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Pulmonary surfactant stabilizes the alveoli by lining the air-fluid interface with films that reduce surface tension to near 0 mN/m (gamma(min)). Surfactant protein B (SP-B) enhances the surface activity of surfactant phospholipids. A captive bubble tensiometer (CBT) was used to study the properties of adsorbed films of dipalmitoylphosphatidylcholine (DPPC) with acidic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or neutral 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine with (7:3) and without 1% dimeric SP-B. SP-B enhanced the adsorption rate of DPPC-containing neutral or acidic lipid suspensions (1 mg/ml) to a similar extent. Quasi-static cycling of these films revealed that SP-B significantly decreased the film area reduction required to reach gamma(min) for the acidic but not for the neutral system. The results obtained with DPPC-phosphatidylglycerol (PG)-SP-B were consistent with selective DPPC adsorption into the surface monolayer during film formation. Film area reduction required to reach gamma(min) with this system (with and without calcium) approached that of pure DPPC, suggesting selective DPPC insertion and PG squeeze-out. Dynamic cycling of such films showed that larger film area reductions were required to reach gamma(min) for the neutral than for acidic system, even after 20 cycles. Fluorescence microscopy of solvent-spread DPPC-POPG-SP-B planar films revealed highly condensed structures at approximately 25 mN/m, although no specific PG phase-segregated structures could be identified. The study suggests that specific interactions of SP-B with acidic phospholipids of surfactant may be involved in the generation and maintenance of DPPC-rich films in the alveoli.

Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1-4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (>/=2-fold); mRNA levels in fetuses delivered 1-3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational.

Biological membranes exist in many forms, one of which is known as tubular myelin (TM). This pulmonary surfactant membranous structure contains elongated tubes that form square lattices. To understand the interaction of surfactant protein (SP) A and various lipids commonly found in TM, we undertook a series of transmission-electron-microscopic studies using purified SP-A and lipid vesicles made in vitro and also native surfactant from bovine lung. Specimens from in vitro experiments were negatively stained with 2% uranyl acetate, whereas fixed native surfactant was delipidated, embedded, and sectioned. We found that dipalmitoylphosphatidylcholine-egg phosphatidylcholine (1:1 wt/wt) bilayers formed corrugations, folds, and predominantly 47-nm-square latticelike structures. SP-A specifically interacted with these lipid bilayers and folds. We visualized other proteolipid structures that could act as intermediates for reorganizing lipids and SP-As. Such a reorganization could lead to the localization of SP-A in the lattice corners and could explain, in part, the formation of TM-like structures in vivo.

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.

To test the hypothesis that chronic placental insufficiency resulting in fetal growth restriction causes an increase in fetal lung surfactant-associated protein (SP) gene expression, we embolized chronically catheterized fetal sheep (n = 6) daily using nonradioactive microspheres in the abdominal aorta for 21 days (between 0.74 and 0.88 of gestation) until fetal arterial oxygen content was reduced by approximately 40-50%. Control animals (n = 7) received saline only. Basal fetal plasma cortisol concentration was monitored. At the end of the experiment, fetal lung tissues were collected, and ratios of tissue levels of SP-A, SP-B, and SP-C mRNA to 18S rRNA were determined by standard Northern blot analysis. Total DNA content of fetal lungs was reduced by 30% in the embolized group compared with control group (P = 0.01). There was a 2.7-fold increase in fetal lung SP-A mRNA (P < 0.05) and a 3.2-fold increase in SP-B mRNA (P < 0.01) in the chronically embolized group compared with those in the control group. SP-A and SP-B mRNA tissue levels were highly correlated with the mean fetal plasma cortisol levels on days 20-21 (r = 0.90, P < 0.01 for SP-A mRNA and r = 0.94, P < 0.01 for SP-B mRNA). SP-C mRNA tissue levels were not significantly affected by placental insufficiency. We conclude that fetal growth restriction due to placental insufficiency is associated with alterations in fetal lung SP, suggesting enhanced lung maturation that was highly dependent on the degree of increase in fetal plasma cortisol levels.

