Studies on pulmonary surfactant. Effects of cortisol administration to fetal rabbits on lung phospholipid content, composition and biosynthesis.

Corticosteroids are known to accelerate maturation of the fetal lung and production of surfactant. We examined the effect of cortisol administration to fetal rabbits on the phospholipid content and composition of lung lavage and lung tissue, as well as on the activities of enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol, the major surface-active components of surfactant. Cortisol was administered by intrauterine injection at 25 days' gestation and the fetuses were delivered at 27 days (full term, 31 days). Saline-injected fetuses, littermates of the cortisol-treated as well as non-littermates, were used as controls. The amount of phospholipid in lung lavage from the hormone-treated fetuses was almost double that of the saline-injected controls and was similar to that of an untreated fetus of more than 30 days' gestation. Similarly, the phospholipid composition of lung lavage from the hormone-treated fetuses was similar to that of an untreated fetus at a greater gestational age. These data, therefore, suggest that cortisol acts by accelerating physiological development. Cortisol administratration stimulated the activity of cholinephosphate cytidylyltransferase and lysolecithin acyltransferase to a small, but statistically significant extent. This is also consistent with an acceleration of normal development. The stimulation of lysolecithin acyltransferase is of interest, since this enzyme is believed to be involved in the synthesis of dipalmitoylglycerophosphocholine, the major surface-active species of phosphatidylcholine. Cortisol administration had no effect on the activities of pulmonary choline kinase, cholinephosphotransferase, lysophosphatidic acid acyltransferase and glycerolphosphate phosphatidyltranferase, although we have previously shown the latter enzyme to be stimulated following a longer period of exposure to the hormone. Saline injection produced some maturational effects presumably as a result of stress, which may be mediated by corticosteroids or other hormones.     

Effects of oligohydramnios on lung growth and maturation in the fetal rat

Oligohydramnios (OH) retards fetal lung growth by producing less lung distension than normal. To examine effects of decreased distension on fetal lung development, we produced OH in rats by puncture of uterus and fetal membranes at 16 days of gestation; fetuses were delivered at 21 or 22 days of gestation. Controls were position-matched littermates in the opposite uterine horn. OH lungs had lower weights and less DNA, protein, and water, but no differences in saturated phosphatidylcholine, surfactant proteins (SP)-A and -B, and mRNA for SP-A, -B, -C, and -D. To evaluate effects on epithelial differentiation, we used RTI(40) and RTII(70), proteins specific in lung to luminal surfaces of alveolar type I and II cells, respectively. At 22 days of gestation, OH lungs had less RTI(40) mRNA (P < 0.05) and protein (P < 0.001), but RTII(70) did not differ from controls. With OH, type I cells (in proportion to type II cells) covered less distal air space perimeter (P < 0.01). We conclude that OH, which retards lung growth, has little effect on surfactant and impedes formation of type I cells relative to type II cells.     

The influence of oligohydramnios on thoracic dimensions of fetal sheep.

Oligohydramnios frequently leads to lung hypoplasia in the fetus, but the underlying mechanisms are incompletely understood. Our aim was to determine the effects of oligohydramnios on the dimensions of the fetal thorax. Using pairs of implanted ultrasound transducers in 6 fetal sheep, we measured 4 thoracic dimensions (transverse, anterior-posterior, manubrium to left and right hand sides of the diaphragmatic dome) for 2 control days, 3 days of amniotic and allantoic fluid drainage (oligohydramnios), and 2 days after the return of drained fluids. The effect of oligohydramnios, which began at 121-2 days of gestation (term being c.145 days), on each dimension was quantified daily as the difference between the measured value and the value predicted from the growth of that dimension over the study period. Oligohydramnios led, within 48 hours, to significant reductions in the transverse dimension (5.9-6.1%) and in the distance between the manubrium and the dome of the diaphragm (1.7-2.2%). There was no change in the anterior-posterior dimension. We conclude that oligohydramnios causes alterations, within 48 hours, in the dimensions of the fetal thorax which can be reversed, at least partially, by re-expansion of the fluid sacs. These changes, which are expected to produce reductions in thoracic volume, may, if prolonged, lead to lung hypoplasia.     

