Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.

Transforming growth factor-beta(1) regulation of surfactant protein B gene expression is mediated by protein kinase-dependent intracellular translocation of thyroid transcription factor-1 and hepatocyte nuclear factor 3.

The transforming growth factor-beta (TGF beta) polypeptides control a variety of cellular processes including organogenesis and cellular proliferation and differentiation. In the developing lung, TGF beta(1) treatment inhibits airway branching and expression of the genes for surfactant proteins (SP). Many effects of TGF beta are mediated at the level of gene transcription but there is limited information regarding signaling pathways and target transcription factors. In this study with human pulmonary adenocarcinoma H441 cells, we investigated TGF beta(1) effects on SP-B, a protein which is essential for normal function of pulmonary surfactant. TGF beta(1) (10 ng/ml) reduced SP-B mRNA content in a time-dependent fashion, and transient transfection studies localized responsiveness to the region of the SP-B promoter (-112/-72 bp) containing binding sites for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor 3 (HNF3), transcription factors that are important enhancers of SP gene expression. Using electrophoretic mobility shift assay and immunofluorescence, we demonstrated rapid accumulation of these transcription factors in the cytoplasm and subsequent loss from the nucleus on TGF beta(1) treatment of both adenocarcinoma cells and cultured human fetal lung. TGF beta(1) treatment caused intracellular translocation of protein kinase C and effects of TGF beta(1) were mostly abrogated in the presence of the protein kinase inhibitor calphostin C. We conclude that TGF beta(1), acting via protein phosphorylation, blocks nuclear translocation of TTF-1 and HNF3 which results in down-regulation of the SP-B gene and presumably other pulmonary genes which are transactivated by these factors.

Pulmonary surfactant metabolism in infants lacking surfactant protein B.

Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.

Intracellular localization of processing events in human surfactant protein B biosynthesis.

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.

Hormonal regulation and cellular localization of fatty acid synthase in human fetal lung.

Fatty acid synthase (FAS; EC 2.3.1.85) supplies de novo fatty acids for pulmonary surfactant synthesis, and FAS gene expression is both developmentally and hormonally regulated in the fetal lung. To further examine hormonal regulation of FAS mRNA and to determine the cellular localization of FAS gene expression, we cultured human fetal lungs (18-22 wk gestation) as explants for 1-4 days in the absence (control) or presence of glucocorticoid [dexamethasone (Dex), 10 nM] and/or cAMP agents (8-bromo-cAMP, 0.1 mM and IBMX, 0.1 mM). FAS protein content and activity increased similarly in the presence of Dex (109 and 83%, respectively) or cAMP (87 and 111%, respectively), and responses were additive in the presence of both hormones (230 and 203%, respectively). With a rabbit anti-rat FAS antibody, FAS immunoreactivity was not detected in preculture lung specimens but appeared in epithelial cells lining the tubules with time in culture. Dex and/or cAMP markedly increased staining of epithelial cells, identified as type II cells, whereas staining of mesenchymal fibroblasts was very low under all conditions. With in situ hybridization, FAS mRNA was found to be enriched in epithelial cells lining the alveolar spaces, and the reaction product increased in these cells when the explants were cultured with the hormones. The increased FAS mRNA content in the presence of Dex and/or cAMP is primarily due to increased stabilization of mRNA, although Dex alone increased the transcription rate by approximately 30%. We conclude that hormonal treatment of cultured human fetal lungs increases FAS gene expression primarily by increasing stability of the message. The induction of FAS during explant culture and by hormones occurs selectively in type II epithelial cells, consistent with the regulatory role of this enzyme in de novo synthesis of fatty acid substrate for surfactant synthesis in perinatal lungs.

TGF-beta1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.

Enhanced distribution of adenovirus-mediated gene transfer to lung parenchyma by perfluorochemical liquid.

