A field study of the ventilatory response to ambient temperature and pressure in sport diving

We measured alveolar-to-vascular leakage of surfactant protein A (SP-A) in immature newborn rabbits delivered at a gestational age of 27 days. Experimental animals received, via a tracheal cannula, 2 ml/kg of a mixture of modified porcine surfactant (Curosurf, 80 mg/ml) and human recombinant SP-A (4 mg/ml). Littermate controls received the same volume of human SP-A in saline (4 mg/ml). After 30 min of artificial ventilation with a frequency of 40/min and an inspiration time of either 0.75 or 0.45 s, blood was sampled from the right ventricle and the lungs were lavaged. The content of human SP-A in serum and lung lavage fluid was determined with ELISA kits, and the alveolar-to-vascular leak expressed as the quotient of total SP-A in serum and lavage fluid. The leak in control animals amounted to about 2% of SP-A in lung wash and was several times higher in these animals than in those receiving surfactant. The leak was of the same order irrespective of whether the animals were ventilated with long or short inspiration time. We speculate that serum levels of SP-A may reflect the degree of lung injury in various forms of respiratory failure.     

Site specificity of surfactant protein expression in airways of baboons during gestation

BACKGROUND: There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP-A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology. METHODS: In situ hybridization studies were done to determine the distribution of SP-A, SP-B, and SP-C in baboon lung specimens from 60, 90, 120, 140, 160, and 180 (term) days of gestation and adults. Lungs from gestation controls were obtained at the time of hysterotomy and adult lungs at necropsy. Riboprobes used for in situ hybridization contained the entire coding regions for human SP-A, SP-B, and SP-C. RESULTS: At 60 days, SP-C mRNA expression was evident in focal portions of primitive tubular epithelium but not bronchi. This distal pattern of SP-C mRNA expression persisted and was present in some epithelial cells of respiratory bronchioles at term. At 90 days, SP-A mRNA expression was present in the epithelium of trachea and large bronchi. SP-B mRNA expression was found in small bronchi, bronchioles, and distal tubular epithelium at 120 days of gestation. SP-A mRNA bronchiolar localization became evident at 140 days of gestation and alveolar type 2 cellular expression at 160 days of gestation. Abrupt transitions of surfactant protein expression were identified (e.g., SP-A mRNA-positive cells in the epithelium of large bronchi with adjoining SP-B mRNA expression in small bronchi and bronchioles). CONCLUSIONS: Findings in the baboon indicate that there are well-delineated sites of surfactant protein mRNA expression in bronchial and bronchiolar epithelia. mRNA expressions of SP-A and SP-B are present in both bronchial and bronchiolar epithelium but at different sites, whereas SP-C expression is seen in loci of epithelial cells in respiratory bronchioles.     

Immunohistochemical distribution of surfactant apoproteins in hypoplastic lungs of nonimmunologic hydrops fetalis

Forty-three patients with nonimmunologic hydrops fetalis (NIHF), including 32 patients (74%) with hypoplastic lung, were immunohistochemically examined for the expression of surfactant apolipoproteins (SPs), using anti-gamma G immunoglobulins against human SP-A with a molecular weight (MW) of 35 K and SP-B with a MW of 5 K compared with that in 59 patients in a control group and 45 patients with hypoplastic lung induced by causes other than NIHF. In the control group, SP-A was expressed in the lungs from 23 gestational weeks and became more numerous and intense in alveolar type II cells after 31 gestational weeks, whereas SP-B began to be expressed from 20 gestational weeks, and almost all patients showed a diffuse positivity after 26 gestational weeks. In the NIHF group, SP-A expression was generally weak, even after 31 gestation weeks. Moreover, most of the patients showing a weak expression of SP-A were also associated with hypoplastic lung and had a clinical history of persistent intrauterine pleural effusion of more than 2 weeks. Conversely, the immunoreactivity of SP-B was well preserved in NIHF cases either with or without hypoplastic lung. These results suggest that in the NIHF lung, there is a possible delay in the functional maturation or development of SP-A synthesis by alveolar type II cells, and this retardation of the functional maturation in type II cells also participates in the postnatal respiratory insufficiency in NIHF.     

