The effect of 3-methylindole on the quantity and functional quality of lung surfactant

Acute bovine pulmonary edema is a naturally occurring lung disease caused by 3-methylindole (3MI), a ruminal fermentation product of tryptophan. Morphological and in vitro studies have suggested that 3MI causes abnormalities in phospholipid synthesis. The present study was designed to investigate the effect of 3MI on the quantity and functional quality of surfactant using the goat as an experimental model. Following intravenous infusion of 3MI, goats were killed at 6-, 18-, and 30-h intervals. The lungs were removed and intracellular surfactant, in the form of lamellar bodies, and extracellular surfactant from alveolar lavage were quantified. 3MI treatment did cause modest changes in the lamellar body phospholipid pools, decreasing the quantity of phosphatidylcholine and the proportion of palmitate in this fraction. The quantity of lavage phospholipids was not significantly affected. There was an increase in the protein content of the lavage, reflecting the presence of edema. The functional quality of the surfactant isolated from the lavage fraction was tested in vitro using a pulsating bubble surfactometer. 3MI infusion decreased the ability of surfactant to lower the surface tension of an air bubble at maximum radius and during compression.     

Surfactant dysfunction in diabetic offspring: inhibitors and fatty acid lecithin content

We have previously demonstrated that the lungs of fetuses of alloxan-diabetic rabbits are functionally immature when compared with controls. In this study we have examined pulmonary lavage fluid from fetuses of diabetic and control does for the presence of a surfactant inhibitor, cholesterol content, and phosphatidylcholine (lecithin) fatty acid content. When lavage fluids from fetuses of control and diabetic does were mixed and examined on the surface balance, the measured surfactant content corresponded to the arithmetic average of the surfactant content of the individual fluids, thus ruling out the presence of an excess of a surfactant inhibitor. We were able to demonstrate inhibition of surfactant function when exogenous cholesterol was added to lavage samples, but cholesterol content of lavage fluid from fetuses of diabetic and control does was not significantly different. Finally, the fatty acid composition of surfactant phosphatidylcholine was not different between the two groups. These studies indicate that mechanisms other than the presence of a surfactant inhibitor or altered fatty acid composition must explain the functional immaturity seen in offspring of the diabetic pregnancy.     

肺机械扩张增进表面活性物质分泌机制的研究

采用大鼠离体肺灌流模型,观察肺泡灌洗液中肺表面活性物质(PS)含量的变化。结果表明:在增大潮气量机械扩张肺时PS分泌显著增加(p<0.05),该分泌增加现象能被细胞松弛素B及硝苯吡啶所抑制(p<0.05),但不为细胞外缺钙所抑制(p>0.05)。从而提示扩张所引起的PS分泌增加可能与细胞内的收缩结构有关,而与细胞外钙离子无关。     

Alteration of surfactant function due to protein leakage: special interaction with fibrin monomer

In isolated rabbit lungs standardized amounts of edema were induced. Stimulation with the Ca ionophore A23187, leukotriene C4, Pseudomonas aeruginosa cytotoxin and human serum (activated complement) all resulted in protein leakage into the alveolar space with no change in the total phospholipid content. The pressure-volume characteristics of the lungs and the characteristics of the lavage surfactant (Wilhelmy balance) were markedly altered, correlating to the lavage protein content. The surfactant alterations were reproduced by addition of perfusion fluid protein to control surfactant in vitro. All changes were far less expressed or even missing in isolated lungs developing the same amount of edema due to omittance of proteins from the perfusion liquid. Different proteins added to control surfactant in the Wilhelmy balance showed a marked rank order of potency in interfering with surfactant function: immunoglobulins G and M and elastin less than albumin less than fibrinogen less than fibrin monomers. The fibrin monomer effect was reproduced by addition of thrombin to a surfactant fibrinogen mixture and was partly reversed by subsequent incubation with plasmin. In conclusion, high-permeability edema induced by different means results in alterations of lung mechanics and surface activity of lavaged surfactant, presumably due to protein surfactant interaction. Among different proteins, fibrin monomers are especially effective in interfering with surfactant function.     

Surfactant function in children with chronic airway inflammation.

Pulmonary surfactant is necessary to keep the terminal conducting airways patent. It is unknown whether mild to moderate airway inflammation may influence surfactant function and thus contribute to the pathogenesis of chronic airway inflammation in children. To answer this question, 21 children with chronic obstructive bronchitis and 19 asymptomatic children with long-term tracheostomy and increased numbers of neutrophils in their airways were compared with 15 healthy controls. Bronchoalveolar lavage fluid was separated into large surfactant aggregates (LA) and a supernatant containing inhibitory constituents. Surfactant function of LA, recombinations of LA and supernatant, and recombinations of a defined bovine surfactant and supernatant was assessed in a capillary surfactometer. Compared with controls, the function of the LA surfactant was reduced and there was no difference between children with tracheostomy and chronic obstructive bronchitis. The function of LA-supernatant recombinations was poor in all subjects. This may be explained by the well-known protein influx during the lavage procedure. The activity of bovine surfactant-supernatant reconstitutions was impaired in children with tracheostomy. In all surfactant mixtures assessed, surfactant function was inversely correlated to the number of neutrophils in the lavage fluid. Chronic lower airway inflammation with mild or no clinical symptoms is associated with impaired surfactant function. The dysfunction may contribute to airflow restrictions frequently observed in these children.     