Although gene therapy holds great promise for the treatment of inherited and acquired diseases of the lung, a number of issues including efficient delivery and distribution of genes to pulmonary target cells must still be addressed. In this study we evaluated the use of perfluorochemical (PFC) liquid as a vehicle for delivery of recombinant adenovirus (AdCBlacZ) to lungs of juvenile rabbits. Virus was instilled into trachea of rabbits, and 4 days later the lungs were removed, cut into multiple pieces, and assayed for beta-galactosidase (beta-Gal) activity. Total lung expression of the beta-Gal reporter gene was increased two- to three-fold by instillation of the virus (10(11) particles/kg body weight) in saline (1.5 ml/kg) simultaneously with perflubron liquid (15 ml/kg) compared to virus+saline alone (control). Uniformity of beta-Gal activity between lobes was significantly improved by the PFC liquid. In perflubron-treated lungs approximately 45% of the lung pieces had beta-Gal-specific activity values within 50-150% of the mean specific activity for the total lung, compared to only approximately 15% of the pieces in control lungs. More of total lobar beta-Gal activity was recovered in the distal lung tissue (approximately two-fold greater than controls, p < 0.05). Morphological assessment of X-Gal-stained, fresh-frozen lung sections showed increased levels and more complete staining of alveolar wall cells in the PFC group. These data indicate that the PFC liquid perflubron enhances distribution of virus-mediated gene expression to the lung parenchyma in healthy rabbits. PFC liquid may be a useful treatment vehicle for accessing distal spaces of the damaged or diseased lung.

Combined effects of fetal beta agonist stimulation and glucocorticoids on lung function of preterm lambs.

We asked whether a single-dose fetal treatment strategy using betamethasone plus either a long-acting beta 2 agonist (formoterol) or betamethasone plus agents that elevate intracellular cyclic adenosine monophosphate (isobutyl methylxanthine and dibutyryl-cyclic adenosine monophosphate) would augment the effects of prenatal betamethasone on postnatal lung function. Preterm lambs were treated with 0.5 mg/kg beta-methasone or betamethasone plus the other agents and delivered 48 h after treatment. The postnatal lung function as assessed by compliance, ventilatory efficiency, and lung volumes at 40 min of age was improved by prenatal betamethasone and improved further by combination treatment, although the augmented responses were not significantly greater than with betamethasone alone. Fatty acid synthase protein and enzymatic activity were not increased by betamethasone or combined treatments, in contrast to responses reported for other animal models. There were no effects of glucocorticoids or the combined treatments on surfactant. Stimulation of the beta 2 agonist system did not augment postnatal lung function significantly above that noted for betamethasone alone with the agents, doses, and duration of exposures tested.

Regulation of surfactant protein D in human fetal lung.

Surfactant protein D (SP-D) is a collagenous glycoprotein, produced by lung type II cells, that has structural and functional similarities with SP-A. In this study we postulated that SP-D and SP-A gene expression are regulated in a similar fashion to provide a coordinated local immune defense response to pulmonary infection. We determined content of SP-D protein and mRNA in second-trimester fetal lung and in postnatal tissue by protein blotting and hybridization analyses. Low levels of SP-D mRNA and protein were detected at 16 wk gestation, before appearance of SP-A, and levels increased during gestation. The content of SP-D did not change during 5 days of explant culture, whereas SP-A increased manyfold. Dexamethasone treatment during culture increased SP-D mRNA and protein about 2-fold with maximal response after 1 to 3 days' exposure to 100 nM steroid; under the same conditions SP-A mRNA content is inhibited. There was no significant change in SP-D mRNA after treatment of explants with adenosine 3',5'-monophosphate (cAMP) analog or interferon-gamma, agents which increase SP-A gene expression, nor after exposure to phorbol ester, tumor necrosis factor-alpha, or lipopolysaccharide at concentrations that reduced levels of SP-A mRNA by approximately 50%. We conclude that SP-D in the human lung is under developmental and glucocorticoid regulation occurring at a pretranslational level. SP-D is not influenced by inflammatory mediators that regulate SP-A, suggesting that these two proteins are not coordinately regulated in response to lung infection.

Transcriptional regulation of human pulmonary surfactant proteins SP-B and SP-C by glucocorticoids.