Surfactant proteins A and D: disease markers

The abundant and restricted expression of surfactant proteins SP-A and SP-D within the lung makes these collectins specific markers for lung diseases. The measurement of SP-A and SP-D in amniotic fluids and tracheal aspirates reflects lung maturity and the production level of the lung surfactant in infants with respiratory distress syndrome (RDS). The SP-A concentrations in bronchoalveolar lavage (BAL) fluids are significantly decreased in patients with acute respiratory distress syndrome (ARDS) and also in patients at risk to develop ARDS. The prominent increase of these proteins in BAL fluids and sputum is diagnostic for pulmonary alveolar proteinosis (PAP). The concentrations of SP-A and SP-D in BAL fluids from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia with collagen vascular diseases (IPCD) are rather lower than those in healthy controls and the SP-A/phospholipid ratio may be a useful marker of survival prediction. SP-A and SP-D appear in the circulation in specific lung diseases. Their serum concentrations significantly increase in patients with PAP, IPF and IPCD. The successive monitoring of serum levels of SP-A and SP-D may predict the disease activity. The serum SP-A levels increase in patients with ARDS. SP-A is also a marker for lung adenocarcinomas and can be used to differentiate lung adenocarcinomas from other types and metastatic cancers from other origins, and to detect metastasis of lung adenocarcinomas.     

Surfactant protein A (SP-A) gene targeted mice

Mice lacking surfactant protein A (SP-A) mRNA and protein in vivo were generated using gene targeting techniques. SP-A (-/-) mice have normal levels of SP-B, SP-C and SP-D mRNA and protein and survive and breed normally in vivarium conditions. Phospholipid composition, secretion and clearance, and incorporation of phospholipid precursors are normal in the SP-A (-/-) mice. Lungs of SP-A (-/-) mice have markedly decreased tubular myelin figures and clear Group B streptococci and Pseudomonas aeruginosa less efficiently than SP-A wild type mice. These studies of SP-A (-/-) mice demonstrate that SP-A has an important role in the innate immune system of the lung in vivo.     

Exposure to crystalline silica or treatment with chlorphentermine increases vitamin E levels in rat alveolar lavage materials

Previous studies have shown that vitamin E may be an integral part of lung surfactant and may function to protect this material from oxidant damage. Therefore, we measured the vitamin E levels in alveolar lavage materials from rats exposed to crystalline silica or treated with chlorphentermine (CP), two treatments that are known to increase surfactant phospholipids (PL) by different mechanisms. Silica exposure leads to increased PL synthesis, and CP treatment causes a reduction in PL degradation. Two different silica preparations, HCL-washed and unwashed silica, were used because exposure to each of them leads to different degrees of phospholipidosis. Exposure to HCL-washed silica results in a more than 17-fold increase in lavage PL and protein levels and a 12.2-fold increase in the amount of vitamin E. Exposure to unwashed silica leads to an approximately 7-fold increase in PL and proteins and a 5.8-fold increase in lavage vitamin E. Following treatment of rats with CP, there is a 15- to 19-fold increase in lavage PL and proteins and a 13.6-fold increase in vitamin E. When the results are expressed as micrograms vitamin E per milligram of lavage PL or protein, there is not much difference between controls and each treatment group. Because surfactant synthesis occurs in the endoplasmic reticulum, we also measured vitamin E in lung microsomes. Both silica exposure and CP treatment also lead to 1.8- to 2.5-fold increases, respectively, in the lung microsomal levels of vitamin E. These results demonstrate that alveolar lavage vitamin E levels are elevated along with lavage PL and proteins, and lung microsomal vitamin E levels are increased following exposure of rats to silica or treatment of the animals with CP.     

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.     

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha

Pulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration. Dipalmitoylphosphatidylcholine has been identified as the most important component for lowering surface tension at the air-liquid interface. Hydrophobic surfactant apoproteins, SP-B and SP-C, play essential roles in the biophysical functions of the surfactant phospholipids. Hydrophilic surfactant apoproteins (SP-A and SP-D) that are members of C-type lectin superfamily, interact with phospholipids and glycolipids and modulate host defense functions in the lung. SP-A also plays an important role in regulating phospholipid homeostasis in the alveolar spaces. Recent advances in genetics and molecular biology have clarified the structure-function relationship of surfactant apoproteins.     

Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes

Congenital alveolar proteinosis is a recently described cause of lung dysfunction and respiratory distress in term neonates. In several cases a deficiency or insufficiency of surfactant apoprotein B (SP-B) has been caused by a frameshift mutation in the gene encoding SP-B. Five full-term children in three unrelated families from The Netherlands are reported. Immunohistochemistry demonstrated large amounts of surfactant proteins A and C (SP-A and SP-C) and precursors in alveolar cells and in intra-alveolar material. Results were positive for antibovine SP-B antibody but negative for antipig SP-B1 antibody, most probably reflecting differences in the antibody specificity. The findings suggest abnormal SP-B function. In two sibs, no pre-SP-C was demonstrated in the alveoli, although it was found in considerable amounts in alveolar cells. One such case has previously been reported. In two families, the parents were heterozygous for the 121 ins 2 mutation in the SP-B gene. Our findings suggest that congenital alveolar proteinosis may result from abnormalities in one or more of the surfactant proteins.     

Short-term ozone exposure affects the surface activity of pulmonary surfactant

The effects of short-term ozone exposure on the lung function and surface activity of surfactant subtypes isolated from rat lung lavage were studied. Rats were exposed to 0.8 ppm ozone for 2 or 12 hr. The surface activity of surfactant was affected by ozone exposure, whereas distinct morphological changes in bronchoalveolar lavage or in the surfactant subtypes were not observed. Adsorption experiments indicated that bronchoalveolar lavage from rats exposed for 12 hr to ozone remained at lower equilibrium surface pressures than lavage from control rats. These observations suggest interference of inflammatory proteins with the surface film. Extracted surfactant, containing only lipids and surfactant proteins B and C, had a decreased adsorption rate after ozone exposure. These results suggest that the activity of one or both of the hydrophobic surfactant proteins (SP-B and SP-C) was affected by ozone.     

Effect of acidic pH on the structure and lipid binding properties of porcine surfactant protein A. Potential role of acidification along its exocytic pathway

Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules (lamellar bodies) together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C). We asked whether the progressive decrease in pH along the exocytic pathway could influence the secondary structure and lipid binding and aggregation properties of porcine SP-A. Conformational analysis from CD spectra of SP-A at various pH values indicated that the percentage of alpha-helix progressively decreased and that of beta-sheet increased as the pH was reduced. The protein underwent a marked self-aggregation at mildly acidic pH in the presence of Ca2+, conditions thought to resemble those existing in the trans-Golgi network. Protein aggregation was greater as the pH was reduced. We also found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation. However, the binding of acidic but not neutral vesicles to SP-A led to 1) a striking change in the CD spectra of the protein, which was interpreted as a decrease of the level of SP-A self-aggregation, and 2) a protection of the protein from endoproteinase Glu-C degradation at pH 4.5. SP-A massively aggregated acidic vesicles but poorly aggregated neutral vesicles at acidic pH. Aggregation of dipalmitoylphosphatidylcholine (DPPC) vesicles either with or without SP-B and/or SP-C strongly depended on pH, being progressively decreased as the pH was reduced and markedly increased when pH was shifted back to 7.0. At the pH of lamellar bodies, SP-A-induced aggregation of DPPC vesicles containing SP-B or a mixture of SP-B and SP-C was very low, although SP-A bound to these vesicles. These results indicate that 1) DPPC binding and DPPC aggregation are different phenomena that probably have different SP-A structural requirements and 2) aggregation of membranes induced by SP-A at acidic pH is critically dependent on the presence of acidic phospholipids, which affect protein structure, probably preventing the formation of large aggregates of protein.     