Obstruction of the fetal trachea is a potent stimulus for fetal lung growth, and it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. However, little is known about the effects of increased lung expansion on other aspects of lung development. Our aim was to determine the effect of increased and decreased lung expansion on the mRNA levels encoding surfactant protein (SP) A, SP-B, and SP-C in ovine fetal lungs. Lung tissue samples were collected from fetuses exposed to 2, 4, or 10 days of increased lung expansion caused by tracheal obstruction. The mRNA levels for SP-A, SP-B, and SP-C were determined by Northern blot analysis with specific ovine cDNA probes; SP-A protein levels were determined by Western blot analysis. Compared with age-matched (128-day gestational age) control fetuses, SP-A, SP-B, and SP-C mRNA levels in fetal lung tissue were significantly reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. However, SP-A protein levels were not reduced at 2 days of tracheal obstruction, tended to be reduced at 4 days, and were almost undetectable at 10 days. In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased SP-C, but not SP-A, mRNA levels in fetal lung tissue compared with age-matched control fetuses. Our results demonstrate that increases in fetal lung expansion, induced by obstruction of the fetal trachea, cause large simultaneous reductions in SP-A, SP-B, and SP-C mRNA levels in the fetal lung as well as a decrease in SP-A protein levels. These data suggest that expression of the genes encoding SPs in the fetal lung are specifically responsive to the degree of lung expansion.

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

A serine-active enzyme, "surfactant convertase", is required for the conversion of surfactant from the tubular myelin (TM) form to the small vesicular (SV) form. This transformation involves at least two steps, the conversion of TM to a surface-active film at the air-fluid interface and the reorientation of the film into the surface-inactive SV form; we asked if convertase was required for the first of these steps. Rat and mouse TMs were pretreated with diisopropyl fluorophosphate (DFP) to inactivate endogenous convertase activity or with vehicle and then were analyzed for their ability to lower surface tension in vitro as an index of the conversion of TM to a surface film. DFP pretreatment did not alter the ability of TM preparations to lower surface tension, as assessed by pulsating bubble, and it did not affect the behavior of TM in a surface balance. In an experiment designed to test the ability of TM to feed a surface film to exhaustion, TMs that had been pretreated with DFP or vehicle performed similarly. These experiments show that convertase activity is not required for the conversion of TM to a monolayer and suggest, instead, that convertase acts at a post surface film stage.

The effect of palmitoylation of pulmonary surfactant-associated protein C (SP-C) on the surface activity of phospholipid mixtures of dipalmitoylphosphatidylcholine and phosphatidylglycerol was studied. Phospholipids reconstituted with palmitoylated or depalmitoylated bovine SP-C were examined at neutral and acidic pH using a captive bubble surfactometer. At low pH, effective lipid adsorption and near zero surface tensions upon compression were obtained even with protein-free samples. At physiological pH, only SP-C-containing samples achieved such properties. Lipid adsorption was decreased by prior SP-C depalmitoylation. Bubbles with palmitoylated SP-C were more mechanically stable and required less compression to reach low surface tensions. Subphase depletion experiments showed that dynamically cycled surface layers containing palmitoylated SP-C maintained their surface activity after subphase lipid depletion. In contrast, surface activity was rapidly lost where depalmitoylated SP-C or SP-B was included. Our results indicate that although SP-C palmitoylation has little effect on its ability to enhance lipid adsorption and surface tension reduction, it greatly enhances lipid respreading and film stability and is therefore important for surfactant function.

The effect of surfactant concentration and supplementation with surfactant-associated protein A (SP-A) on the surface activity of lipid extract surfactant (LES) was examined using a captive bubble technique. Adsorption of LES is strongly concentration dependent over the range of 50-1,000 micrograms/ml. Addition of SP-A to LES at low concentrations in the presence of calcium dramatically increases the rate of adsorption. In quasistatic cycling experiments, samples containing SP-A require less compression to achieve low surface tensions even during the first compression cycle. Calculated film compressibilities at 15 mN/m indicate that SP-A alters the surfactant monolayer behavior such that in a small number of cycles the compressibility is indistinguishable from that of pure DPPC. Furthermore, SP-A reduces the incidence of bubble "clicking," suggesting a stabilization of the monolayer at low surface tensions. In dynamic cycling experiments, SP-A reduces compression of the film area required to achieve a low surface tension of approximately 1 mN/m. SP-A eliminated the plateau just below 25 mN/m normally observed during the compression phase with low concentrations of LES and the shoulder observed at approximately 35 mN/m during expansion. In the presence of SP-A and, to a lesser extent with high concentrations of LES, there is a marked lag in the increase in surface tension during the initial part of the dynamic expansion loop, with surface tensions remaining near 1 mN/m for approximately 10% of the increase in bubble area. The results indicate that SP-A enhances phospholipid adsorption during dynamic cycling and may enhance elimination of non-DPPC lipids during cycling.(ABSTRACT TRUNCATED AT 250 WORDS)