease. Keratinocyte growth factor (KGF) is a potent survival factor unique to the epithelium that promotes lung epithelial cell survival, accelerates wound closure, and reduces fibrosis. We therefore hypothesized that KGF preserves lung function by inhibiting apoptosis through activation of a signal transduction pathway responsible for cell survival. To test this hypothesis we determined that KGF inhibits death following Fas activation, a relevant apoptosis pathway, and then determined that cell survival is mediated through activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt kinase signal transduction pathway. We found that KGF induces a dose- and time-dependent increase in Akt kinase activity and that, as expected, activation of Akt via KGF is PI3K dependent. KGF inhibited Fas-induced apoptosis as measured by a reduction in apoptotic cells and caspase-3 activity. This investigation supports our original hypothesis that KGF protects the lung epithelium by inhibiting apoptosis and that protection occurs through activation of PI3K/Akt-mediated cell survival pathway. Our results are in agreement with other reports that identify the PI3K/Akt axis as a key intracellular pathway in the lung epithelium that may serve as a therapeutic target to preserve epithelial integrity during inflammation.     

Extracellular heat shock protein 72 is a marker of the stress protein response in acute lung injury

Previous studies have shown that heat shock protein 72 (Hsp72) is found in the extracellular space (eHsp72) and that eHsp72 has potent immunomodulatory effects. However, whether eHsp72 is present in the distal air spaces and whether eHsp72 could modulate removal of alveolar edema is unknown. The first objective was to determine whether Hsp72 is released within air spaces and whether Hsp72 levels in pulmonary edema fluid would correlate with the capacity of the alveolar epithelium to remove alveolar edema fluid in patients with ALI/ARDS. Patients with hydrostatic edema served as controls. The second objective was to determine whether activation of the stress protein response (SPR) caused the release of Hsp72 into the extracellular space in vivo and in vitro and to determine whether SPR activation and/or eHsp72 itself would prevent the IL-1beta-mediated inhibition of the vectorial fluid transport across alveolar type II cells. We found that eHsp72 was present in plasma and pulmonary edema fluid of ALI patients and that eHsp72 was significantly higher in pulmonary edema fluid from patients with preserved alveolar epithelial fluid clearance. Furthermore, SPR activation in vivo in mice and in vitro in lung endothelial, epithelial, and macrophage cells caused intracellular expression and extracellular release of Hsp72. Finally, SPR activation, but not eHsp72 itself, prevented the decrease in alveolar epithelial ion transport induced by exposure to IL-1beta. Thus SPR may protect the alveolar epithelium against oxidative stress associated with experimental ALI, and eHsp72 may serve as a marker of SPR activation in the distal air spaces of patients with ALI.     

Does interstitial lung edema compress airways and arteries? A morphometric study

We compared areas and diameters of small airways and arteries in three groups of anesthetized dogs: 1) control (n = 5), 2) hydrostatic edema induced by fluid overload (n = 13), and 3) increased permeability edema induced with alpha-naphthylthiourea (n = 5). We measured pulmonary arterial and wedge pressures in all groups and cardiac output in the hydrostatic edema group. Postmortem, lobes were frozen at functional residual capacity and samples taken for measurements of extravascular lung water (Qwl/dQl) and for light microscopy. We also examined lobes from hydrostatic edema experiments fixed at transpulmonary pressures of 5 and 27 cmH2O. From the histology slides, bronchovascular bundles with respiratory bronchioles (n = 706) and bronchioles (n = 467) were photographed and airway and vessel areas and diameters measured. Alveolar and airway luminal edema were graded. We found that only in hydrostatic edema, pulmonary arterial and wedge pressures increased and vascular resistance fell with fluid infusion. Mean Qwl/dQl values were 3.80 +/- 0.17, 6.81 +/- 0.96, and 9.34 +/- 0.62 (SE) in control, hydrostatic, and increased permeability edema groups, respectively. By quantitative histology, airway and arterial areas and diameters did not decrease in edema and rose with increasing transpulmonary pressure. Variable quantities of air-space edema were seen. We conclude that interstitial edema does not compress small airways or arteries and that other mechanisms, including alveolar and airway luminal edema, may explain reported increases in airway resistance.     

Physiological aspects of high-altitude pulmonary edema.

High-altitude pulmonary edema (HAPE) develops in rapidly ascending nonacclimatized healthy individuals at altitudes above 3,000 m. An excessive rise in pulmonary artery pressure (PAP) preceding edema formation is the crucial pathophysiological factor because drugs that lower PAP prevent HAPE. Measurements of nitric oxide (NO) in exhaled air, of nitrites and nitrates in bronchoalveolar lavage (BAL) fluid, and forearm NO-dependent endothelial function all point to a reduced NO availability in hypoxia as a major cause of the excessive hypoxic PAP rise in HAPE-susceptible individuals. Studies using right heart catheterization or BAL in incipient HAPE have demonstrated that edema is caused by an increased microvascular hydrostatic pressure in the presence of normal left atrial pressure, resulting in leakage of large-molecular-weight proteins and erythrocytes across the alveolarcapillary barrier in the absence of any evidence of inflammation. These studies confirm in humans that high capillary pressure induces a high-permeability-type lung edema in the absence of inflammation, a concept first introduced under the term "stress failure." Recent studies using microspheres in swine and magnetic resonance imaging in humans strongly support the concept and primacy of nonuniform hypoxic arteriolar vasoconstriction to explain how hypoxic pulmonary vasoconstriction occurring predominantly at the arteriolar level can cause leakage. This compelling but as yet unproven mechanism predicts that edema occurs in areas of high blood flow due to lesser vasoconstriction. The combination of high flow at higher pressure results in pressures, which exceed the structural and dynamic capacity of the alveolar capillary barrier to maintain normal alveolar fluid balance.     