Elevated levels of lung surfactant protein A in sera from patients with idiopathic pulmonary fibrosis and pulmonary alveolar proteinosis

An enzyme-linked immunosorbent assay using monoclonal antibodies to human lung surfactant protein A (SP-A) was applied to sera from patients with lung diseases. We examined whether SP-A appears in the sera of patients with diseases that are known to cause alterations in surfactant composition in bronchoalveolar lavage fluids, and we characterized the SP-A that was found. The level of SP-A in sera from 57 healthy volunteers was 45 +/- 3 ng/ml (mean +/- SEM). The levels in patients with idiopathic pulmonary fibrosis (IPF) (205 +/- 23 ng/ml, n = 32) and pulmonary alveolar proteinosis (PAP) (285 +/- 23 ng/ml, n = 6) were significantly higher than those in healthy control subjects (p < 0.01), whereas those of sarcoidosis (n = 16), pneumonia (n = 14), and tuberculosis (n = 14) were 52 +/- 27 ng/ml, 65 +/- 11 ng/ml, and 49 +/- 23 ng/ml, respectively. Electrophoresis and immunoblotting analysis demonstrated that the fraction isolated from serum of a patient with PAP or IPF by anti-SP-A immunoaffinity column chromatography consisted chiefly of human IgG and IgM, and that it also contained SP-A. Furthermore, IgG was found in preparation of purified human SP-A. SP-A was demonstrated to bind to nonimmune IgG coated onto microtiter wells. Gel filtration analysis revealed that serum SP-A was eluted at fractions of larger molecular size than was the purified SP-A. These findings suggest that SP-A appears in the bloodstream as a complex with immunoglobulin in IPF and in PAP.     

Lung surfactant in a cystic fibrosis animal model: increased alveolar phospholipid pool size without altered composition and surface tension function in cftrm1HGU/m1HGU mice

BACKGROUND: Progressive pulmonary dysfunction is a characteristic symptom of cystic fibrosis (CF) and is associated with functional impairment and biochemical alterations of surfactant phospholipids in the airways. However, the fundamental question of whether surfactant alterations in the CF lung are secondary to the pulmonary damage or are present before initiation of chronic infection and inflammation has yet to be resolved in patients with cystic fibrosis but can now be addressed in CF mice that exhibit the basic defect in the airways. A study was therefore undertaken to investigate the pool sizes, composition, and function of lung surfactant in the non-infected cftrm1HGU/m1HGU mouse. METHODS: The amount and composition of phospholipid classes and phosphatidylcholine molecular species were determined in bronchoalveolar lavage (BAL) fluid and lavaged lungs by high performance liquid chromatography (HPLC). Surfactant protein A (SP-A) levels in BAL fluid were determined by ELISA and surfactant for functional measurements was isolated from BAL fluid by differential ultracentrifugation. Equilibrium and minimal surface tension of surfactant was assessed by the pulsating bubble surfactometer technique. MF1, BALB/c, C57/BL6, and C3H/He mice served as controls. RESULTS: BAL fluid of cftrm1HGU/m1HGU mice contained 1.02 (95% confidence interval (CI) 0.89 to 1.16) mumol phospholipid and 259 (239 to 279) ng SP-A. BAL fluid of MF1, BALB/c, C57BL/6, and C3H/He mice contained 0.69 (0.63 to 0.75), 0.50 (0.42 to 0.57), 0.52 (0.40 to 0.64), and 0.45 (0.27 to 0.63) mumol phospholipid, respectively. After correction for the different body weights of mouse strains, phospholipid levels in BAL fluid of cftrm1HGU/m1HGU mice were increased by 64 (52 to 76)%, 60 (39 to 89)%, 72 (45 to 113)%, and 92 (49 to 163)%, respectively, compared with controls. The amount of SP-A in BAL fluid and the composition of phospholipid as well as phosphatidylcholine molecular species in BAL fluid and lung tissue was unchanged in cftrm1HGU/m1HGU mice compared with controls. The increase in phospholipids in BAL fluid of cftrm1HGU/m1HGU mice resulted from an increased fraction of large aggregates which exhibited normal surface tension function. CONCLUSION: In cftrm1HGU/m1HGU mice surfactant homeostasis is perturbed by an increased phospholipid pool in the alveolar compartment.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca(2+), of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline beta-deuterons but had little effect on choline alpha-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Novel bacterial rhodopsins from Haloarcula vallismortis