Intra- and extracellular compartmentalization of the surface-active fraction in dog lung

Adult mongrel dogs were killed at various times after injection of (3)H-labeled palmitate. The lungs were removed and subjected to an extensive saline lavage. The surface-active fraction was isolated from the lavage and from homogenized residual lung by a procedure based upon differential centrifugation in sucrose solutions. The material isolated from the lavage was designated extracellular surfactant; material from the residual lung was designated intracellular surfactant. Both had similar chemical composition and surface activity. The results of the isotopic labeling studies demonstrate that the two fractions have distinctly different specific activity curves. Label was incorporated into the intracellular surfactant rapidly and reached a peak at 1 hr. No radioactivity was found in the extracellular surfactant for the first 15 min, and the specific activity increased much more slowly than in the intracellular surfactant. These results demonstrate at least two anatomically distinct metabolic "pools" of pulmonary surfactant in the lung. While our data are not conclusive, one possible interpretation is that the biosynthesis of pulmonary surfactant takes place intracellularly with a subsequent secretion onto the alveolar surface.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

Hydrophobic proteins of lamellated osmiophilic bodies isolated from pig lung.

1. In addition to proteins that are insoluble in organic solvents, lamellated bodies isolated from pig lung and surfactant prepared from bronchopulmonary lavage fluid contain another group of proteins that are extracted together with lipid into the organic phase. 2. These hydrophobic proteins constitute about 40% of the total protein of lamellated bodies and about 13% of the total protein of surfactant isolated from lavage fluid, whereas less than 1% of the total protein of pig lung microsomal fraction and mitochondria is extracted by organic solvents. 3. The hydrophobic proteins of lamellated bodies were separated into four fractions and freed from phospholipid by chromatographic procedures. Their apparent molecular weights vary between 11 500 and 16 500, they contain 72--79% of hydrophobic residues and 16--22% of sulphur-containing amino-acids, and leucine is the major N-terminal amino acid in each case.     

Altered function of pulmonary surfactant in fatty acid lung injury

To determine whether acute fatty acid lung injury impairs pulmonary surfactant function, we studied anesthetized ventilated rabbits given oleic acid (55 mg/kg iv, n = 11) or an equivalent volume of saline (n = 8). Measurements of pulmonary mechanics indicated a decrease in dynamic compliance within 5 min of injury and a decrease in lung volume that was disproportionately large at low pressures, consistent with diminished surfactant activity in vivo. Bronchoalveolar lavage fluid obtained 1 h after injury had significantly increased erythrocytes and total leukocytes, largely polymorphonuclear cells. The phospholipid content and composition of the cell-free fraction had only minor changes from those of controls, but the protein content was increased 35-fold. Measurements of lavage surface activity in vitro showed an increase in average minimum surface tension from 1.3 +/- 0.4 (SE) dyn/cm in controls to 20.2 +/- 3.9 dyn/cm in injured animals. The alterations in static pressure-volume curves and decrease in lavage surface activity suggest a severe alteration of surfactant function in this form of lung injury that occurs despite the presence of normal amounts of surfactant phospholipids.     

Uptake of lung surfactant subfractions into lamellar bodies of adult rabbit lungs

The goals of this investigation were to determine whether subfractions of alveolar surfactant that have different physical and biochemical properties are preferentially taken up from the alveolar air space into lamellar bodies and to correlate the magnitude of the uptake with the properties of the fractions. Radiolabeled subfractions were obtained by differential centrifugation of lavage fluid from rabbits that had been intravenously injected with radioactive palmitate. The subfractions were P (pellet) 3 (1,000 g, 20 min), P4 (60,000 g, 60 min), P5 (100,000 g, 16 h). Subfractions were instilled into the lungs of anesthetized spontaneously breathing adult rabbits, and lavage and lamellar body fractions were isolated at later times. P3 and P4 were taken up to a larger extent than was P5 or liposomes prepared from a P4 lipid extract. The fractions that were preferentially taken up (P3 and P4) contained surfactant apoprotein (APO) 36, tubular myelin, multilamellar vesicles, and were rapidly adsorbed to an air-water interface. P3 also contained APO 10. These results demonstrate that different forms of surfactant are recycled at different rates and suggest that there is specificity in the recycling process.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Sequential analysis of surfactant,lung function and inflammation in cystic fibrosis patients

Background

In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.

