Surfactant Phospholipid Composition Research Articles

Surfactant Phospholipid Kinetics in Ventilated Children after Therapeutic Surfactant Supplementation

Acute lung Injury leads to alterations in surfactant lipid composition and metabolism. Although several mechanisms contribute to dysregulated surfactant metabolism, studies investigating in vivo surfactant metabolism are limited. The aim of this study is to characterise surfactant phospholipid composition and flux utilising a stable isotope labelling technique in mechanically ventilated paediatric patients. Paediatric patients (<16 years of age) received 3.6 mg/kg intravenous methyl-D9-choline chloride followed by the endotracheal instillation of 100 mg/kg of exogenous surfactant after 24 h. Bronchioalveolar fluid samples were taken at baseline and 12, 24, 36, 48, 72 and 96 h after methyl-D9-choline infusion. Nine participants (median age of 48 days) were recruited. The primary phosphatidylcholine (PC) composition consisted of PC16:0/16:0 or DPPC (32.0 ± 4.5%). Surfactant supplementation resulted in a 30% increase in DPPC. Methyl-D9 PC enrichment was detected after 12 h and differed significantly between patients, suggesting variability in surfactant synthesis/secretion by the CDP-choline pathway. Peak enrichment was achieved (0.94 ± 0.15% of total PC) at 24 h after methyl-D9-choline infusion. There was a trend towards reduced enrichment with the duration of mechanical ventilation prior to study recruitment; however, this was not statistically significant (p = 0.19). In this study, we demonstrated the fractional molecular composition and turnover of surfactant phospholipids, which was highly variable between patients.

Magnitude of Obesity Alone does not Alter the Alveolar Lipidome.

Obesity may lead to pulmonary dysfunction through complex and incompletely understood cellular and biochemical effects. Altered lung lipid metabolism has been identified as a potential mechanism of lung dysfunction in obesity. While murine models of obesity demonstrate changes in pulmonary surfactant phospholipid composition and function, data in humans are lacking. We measured untargeted shotgun lipidomes in two bronchoalveolar lavages (BAL) from apical and anteromedial pulmonary subsegments of 14 adult subjects (7 males and 7 females) with body mass indexes ranging from 24.3 to 50.9 kg/m2. The lipidome composition was characterized at the class, species, and fatty acyl / alkyl level using total lipid molecular ion signal intensities normalized to BAL protein concentration and epithelial lining fluid volumes. Multivariate analyses were conducted to identify potential changes with increasing BMI. The alveolar lipidomes contained the expected composition of surfactant associated phospholipids, sphingolipids, and sterols in addition to cardiolipin and intracellular signaling lipid species. No significant differences in lipidomes were detected between the two BAL regions. Though a small number of lipid species were associated with BMI in multivariate analyses, no robust differences in lipidome composition or specific lipid species were identified over the range of body habitus. The magnitude of obesity alone does not substantially alter the alveolar lipidome in patients without lung disease. Differences in lung function in patients with obesity and no lung disease are unlikely related to changes in alveolar lipid composition.

Surfactant system of the lungs in antiphospholipid syndrome under the conditions of administration of the immunosuppressive agent FTY-720