Regeneration of alveolar epithelial cells is one of the important repair processes in many types of lung injury, including the adult respiratory distress syndrome (ARDS). We have examined the effects of growth factors and cytokines on alveolar epithelial cells in vitro, and also examined histopathology and surfactant protein gene expressions in alveolar epithelial cells in rats with endotoxin-induced lung injury. Keratinocyte growth factor only induced a marked increase in levels of mRNAs for SP-A and SP-B, accompanied by an increase in SP-A protein. Intratracheal administration of endotoxin induced the marked proliferation of alveolar type II cells in association with the increased surfactant protein mRNAs, and SP-A production. Proliferation and differentiation of alveolar epithelial cells, and KGF may play important roles of repair process of the damaged alveoli after acute lung injury.     

Differential effects in vivo of thyroid hormone on the expression of surfactant phospholipid, surfactant protein mRNA and antioxidant enzyme mRNA in fetal rat lung

Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significantly increased TPL and DSPC content, and significantly decreased the expression of SP-A, SP-C, CuZnSOD, and catalase genes. Because of a crucial interplay of these factors for normal lung function at the time of birth, the molecular mechanisms by which these apparently opposing changes are accomplished warrant further investigation.     

Method of purification affects some interfacial properties of pulmonary surfactant proteins B and C and their mixtures with dipalmitoylphosphatidylcholine

Two methods were employed for preparation of lipid extracts from porcine lung surfactant. Pulmonary surfactant proteins SP-B and SP-C were isolated from the extracts using gel-exclusion chromatography on LH-60 with chloroform:methanol acidified with hydrochloric acid. Monolayers of pure SP-B or SP-C isolated from butanol lipid extracts spread at the air-water interface showed larger molecular areas than those determined in films of SP-B or SP-C isolated from chloroform surfactant extracts. Aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) supplemented with 2.5 and 5.0 wt% of SP-B or SP-C obtained from butanol extracts adsorbed faster to the air-water interface than their counterparts reconstituted with proteins isolated from chloroform extracts. Surface pressure-area characteristics of spread monolayers of DPPC plus SP-B or SP-C did not depend on the method of isolation of the proteins. The diagrams of the mean molecular areas vs. composition for the monolayers of DPPC plus SP-B or SP-C showed positive deviations from the additivity rule, independently of the procedure used for preparation of lipid extract surfactant. Matrix-assisted laser desorption/ionization spectrometry of the proteins isolated from different extraction solvents was consistent with some differences in the chemical compositions of SP-Bs. Butylation of SP-B during extraction of surfactant pellet with butanol may account for the differences observed in the molecular masses of SP-Bs isolated by the two different extraction protocols. The study suggests that the method of purification of SP-B and SP-C may modify their ability to enhance the adsorption rates of DPPC/protein mixtures, and this may be relevant to the formulation of protein-supplemented lipids for exogenous treatment of pulmonary surfactant insufficiency.     

Neurobiological basis of creativity

We have used a previously described model of bilateral radiation-induced lung disease in the rat (Ward et al., Radiat. Res., 136, 15-21, 1993) to study the role of hyaluronan in this process. Hyaluronan was measured in the bronchoalveolar lavage fluid, serum and lung tissue of rats after gamma irradiation or sham irradiation. Four weeks after irradiation, during peak alveolitis (12-fold increase in protein in the lavage, 7-fold increase in lavaged cells) hyaluronan was elevated 5.5-fold in serum and 1.5-fold in the bronchoalveolar lavage fluid. Histochemical staining demonstrated hyaluronan was in the intra-alveolar edema fluid but was not increased in the alveolar walls; hyaluronan, measured by high-performance liquid chromatography, also was not elevated in lavaged lung tissue. Hyaluronan was not increased in bron-choalveolar lavage fluid, serum or lung tissue during pulmonary edema (2 weeks) or fibrosis (6 to 20 weeks). The administration of methylprednisolone significantly decreased the alveolitis, including the increase in hyaluronan in the alveolar space and serum, but did not suppress fibrosis. It appears that hyaluronan is a marker of inflammation and cannot be used as a serum marker to predict the onset of radiation pneumonitis. Furthermore, an increase in interstitial hyaluronan does not appear to be a necessary precursor in the evolution of radiation fibrosis.