Expression of the pulmonary surfactant-associated proteins SP-B and SP-C is under both developmental and hormonal regulation. We used human fetal lung to investigate developmental changes and the mechanism of glucocorticoid stimulation of SP-B and SP-C gene expression. There were similar approximately 3-fold increases in SP-B cytoplasmic mRNA content and transcription rate comparing lung samples of 24 wk versus 16 wk gestation. During 5 days of lung explant culture without hormones, the transcription rate increased for SP-B and decreased for SP-C, paralleling changes in mRNA content. Treatment with 100 nM dexamethasone maximally increased transcription of the SP-B gene (approximately 3-fold) and SP-C gene (approximately 11-fold) after 2 and 8 h, respectively, similar to changes in mRNA content. In dose-response studies, the maximal increase in transcription rate occurred at approximately 10 nM dexamethasone for SP-B and at > or = 100 nM for SP-C. Induction of SP-B mRNA content and transcription rate were not affected by prior cycloheximide exposure, whereas induction of SP-C mRNA was decreased by as little as 1 h exposure to inhibitor. We conclude that glucocorticoids, acting directly in type II cells, regulate the SP-B and SP-C genes primarily at the level of transcription. Induction of SP-C, but not SP-B, requires ongoing protein synthesis which likely reflects involvement of a labile transcription factor. The difference in glucocorticoid sensitivity may indicate that the two surfactant protein genes contain glucocorticoid response elements with different affinities for receptor.

Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.

Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18-20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (< 2% of adult), appeared during culture (26% of adult), and was further increased approximately 3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7-9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19-24-wk preculture tissue at 0.2-2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19- and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B.

Elemental composition of lamellar bodies from fetal and adult human lung.

Previous studies in the rat using electron probe microanalysis have suggested that most of the Ca2+ in alveolar space is probably derived from secreted lamellar bodies (LB). However, the LB Ca2+ content in cultured rat type II cells is low and unaltered by dexamethasone supplementation. In this study, we examined LB Ca2+ content in adult human lung and cultured explants of fetal lung treated with hormones to promote type II cell differentiation. Lung tissue of 20 to 24 wk of gestation was cultured for 4 to 6 days without hormone and with dexamethasone (10 nM), triiodothyronine (2 nM), 8-bromo-cyclic adenosine monophosphate (0.1 mM), or their combinations. The cultured tissue samples were processed for light and electron microscopy or rapidly frozen and stored in freon-22 under liquid nitrogen. Thin cryosections from the frozen samples were prepared and examined using the electron probe microanalysis. Human lung tissue up to 24 wk of gestational age had no detectable LB before explant culture. Explants cultured for 4 days without hormone supplementation (control) had no detectable LB, single-hormone treatments resulted in small LB, and combination treatments resulted in the formation of many large LB in explant type II cells. Despite such morphologic changes, LB Ca2+ in both control and hormone-treated explants was low (overall mean, 4.1 +/- 0.6; P < 0.05) compared with LB of in situ rat and adult human lung (30 +/- 2 and 27 +/- 1.5 mmol/kg dry wt, respectively) and was similar to that found in cultured rat type II cells. LB phosphorus and sulfur under all explant culture conditions were comparable. These observations indicate that human and rat LB elemental compositions are similar and that optimal in vitro conditions with respect to LB Ca2+ metabolism have not been established.

Glucocorticoid and cAMP increase fatty acid synthetase mRNA in human fetal lung explants.

During late fetal development, synthesis of surfactant phospholipid requires a large supply of fatty acid precursor. Fatty acid synthetase is a regulatory enzyme for de novo fatty acid synthesis in lung as well as other lipogenic tissues. In this study, we report hormonal induction of FAS mRNA in human fetal lung explants (16-23 week gestation) cultured up to 7 days in Waymouth's medium (no serum) supplemented with dexamethasone (Dex, 10 nM) or agents that increase cAMP (8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM; forskolin, 0.01 mM; PGE1, 0.01 mM). Exposure of explants to Dex or cAMP agents increased FAS mRNA content by 6 h and maximal stimulation occurred at 72 h for Dex (approx. 3-fold increase) and 24 h for cAMP (approx. 2-fold increase). In the presence of both Dex and cAMP there was a synergistic increase in FAS mRNA content at all times (approx. 11-fold increase at 72 h). Induction of FAS mRNA was specific for steroids with glucocorticoid activity, reversible on removal of hormone, and was half-maximal at 2-3 nM Dex consistent with receptor mediation. Actinomycin D blocked induction by Dex but not by cAMP suggesting a transcriptional effect by glucocorticoid but not by cAMP. T3, which increases phosphatidylcholine synthesis, did not induce FAS mRNA. The findings indicate that both glucocorticoid and cAMP increase FAS gene expression consistent with an important role for FAS in regulating the supply of fatty acid for surfactant phospholipid synthesis.