Antiphospholipid syndrome (APS) is a thrombophilic disease in the pathogenesis of which the leading role belongs to antibodies reacting with antigenic determinants of phospholipids. APS is a systemic autoimmune pathology that involves many organs and systems in the pathological process, including the respiratory system. Given the fact that the lung surfactant is mainly represented by phospholipids, it can be presented that the surfactant is damaged and its functioning is impaired in APS. The results of our experiments showed that in antiphospholipid syndrome, there was a decline in the functional activity of the surfactant and a change in its biochemical composition. The introduction of FTY-720 normalized the parameters of the lung surfactant system, which were changed during the modeling of antiphospholipid syndrome. The work shows the effectiveness of the therapy of this autoimmune pathology with immunosuppressor FTY-720, which is a structural analogue of endogenous sphingosine. The effect of the drug is based on the modulation of sphingosine-1 phosphate receptors of lymphocytes. The main effect of the immunosuppressor FTY-720 as a result of receptor interaction is a decrease in the number of circulating lymphocytes. The research involved 85 white random bred male rats: Group 1 (APS modeling) consisted of 30 rats who were intravenously injected with cardiolipin antigen at a total dose of 0.2-0.4 mg per rat every other day for three weeks; Group 2 (control) consisted of 25 rats who were injected with 0.9% NaCl solution according to the same scheme; and Group 3 including 30 rats in which APS was combined with the administration of the immunosuppressive agent FTY-720 (intraperitoneally 1 mg/kg of animal weight). Three weeks later, there was an operation conducted with the purpose of extraction of the bronchopulmonary complex. After extraction of the bronchopulmonary complex, its triple lavage was carried out with 0.9% NaCl solution. The material of the experimental study was a lavage liquid, in which the biophysical and biochemical parameters of the surfactant were studied. Langmuir–Blodgett method was used to identify static, minimal and maximal surface tension. These figures were used to identify integrative indicator reflecting surfactant characteristics – the Clements stability index. The fractional composition of surfactant phospholipids was determined by thin-layer chromatography. The results of our experiments showed that in antiphospholipid syndrome, there was a decline in the functional activity of the surfactant and a change in its biochemical composition. The introduction of FTY-720 normalized the parameters of the lung surfactant system, which were changed during the modeling of antiphospholipid syndrome.

Phospholipid composition and kinetics in different endobronchial fractions from healthy volunteers

BackgroundAlterations in surfactant phospholipid compositions are a recognized feature of many acute and chronic lung diseases. Investigation of underlying mechanisms requires assessment of surfactant phospholipid molecular composition and kinetics of synthesis and turnover. Such studies have recently become possible in humans due to the development of stable isotope labelling combined with advances in analytical methods in lipidomics. The objectives of this study are to compare phospholipid molecular species composition and phosphatidylcholine synthesis and turnover in surfactant isolated from various endobronchial compartments in healthy adults.MethodsHealthy adults (N = 10) were infused with methyl-D9-choline chloride and samples of induced sputum, tracheal wash and small volume bronchoalveolar lavage fluid were obtained subsequently at intervals up to 96 hours. Surfactant phospholipid composition and incorporation of stable isotope into surfactant phosphatidylcholine were determined by electrospray ionisation mass spectrometry.ResultsWhile molecular species compositions of phospholipids were similar for all three sample types, dipalmitoylphosphatidylcholine content was highest in lavage, intermediate in tracheal wash and lowest in sputum. Methyl-D9-choline incorporation into surfactant phosphatidylcholine was lower for sputum at 24 hours but reached equilibrium with other sample types by 48 hours. Fractional methyl-D9-dipalmitoylphosphatidylcholine incorporation for all sample types was about 0.5% of the endogenous composition. Lysophosphatidylcholine enrichment was twice than that of phosphatidylcholine.ConclusionsTracheal secretions may be of value as a surrogate to assess bronchoalveolar lavage fluid surfactant molecular composition and metabolism in healthy people. Despite minor differences, the phospholipid molecular composition of induced sputum also showed similarities to that of bronchoalveolar lavage fluid. Detailed analysis of newly synthesized individual phosphatidylcholine species provided novel insights into mechanisms of surfactant synthesis and acyl remodelling. Lysophosphatidylcholine methyl-D9 incorporation patterns suggest that these species are secreted together with other surfactant phospholipids and are not generated in the air spaces by hydrolysis of secreted surfactant phosphatidylcholine. Application into patient populations may elucidate potential underlying pathophysiological mechanisms that lead to surfactant alterations in disease states.

Does lung development differ in male and female fetuses?