Apoptosis in microvascular endothelial cells of perfused rabbit lungs with acute hydrostatic edema.

We test the hypothesis that microvascular endothelial cells may undergo apoptosis in response to acute pulmonary venous hypertension. The isolated rabbit lungs were perfused in situ for 4 h with left atrial pressure of 0, 10, or 20 mmHg at a constant blood flow. Edema formation was monitored by lung weight gain. To assay for apoptosis, we performed agarose gel electrophoresis of DNA, in situ nick end labeling of DNA strand breaks, and electron microscopy. We also examined the levels of expression of Bcl-2, a suppressor of apoptosis, in microvascular endothelial cells using an immunohistochemical technique. In a vascular pressure-dependent fashion, we found apoptosis in endothelial cells of alveolar septal capillaries, as well as expression of Bcl-2 in arteriolar and venular endothelial cells. We conclude that acute pulmonary venous hypertension induces apoptosis in capillary endothelial cells but not in arteriolar and venular endothelial cells, suggesting that microvascular endothelial cell apoptosis is dependent on the levels of Bcl-2 expression and influences the formation or resolution of acute hydrostatic lung edema.     

Effect of regional alveolar hypoxia on permeability pulmonary edema formation in dogs

We studied the effects of regional alveolar hypoxia on permeability pulmonary edema formation. Anesthetized dogs had a bronchial divider placed so that the left lower lobe (LLL) could be ventilated with a hypoxic gas mixture (HGM) while the right lung was continuously ventilated with 100% O2. Bilateral permeability edema was induced with 0.05 ml/kg oleic acid and after 4 h of LLL ventilation with an HGM (n = 9) LLL gross weight was 161 +/- 13 (SE) g compared with 204 +/- 13 (SE) g (P less than 0.05) in the right lower lobe (RLL). Bloodless lobar water and dry weight were also significantly lower in the LLL as compared with the RLL of the study animals. In seven control animals in which the LLL fractional inspired concentration of O2 (FIO2) was 1.0 during permeability edema, there were no differences in gravimetric variables between LLL and RLL. In eight additional animals, pulmonary capillary pressure (Pc), measured by simultaneous occlusion of left pulmonary artery and vein, was not significantly different between LLL FIO2 of 1.0 and 0.05 either before or after pulmonary edema. We conclude that, in the presence of permeability pulmonary edema, regional alveolar hypoxia causes reduction in edema formation. The decreased edema formation during alveolar hypoxia is not due to a reduction in Pc.     

Endogenous plasma proteins in edematous lungs and alveolar fluid in rabbits

In this study, we compared two methods of differentiating hydrostatic and permeability types of pulmonary edema. The first method entailed measurement of protein concentrations directly in samples of alveolar fluid (AF); the second method was an indirect technique in which protein concentration in extravascular extracellular water (EVECW) was calculated on the basis of separate measurements of the quantity of protein in the lung and the volume of EVECW. The concentration of albumin (Alb) and gamma-G-globulin was measured in EVECW and alveolar fluid in excised edematous rabbit lungs. Edema was caused by elevation of left ventricular end-diastolic pressure to 25 Torr (hydrostatic edema, HE) or by intravenous oleic acid, 0.09 ml/kg (permeability edema, PE). The volume of distribution of Na+ was utilized as a measure of EVECW in the lung. Protein concentration in EVECW and AF relative to plasma (EV/PL and AF/PL, respectively) was compared in the two types of edema. The EV/PL was 0.61 +/- 0.12 (SD) for Alb in He compared with 1.18 +/- 0.47 in PE (P less than 0.02). The AF/PL was 0.54 +/- 0.12 and 1.25 +/- 0.33 in HE and PE, respectively (P less than 0.001). There was good correlation between EV/PL and AF/PL for Alb (r = 0.74, P less than 0.001) but not for gamma-G-globulin. Thus EV/PL for Alb, AF/PL for Alb, and gamma-G-globulin all differentiated hydrostatic from permeability edema.     

Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.     

Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung injury. Statins exert beneficial effects in septic patients although the mechanisms remain elusive. This study examined effects of simvastatin on M1 protein-provoked pulmonary inflammation and tissue injury. Male C57BL/6 mice were pretreated with simvastatin or a CXCR2 antagonist before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for determination of neutrophil infiltration, formation of edema, and CXC chemokines. Flow cytometry was used to determine Mac-1 expression on neutrophils. Gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative RT-PCR. M1 protein challenge caused massive infiltration of neutrophils, edema formation, and production of CXC chemokines in the lung as well as upregulation of Mac-1 on circulating neutrophils. Simvastatin reduced M1 protein-induced infiltration of neutrophils and edema in the lung. In addition, M1 protein-induced Mac-1 expression on neutrophils was abolished by simvastatin. Furthermore, simvastatin markedly decreased pulmonary formation of CXC chemokines and gene expression of CXC chemokines in alveolar macrophages. Moreover, the CXCR2 antagonist reduced M1 protein-induced neutrophil expression of Mac-1 and accumulation of neutrophils as well as edema formation in the lung. These novel findings indicate that simvastatin is a powerful inhibitor of neutrophil infiltration in acute lung damage triggered by streptococcal M1 protein. The inhibitory effect of simvastatin on M1 protein-induced neutrophil recruitment appears related to reduced pulmonary generation of CXC chemokines. Thus, simvastatin may be a useful tool to ameliorate acute lung injury in streptococcal infections.