Hormonal regulation of cholinephosphate cytidylyltransferase in human fetal lung.

Cytidylyltransferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15, CYT) is a regulatory enzyme for synthesis of pulmonary surfactant phosphatidylcholine (PC). The effects of glucocorticoid, T3, and cAMP on CYT activity were studied in explants of human fetal lung (18-22 weeks gestation) cultured for 1-6 days in serum-free medium. Dexamethasone (Dex, 10 nM) treatment for 5 days increased homogenate CYT activity (+115%, P < 0.02) when assayed in the presence of added lipid co-factor (L-alpha-phosphatidylglycerol, PG, 1.1 mM) and tended to increase activity in its absence (+77%, P = 0.12). Cytosolic activity was also significantly elevated in the presence of added co-factor (+124%, P < 0.01), but there was no effect of Dex on microsomal specific activity. Dex increased the recovery of CYT activity in the cytosolic fraction (75% vs. 43% (control) of the homogenate activity), but not in the microsomal, nuclear or mitochondrial fractions. Assayed in the presence of added co-factor, stimulation of CYT by Dex was apparent after 48 h exposure and maximal by 5-6 days exposure to < or = 30 nM concentration. T3 or agents that increase endogenous cAMP stimulated cytosolic activity by 40% and 36-74%, respectively, after 4-6 days exposure, but none produced an additive increase in the presence of Dex. We conclude that stimulation of CYT activity contributes to hormonal induction of surfactant lipids by each of these hormones. Glucocorticoids may increase the amount of CYT enzyme as well as activate the enzyme via increased synthesis of lipid co-factor.

Secretion of surfactant protein A and phosphatidylcholine from type II cells of human fetal lung.

Surfactant protein A (SP-A) appears to have a role in lung immune defense as well as generation and metabolism of the alveolar surface-active film. Previous studies indicated that lamellar bodies isolated from lung tissue had a relatively low content of SP-A and that exogenous SP-A was needed for rapid formation of a surface-active film in vitro. We therefore tested the hypothesis that SP-A was secreted from type II cells primarily by a pathway separate from lamellar bodies. Cells were isolated from explants of human fetal lung that had been cultured with hormones to promote differentiation of type II cells, and secretion of surfactant lipid and SP-A were compared. Cultured cells secreted labeled phosphatidylcholine in a nearly linear fashion for 48 h. Basal secretion of SP-A, assayed by enzyme-linked immunosorbent assay, was linear for only 12 h after plating of cells; during this time, there was no accumulation of intracellular SP-A. Addition of secretagogues (phorbol ester, calcium ionophore, and beta-adrenergic agonist) stimulated phosphatidylcholine secretion approximately 4-fold. In contrast, the secretion rate of SP-A was not significantly affected by secretagogues. These findings indicate that a relatively small amount of secreted SP-A (approximately 10%) is released with lamellar bodies. Most SP-A is released by constitutive secretion and may be important for both surfactant- and nonsurfactant-related functions.

Differentiation of type II cells during explant culture of human fetal lung is accelerated by endogenous prostanoids and adenosine 3',5'-monophosphate.

Explant culture of the fetal lung, in the absence of serum or hormones, results in precocious biochemical and morphological differentiation of alveolar epithelial cells. In this study we tested the hypothesis that these maturational events are induced by endogenous cAMP. During culture of human fetal lung the content of tissue cAMP increased 140% from days 3-6. Treatment with isobutylmethylxanthine caused a further doubling of cAMP content, and indomethacin blocked most of the increase in cAMP. Isobutylmethylxanthine accelerated and indomethacin inhibited the increases during culture in surfactant protein-A (SP-A), SP-A mRNA, SP-B mRNA, phosphatidylcholine content, and activity of fatty acid synthetase. There was no effect of these treatments on the content of SP-C mRNA, which did not increase during culture. Increasing concentrations of prostaglandins E1 and E2 in the presence of indomethacin produced a parallel stimulation of cAMP content, SP-A, and fatty acid synthetase activity. The temporal increase in SP-A was also blocked by inhibitors of protein kinase-A. In morphological studies, indomethacin-treated explants appeared less mature, with decreased intralumenal volume and more columnar epithelial cells. We conclude that increased tissue cAMP levels, stimulated by endogenous prostaglandins, accelerate differentiation of alveolar epithelial cells during explant culture.

Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro.

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.

Interferon-gamma and synthesis of surfactant components by cultured human fetal lung.

We examined the effects of interferon-gamma (IFN-gamma) on development of the surfactant system in alveolar epithelial cells of fetal lung. Explants of second-trimester human fetal lung were cultured for 1 to 6 days in serum-free medium containing recombinant human IFN-gamma (0.03 to 30 ng/ml) and/or dexamethasone (10 or 100 nM). Treatment for 3 days with IFN-gamma alone, dexamethasone alone, and IFN plus dexamethasone increased the content of surfactant protein A (SP-A, 28 to 36 kD) by approximately 3-, 2.5-, and 10-fold, respectively. The biphasic response pattern of SP-A to dexamethasone (stimulation initially and inhibition with continued culture) was not altered by the presence of IFN-gamma. IFN-gamma also stimulated accumulation of SP-A mRNA (2.7-fold at 24 h) but did not affect the levels of mRNAs for surfactant protein B (18 kD) and surfactant protein C (5 kD). To assess the effect of IFN-gamma on synthesis of surfactant lipids, we determined the content of phosphatidylcholine, the rate of labeled choline incorporation into phosphatidylcholine, saturation of newly synthesized phosphatidylcholine, and the activity of fatty acid synthetase, a glucocorticoid-inducible enzyme. Treatment of explants for 5 days with IFN-gamma had no effect on these parameters. Studies by light and electron microscopy revealed little difference between control and IFN-treated explants with regard to cell viability and epithelial cell differentiation. We conclude that IFN-gamma has a selective stimulatory effect on SP-A among surfactant components.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid stimulation of fatty acid synthesis in explants of human fetal lung.

We examined the effects of glucocorticoids and thyroid hormone (T3) on fatty acid synthesis, fatty acid composition and fatty acid synthetase activity in explants of human fetal lung (16-23 wk gestation). Explants were cultured 1-7 days in the absence (control) or presence of dexamethasone (10 nM) and/or T3 (2 nM). In control explants fatty acid synthesis and fatty acid synthetase activity increased 200% and 455%, respectively, between 1 and 5 days. Dexamethasone (10 nM) stimulated fatty acid synthesis (tritiated water incorporation) 155% and fatty acid synthetase activity 117% after 5 days in culture. T3 (2 nM) was not stimulatory, either alone or in the presence of dexamethasone. Dexamethasone increased the proportion of newly synthesized fatty acid recovered in phosphatidylcholine from 72% (control) to 90% (P less than 0.02) of total fatty acid. Dexamethasone stimulation of fatty acid synthetase activity was consistent with a receptor-mediated process: (1) stimulation was saturable and dose-dependent (Kd = 1.5 +/- 0.3 nM); (2) the potency of glucocorticoid analogs and other steroids reflected their glucocorticoid activity; (3) stimulation was reversible when cortisol was removed from the medium. Stimulation by dexamethasone was apparent within 24 h of hormone exposure, and increased to a maximum between 4 and 6 days. Fatty acid synthetase activity was higher in Type II cells (3.54 +/- 0.58 nmol malate/min per mg protein) than in fibroblasts from treated explants. Although both cell types responded to hormone treatment the stimulation was greater for Type II cells (200% vs. 75% increase). The fatty acid composition of PC showed increases in 14:0 and 16:1 with culture alone which were further stimulated by dexamethasone but not T3. These results indicate glucocorticoid stimulation of fatty acid synthesis and are consistent with a key role for fatty acid synthetase in the hormonal induction of pulmonary surfactant phosphatidylcholine synthesis in cultured fetal lung.