ABSTRACTPreterm male infants have a higher incidence of morbidity and mortality due to respiratory insufficiency than females of the same gestational age. This male disadvantage could be due to differences in lung architecture; however, few studies have compared lung architecture in male and female fetuses during late gestation. Our principal objectives were to compare the morphology of the fetal lung and the maturity of the surfactant system in preterm male and female fetuses. Lungs from male (n = 9) and female (n = 11) fetal sheep were collected at 0.9 of term (131 days of the 145-day gestation) for morphological and molecular analyses. In separate groups, tracheal liquid was obtained from male (n = 9) and female (n = 9) fetuses at 0.9 of term for determination of surfactant phospholipid composition. We found no sex-related differences in body weight, lung weight, right lung volume, lung tissue and airspace fractions, mean linear intercept, septal crest density, septal thickness, the proportion of proliferating and apoptotic cells, and the percentages of collagen or elastin. The gene expression of surfactant protein -A, -B, -C, and -D and tropoelastin was similar between sexes. There were no differences in the proportion of the major phospholipid classes in the tracheal liquid between sexes; however there was a significantly higher percentage of the phospholipid species phosphatidylinositol 38:5 in males. The greater morbidity and mortality in preterm male lambs do not appear to be related to differences in lung structure or surfactant phospholipid synthesis before birth, but may relate to physiological adaptation to air-breathing at birth.

Effect of Long-Term Simulated Weightlessness on Surfactant and Water Balance in Mouse Lungs

Weightlessness produces adaptive and maladaptive changes in the respiratory system. We assessed the effects of 30-day antiorthostatic hanging as a model of microgravity on the water balance in the lungs and surface activity and phospholipid composition of pulmonary surfactant in C57Bl/6 mice. Long-term antiorthostatic hanging increased water content in the lungs and reduced surface-active properties of the surfactant. This was accompanied by an increase in the content of alveolar phospholipids and changes in their fractional composition (increase in the relative content of lysophosphatidylcholine and phosphatidylethanolamine).

Sex differences in cardiorespiratory transition and surfactant composition following preterm birth in sheep

Male preterm infants are at greater risk of respiratory morbidity and mortality than females but mechanisms are poorly understood. Our objective was to identify the basis for the "male disadvantage" following preterm birth using an ovine model of preterm birth in which survival of females is greater than males. At 0.85 of term, fetal sheep underwent surgery (11 female, 10 male) for the implantation of vascular catheters to monitor blood gases and arterial pressure. After cesarean delivery at 0.90 of term, lambs were monitored for 4 h while spontaneously breathing; lambs were then euthanized and static lung compliance measured. We analyzed surfactant phospholipid composition in amniotic fluid and in bronchoalveolar lavage fluid (BALF) taken at necropsy; we also analyzed surfactant protein (SP) expression in lung tissue. Before delivery male fetuses tended to have lower pH (P = 0.052) compared with females. One hour after delivery, males had significantly lower pH and higher arterial partial pressure of CO(2) (Pa(CO(2))), lactate, glucose, and mean arterial pressure than females. Two males died 1 h after birth. Static lung compliance was 37% lower in males than females (P < 0.05). In BALF, males had significantly more protein, a lower percentage of the phosphatidylcholine (PC) 32:0 (dipalmitoylphosphatidylcholine) and higher percentages of PC34:2 and PC36:2. There were no sex-related differences in lung architecture or expression of SP-A, -B, -C, and -D. The lower lung compliance in male preterm lambs compared with females may be due to altered surfactant phospholipid composition and function. These changes may compromise gas exchange and impair respiratory adaptation after male preterm birth.