Methods

As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV₁ 94% of predicted) at three times over a three year period.

Results

There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV₁, MEF_75/25%VC, and MEF_25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.

Conclusion

Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.     

Expression and localization of lung surfactant protein B in Eustachian tube epithelium

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.     

Determinants of surfactant function in acute lung injury and early recovery

Relationships between lung function and surfactant function and composition were examined during the evolution of acute lung injury in guinea pigs. Lung mechanics and gas exchange were assessed 12, 24, or 48 h after exposure to nebulized lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) fluid was processed for phospholipid and protein contents and surfactant protein (SP) A and SP-B levels; surfactant function was measured by pulsating bubble surfactometry. Lung elastance, tissue resistance, and arterial-alveolar gradient were moderately elevated by 12 h after LPS exposure and continued to increase over the first 24 h but began to recover between 24 and 48 h. Similarly, the absolute amount of 30,000 g pelleted SP-A and SP-B, the phospholipid content of BAL fluid, and surfactant function declined over the first 24 h after exposure, with recovery between 24 and 48 h. BAL fluid total protein content increased steadily over the first 48 h after LPS nebulization. In this model of acute lung injury, the intra-alveolar repletion of surfactant components in early recovery led to improved surfactant function despite the presence of potentially inhibitory plasma proteins.     

Surfactant replacement and open lung concept – Comparison of two treatment strategies in an experimental model of neonatal ARDS

Background

Several concepts of treatment in neonatal ARDS have been proposed in the last years. The present study compared the effects of open lung concept positive pressure ventilation (PPV_OLC) with a conventional ventilation strategy combined with administration of two different surfactant preparations on lung function and surfactant homoeostasis.

Methods

After repeated whole-lung saline lavage, 16 newborn piglets were assigned to either PPV_OLC (n = 5) or surfactant treatment under conventional PPV using a natural bovine (n = 5) or a monomeric protein B based surfactant (n = 6).

Results

Comprehensive monitoring showed each treatment strategy to improve gas exchange and lung function, although the effect on Pa_O2 and pulmonary compliance declined over the study period in the surfactant groups. The overall improvement of the ventilation efficiency index (VEI) was significantly greater in the PPV_OLC group. Phospholipid and protein analyses of the bronchoalveolar lavage fluid showed significant alterations to surfactant homoeostasis in the PPV_OLC group, whereas IL-10 and SP-C mRNA expression was tendentially increased in the surfactant groups.

Conclusion

The different treatment strategies applied could be shown to improve gas exchange and lung function in neonatal ARDS. To which extent differences in maintenance of lung function and surfactant homeostasis may lead to long-term consequences needs to be studied further.     

Liver carboxylesterase cleaves surfactant protein (SP-) B and promotes surfactant subtype conversion

Conversion of the biophysically active large surfactant aggregate subtype of alveolar surfactant into the less surface active small surfactant aggregates occurs in vitro and in vivo, possibly in dependency of a carboxylesterase, entitled surfactant convertase. The substrate has yet not been safely identified. Utilizing the in vitro cycling assay we investigated conversion of an organic rabbit lavage extract reconstituted with SP-A. Porcine liver carboxylesterase, which is closely related to surfactant convertase, induced subtype conversion to a similar degree as compared with native lavage fluid containing endogenous convertase. In addition, we asked for cleavage products of SP-B and identified a approximately 12 kDa band upon cycling with liver carboxylesterase, having the same N-terminus as mature SP-B. A band of same molecular weight was found in native lavage fluid after in vitro conversion mediated by the endogenous convertase. We conclude that SP-B plays a pivotal role during subtype conversion and represents the substrate for surfactant convertase.     

Eustachian tube surfactant is different from alveolar surfactant: determination of phospholipid composition of porcine eustachian tube lavage fluid.

Phosphatidylcholine (PC) is the main phospholipid in lung surfactant and, more specifically, dipalmitoyl PC (PC16:0/16:0) is the major surface-active component. Several studies have tentatively shown that eustachian tube lavage fluid (ETLF) contains surface-active material. The aim of the present study was to determine, using electrospray ionization mass spectrometry, whether the phospholipid molecular species composition of ETLF is similar to that of lung surfactant. PC was the main component of both ETLF and bronchoalveolar lavage fluid (BALF). The concentration of phosphatidylethanolamine was higher and phosphatidylglycerol was undetectable in ETLF compared with BALF. The molecular species composition of PC in ETLF was notably different from that of BALF, palmitoyloleoyl PC being the major component. Importantly, given its predominance in BALF PC, the concentration of PC16:0/16:0 was low in ETLF. As expected on the basis of this molecular species composition of PC, ETLF did not generate low surface tension values under dynamic compression in a pulsating bubble surfactometer.We conclude that the surfactant in ET is different from lung surfactant, and that low surface tension is not a major determinant of ETLF function.     

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.