The effect of betamethasone and fetal sex on the synthesis and maturation of lung surfactant phospholipids in rabbits

In the present study we investigated the maturation of the surfactant phospholipids and the role of fetal sex on the effect of betamethasone in male and female rabbit fetuses. Betamethasone was administered to the doe (0.2 mg/kg intramuscularly) 42 and 18 h prior to killing. The fetuses were studied at 27 and 28 days from conception. Results from the alveolar lavage show that male fetuses tended to have a lower disaturated phosphatidylcholine/sphingomyelin ratio and lower levels of phosphatidylinositol. Phosphatidylglycerol was detected in trace amounts. This was apparently due to the high extracellular levels of myo-inositol inhibiting the synthesis of surfactant phosphatidylglycerol while increasing the synthesis of surfactant phosphatidylinositol. Betamethasone increased the recovery of disaturated phosphatidylcholine and phosphatidylinositol from the lung lavage in both sexes. As studied in lung slices in vitro, the betamethasone treatment decreased the incorporation of glucose into phospholipids, including into the fatty acid moiety of disaturated phosphatidylcholine, although it had no significant effect on the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. However, the addition of palmitate increased the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. The betamethasone treatment did not increase the incorporation of [1-14C]pyruvate into disaturated phosphatidylcholine. Following betamethasone administration, the availability of fatty acids may become rate-limiting for the synthesis of surfactant phospholipids. Betamethasone increased the activities of phosphatidic acid phosphohydrolase and phosphatidate cytidyltransferase in a fraction of microsomal membranes. The present evidence suggests that the glucocorticoid-induced lung maturation and the maturation of the normal lung are associated with an increase in the activity of the enzymes which are involved in metabolizing phosphatidic acid to neutral and acidic surfactant secretion of the male fetus was not explained by possible sex-related differences in the biosynthesis of the phospholipids.     

Clearance of 99mTc-DTPA and experimentally increased alveolar surfactant content.

We measured clearance of 99mTc-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) in rabbits with experimentally increased alveolar surfactant content. In one group of animals, surfactant production was increased by treatment with ambroxol, and another group of animals was treated with tracheal instillation of natural surfactant. A group of untreated control animals and animals treated with instillation of saline were also studied. Clearance was measured during standard conditions of mechanical ventilation and during ventilation with large tidal volumes. In ambroxol- and surfactant-treated groups, clearance rate was reduced compared with untreated control animals. In contrast, clearance rate increased after saline instillation. The differences were observed at both modes of ventilation. The findings indicate that the pulmonary surfactant system is a rate-limiting factor for the clearance of 99mTc-DTPA and that the volume dependence of clearance is not explained by stretching of the alveolar wall only.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Studies on the fate of pulmonary surfactant in the lung.

1. Radioactively labelled pulmonary surfactant was prepared in an isolated perfused lung system provided with [14C]hexadecanoate. 2. After intratracheal administration of pulmonary surfactant radioactively labelled components were rapidly distributed into different lung fractions, including macrophages (free cells), but most of the radioactive label was accumulated by the lung tissue. 3. Alveolar macrophages, maintained in a variety of culture media in the presence and absence of mineral particles, incorporated a low percentage (11%) of radioactively labelled components when incubated with the surfactant, although evolution of labelled CO2 (6% of the original total activity) suggested that some breakdown of the components had taken place. 4. In similar cultures little intracellular accumulation or extracellular release of non-esterified fatty acids was demonstrated, indicating minimal catabolism of the high-molecular-weight lipid components of surfactant (particularly phosphatidylcholine). 5. However, experiments in vitro designed to simulate the lysosomal degradation of endocytosed surfactant indicated that the macrophage had enzymes capable of releasing non-esterified fatty acids, particularly hexadecanoate, from the lipoprotein complex. 6. It is argued that lung cells, other than alveolar macrophages, may also have a role in surfactant turnover.     

Differentiation of the pulmonary surfactant system. Disaturated phosphatidylcholine accumulation in fetal rat lung in vivo and in vitro

Fetal rat lung was placed in organ culture at 15 days gestation (22 days total gestation period), before biochemical and morphological development of the pulmonary surfactant system. At the fifth day of culture numerous Type II cells containing lamellar bodies were present as determined by electron micrography. Phospholipid accumulation in the cultures increased abruptly beginning at 6 days in culture. The phospholipid which accumulated between the sixth and twelfth culture days was composed of 21--27% disaturated phosphatidylcholines. Both the percent of disaturated phosphatidylcholines in the phospholipid fraction and the qualitative pattern of accumulation as a function of time were similar to observations for fetal rat lung developing in vivo. The data presented provide evidence for development of the pulmonary surfactant system in organ culture in vitro.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

Ultrastructural study of mycobacteria in experimentally produced lung lesions of mice.