Secretory Phospholipase A2-Mediated Depletion of Phosphatidylglycerol in Early Acute Respiratory Distress Syndrome

IntroductionSecretory phospholipases A2 (sPLA2) hydrolyze phospholipids in cell membranes and extracellular structures such as pulmonary surfactant. This study tests the hypothesis that sPLA2 are elevated in human lungs during acute respiratory distress syndrome (ARDS) and that sPLA2 levels are associated with surfactant injury by hydrolysis of surfactant phospholipids. MethodsBronchoalveolar lavage (BAL) fluid was obtained from 18 patients with early ARDS (<72 hours) and compared with samples from 10 healthy volunteers. Secreted phospholipase A2 levels were measured (enzyme activity and enzyme immunoassay) in conjunction with ARDS subjects’ surfactant abnormalities including surfactant phospholipid composition, large and small aggregates distribution and surface tension function. ResultsBAL sPLA2 enzyme activity was markedly elevated in ARDS samples relative to healthy subjects when measured by ex vivo hydrolysis of both phosphatidylglycerol (PG) and phosphatidylcholine (PC). Enzyme immunoassay identified increased PLA2G2A protein in the ARDS BAL fluid, which was strongly correlated with the sPLA2 enzyme activity against PG. Of particular interest, the authors demonstrated an average depletion of 69% of the PG in the ARDS sample large aggregates relative to the normal controls. Furthermore, the sPLA2 enzyme activity against PG and PC ex vivo correlated with the BAL recovery of in vivo PG and PC, respectively, and also correlated with the altered distribution of the large and small surfactant aggregates. ConclusionsThese results support the hypothesis that sPLA2-mediated hydrolysis of surfactant phospholipid, especially PG by PLA2G2A, contributes to surfactant injury during early ARDS.

Alcohol exposure during late gestation: multiple developmental outcomes in sheep.

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life.

Effects of prenatal ethanol exposure on the lungs of postnatal lambs

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.

Surfactant alterations in horses with recurrent airway obstruction at various clinical stages

To evaluate the phospholipid composition and function of surfactant in horses with recurrent airway obstruction (RAO) at various clinical stages and compare these properties with findings in horses without RAO. 7 horses with confirmed RAO and 7 without RAO (non-RAO horses). Pairs of RAO-affected and non-RAO horses were evaluated before, during, and after exposure to hay. Evaluations included clinical scoring, lung function testing, airway endoscopy, and bronchoalveolar lavage fluid (BALF) absolute and differential cell counts. Cell-free BALF was separated into crude surfactant pellet and supernatant by ultracentrifugation, and phospholipid and protein concentrations were determined. Phospholipid composition of crude surfactant pellets and surface tension were evaluated with high-performance liquid chromatography and a pulsating bubble surfactometer, respectively. Findings were compared statistically via mixed-effects, repeated-measures ANOVA. Total phospholipid concentration in BALF was lower in RAO-affected versus non-RAO horses at all sample collection times. In the RAO-affected group, total phospholipid concentration was lower during exposure to hay than before or after exposure. There were no significant differences in BALF protein concentration, percentages of phospholipid classes, or surface tension between or within groups of horses. All clinical stages of RAO-affected horses were characterized by low surfactant concentration in BALF. Exacerbation of RAO led to an additional decrease in surfactant concentration. Causes for low surfactant concentration in RAO-affected horses remain to be determined. Low phospholipid concentration may render RAO-affected horses more susceptible than unaffected horses to surfactant alterations and contribute to clinical disease status and progression.

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.

Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60-80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60-100% oxygen, whereas levels of T1alpha, a protein expressed by type I cells, were comparably increased in mice exposed to 40-100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to > or = 60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

Influence of Age on Surfactant Isolated from Healthy Horses Maintained on Pasture

Surfactant alterations are described in horses after exercise, anesthesia, and prolonged transport, in horses with recurrent airway obstruction, and in neonatal foals. The effect of horse age or bronchoalveolar lavage fluid (BALF) sample characteristics on surfactant is unknown. To evaluate surfactant phospholipid composition and function in healthy horses, and to investigate the influence of age and BALF sample characteristics on surfactant. Seventeen healthy horses 6-25 years of age maintained on pasture year-round. BALF was collected by standard procedures and was assessed for recovery volume, nucleated cell count (NCC), and cytology. Cell-free BALF was separated into crude surfactant pellet (CSP) and surfactant supernatant (Supe) by ultracentrifugation. Phospholipid and protein content were determined from both fractions. CSP phospholipid composition was analyzed by high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was evaluated with a pulsating bubble surfactometer. Regression analysis was used to evaluate associations between age, BALF sample characteristics, and surfactant variables. Results and conclusions were derived from 15 horses. Increasing age was associated with decreased phospholipid content in CSP but not Supe. Age did not affect protein content of CSP or Supe, or surfactant phospholipid composition or function. Age-related surfactant changes were unaffected by BALF recovery percentage, NCC, and cytological profile. Older horses have decreased surfactant phospholipid content, which might be because of age-related pulmonary changes. Surfactant composition is unaffected by BALF sample characteristics at a BALF recovery percentage of at least 50%.

Alterations of the composition and metabolism of pulmonary surfactant phospholipids induced by experimental peritonitis in rats

Pulmonary complications often accompany the development of acute peritonitis. In this study, we analyzed the alterations of alveolar surfactant phospholipids in rats with experimentally induced peritonitis. The results showed a reduction of almost all phospholipid fractions in pulmonary surfactant of experimental animals. The most abundant alveolar phospholipids—phosphatidylcholine and phosphatidylglycerol were reduced significantly in surfactant of rats with experimental peritonitis. In addition, analysis of the fatty acid composition of these two phospholipids revealed marked differences between experimental and control animals. The activity of phospholipase A2, which is localized in the hydrophyllic phase of alveolar surfactant, was higher in rats with experimental peritonitis compared to sham-operated ones. Also, a weak acyl-CoA:lysophospholipid acyltransferase activity was detected in alveolar surfactant of rats with experimental peritonitis, whereas in control animals this activity was not detectable. The lipid-transfer activity was quite similar in pulmonary surfactant of control and experimental rats. The total number of cells and the percentage of neutrophils were strongly increased in broncho-alveolar lavage fluid from rats with peritonitis. Thus, our results showed that the development of peritonitis was accompanied by pulmonary pathophysiological processes that involved alterations of the phospholipid and fatty acid composition of alveolar surfactant. We suggest that the increased populations of inflammatory cells, which basically participate in internalization and secretion of surfactant components, contributed to the observed alterations of alveolar phospholipids. These studies would be useful for clarification of the pathogenic mechanisms underlying the occurrence of pulmonary disorders that accompany acute inflammatory conditions, such as peritonitis and sepsis.

Combined activities of secretory phospholipases and eosinophil lysophospholipases induce pulmonary surfactant dysfunction by phospholipid hydrolysis

Surfactant dysfunction is implicated in small airway closure in asthma. Increased activity of secretory phospholipase A(2) (sPLA(2)) in the airways is associated with asthma exacerbations. Phosphatidylcholine, the principal component of pulmonary surfactant that maintains small airway patency, is hydrolyzed by sPLA(2). The lysophosphatidylcholine product is the substrate for eosinophil lysophospholipases. To determine whether surfactant phospholipid hydrolysis by the combined activities of sPLA(2)s and eosinophil lysophospholipases induces surfactant dysfunction. The effect of these enzymes on surfactant function was determined by capillary surfactometry. Thin layer chromatography was used to correlate enzyme-induced changes in surfactant phospholipid composition and function. Phosphatidylcholine and its hydrolytic products were measured by using mass spectrometry. Eosinophils express a 25-kd lysophospholipase and group IIA sPLA(2). Phospholipase A(2) alone induced only a small decrease in surfactant function, and 25-kd lysophospholipase alone degraded lysophosphatidylcholine but had no effect on surfactant function. The combined actions of sPLA(2) and lysophospholipase produced dose-dependent and time-dependent losses of surfactant function, concomitant with hydrolysis of phosphatidylcholine and lysophosphatidylcholine. Lysates of AML14.3D10 eosinophils induced surfactant dysfunction, indicating these cells express all the necessary lipolytic activities. In contrast, lysates of blood eosinophils required exogenous phospholipase A(2) to induce maximal surfactant dysfunction. The combined activities of sPLA(2)s and eosinophil lysophospholipases are necessary to degrade surfactant phospholipids sufficiently to induce functional losses in surfactant activity as reported in asthma. The phospholipases and lysophospholipases expressed by eosinophils or other airway cells may represent novel therapeutic targets for blocking surfactant degradation, dysfunction, and peripheral airway closure in asthma.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Surfactant Composition and Function in Patients with ABCA3 Mutations