Mice were infected by iv injection with a virulent strain of Mycobacterium bovis, Ravenel strain, to prepare specimens for electron microscopical observation of their intracellular morphology. Observation was made with ultra-thin sections of the granulomatous lungs at an advanced stage of infection. Many apparently intact bacterial cells were found intracellulary, and the majority of them had lipoidal inclusions enclosed by a membranous structure. Several layers of mycobacterial cell wall were discernible, including a fairly wide space of the electron-transparent zone just beneath the electrondense outmost layer. Mesosomes, nuclear material, small dense granules and cross wall were found in almost the same appearance as those reported of mycobacteria grown in vitro. The bacilli were located mainly within intact or damaged phagosomes which were often filled with amorphous material of various electron densities.     

Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep

Reduced amniotic fluid volume often results in fetal lung hypoplasia. Our aim was to examine the effects of prolonged drainage of amniotic and allantoic fluids on lung liquid volume (Vl), secretion rate (Vs), and tracheal flow rate (Vtr) in fetal sheep. In five experimental animals, amniotic and allantoic fluids were drained from 107 to 135 days of gestation. The volume of fluid drained from the experimental animals was 411.8 +/- 24.4 ml/day (n = 140). In six control animals, amniotic fluid volume was 747.7 +/- 89.7 ml (n = 15). Wet and dry lung weights were 20-25% lower in experimental fetuses than in control fetuses. Fetal hemoglobin, O2 saturation, arterial PO2, pH, and hematocrit were unchanged by drainage. During the drainage period, Vl was up to 65% lower, Vs was up to 35% lower, and Vtr was up to 40% lower in experimental fetuses than in control fetuses. We conclude that prolonged drainage of amniotic and allantoic fluids decreases Vl, Vs, and Vtr in fetal sheep. These findings indicate that fetal lung hypoplasia associated with oligohydramnios may be the result of a prolonged reduction in Vl.     

Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.     

Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis.

We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure-volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure-volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis.     

Lectin teratogenesis: defects produced by concanavalin A in fetal rabbits.

Concanavalin A (con A) is teratogenic to rabbit embryos during gestational days 12--15. Intracoelomic injections of 40 microliter con A solution (4 microgram/microliter) were performed on rabbit embryos during gestational days 10--15. Control embryos received either 40 microliter of saline, sham injection or no treatment. Con A caused increased fetal resorptions on days 10 and 11, but malformation levels did not differ from controls. On days 12--15, con A produced craniofacial, trunk and limb anomalies. The highest percentage of malformation occurred on day 14. The defects were classified into four groups: (1) malformations of limbs including paw and digital dysplasias as well as fusions of the limbs to the head or body wall; (2) "closure" defects such as umbilical hernia, encephalocoele, exencephaly or ectopia cordis; (3) "contracture" defects such as club paws, extended knees, or clenched digits, which exhibited normal osseous and cartilaginous skeletons; and (4) miscellaneous, non-specific anomalies including fused or dysplastic sternebrae or ribs. Histologic analysis of selected 12-day embryos 4 to 18 hours post-injection was performed to ascertain potential sites of teratogenic action. At 12 hours ectodermal necrosis was observed in the limb buds adjacent to the apical ectodermal ridge. By 18 hours, the ectoderm had eroded, exposing the basal lamina to the amniotic fluid. Focal areas of mesenchymal necrosis were observed in association with the ectodermal erosion. The potential roles of amniocentesis and limb bud repair in the genesis of the malformations are discussed.     

The effect of prolonged oligohydramnios on fetal lung development, maturation and ventilatory patterns in the newborn guinea pig

We drained the amniotic fluid surrounding guinea pig fetuses between days 45 and 65 of gestation (term is 67 days). The fetuses were delivered by Cesarean section and the impact of prolonged oligohydramnios on lung growth, maturation and postnatal ventilatory pattern was measured. Untouched littermate fetuses served as controls. Neither fetal body, liver nor brain weights were significantly affected by the experimental situation. When expressed in percent of control values, lung weight (63%), lung/body weight ratio (70%), lung volume (67%), total lung DNA content (63%) and lung DNA per gram of fetal weight (71%) were all significantly less following amniotic fluid drainage, confirming the diagnosis of lung hypoplasia. Disaturated phosphatidylcholine content per gram of lung tissue and total lung glycogen content were not affected by the procedure, indicating that the maturity of the hypoplastic lungs was not delayed. When measured 4 to 6 hours after birth, tidal volume was significantly less (62%) and respiratory frequency was significantly more (137%); however, minute ventilation per unit of body weight was not significantly changed. This animal model of sublethal lung hypoplasia could become useful to study the potential for, and the kinetics of, postnatal catch-up lung growth about which little is known.