Mutations in the gene encoding the ATP binding cassette transporter member A3 (ABCA3) are associated with fatal surfactant deficiency. ABCA3 lines the limiting membrane of lamellar bodies within alveolar type-II cells, suggesting a role in surfactant metabolism. The objective of this study was to determine the surfactant phospholipid composition and function in patients with mutations in the ABCA3 gene. Bronchoalveolar lavage (BAL) fluid was analyzed from three groups of infants: 1) Infants with ABCA3 mutations, 2) infants with inherited surfactant protein-B deficiency (SP-B), and 3) patients without parenchymal lung disease (CON). Surfactant phospholipid profile was determined using two-dimensional thin-layer chromatography, and surface tension was measured with a pulsating bubble surfactometer. Phosphatidylcholine comprised 41 +/- 19% of the total phospholipid in the BAL fluid of the ABCA3 group compared with 78 +/- 3% and 68 +/- 18%, p = 0.008 and 0.05, of the CON and SP-B groups, respectively. Surface tension was 31.5 +/- 9.3 mN/m and was significantly greater than CON but no different from SP-B. We conclude that mutations in ABCA3 are associated with surfactant that is deficient in phosphatidylcholine and has decreased function, suggesting that ABCA3 plays an important role in pulmonary surfactant phospholipid homeostasis.

Lung Surfactant Function and Composition in Neonatal Foals and Adult Horses

Lung surfactant function and composition are varied and adapted to the specific respiratory physiology of all mammalian species. Lung surfactant function and composition are different in neonatal foals as compared to adult horses. Six adult horses, 7 term foals (<24 hours old), and 4 premature foals were used. Animals were part of the Auburn University teaching herd except for 3 client-owned premature foals. Bronchoalveolar lavage fluid (BALF) was obtained from all animals. Ultracentrifugation of cell-free BALF separated surfactant into crude surfactant pellets (CSP) and supernatant. Both fractions were analyzed for phospholipid and protein content with the Bartlett and bicinchoninic acid method, respectively. Phospholipid composition of the CSP was determined by using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of the CSP was measured with a pulsating bubble surfactometer. Results from term foals (<24 hours old) were compared statistically to those from adult horses. Values of P < .05 were considered significant. BALF phospholipid content was similar between adult horses and term foals, but BALF protein content was significantly decreased in term foals. Phosphatidylglycerol was significantly decreased, phosphatidylinositol was significantly increased, and the minimum surface tension was significantly increased in the CSP from term foals compared to adult horses. Surface tension and phospholipid composition of surfactant in neonatal foals are significantly different compared to adult horses. These changes may influence biophysical and immunologic functions of surfactant.

Reversibility of lung inflammation caused by SP-B deficiency

Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1beta, and macrophage inflammatory protein-2 concentrations were increased after removal of doxycycline, indicating pulmonary inflammation. Restoration of SP-B expression following administration of doxycycline rapidly reversed SP-B-dependent abnormalities in lung mechanics and inflammation. SP-B deficiency is sufficient to cause lung dysfunction and inflammation in adult mice. SP-B reversed inflammation and maintained lung function in vivo, indicating its potential utility for the prevention and treatment of pulmonary injury and surfactant deficiency.