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Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species. Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls. In addition, purified preparations of SPs often contain endotoxin. The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP. Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-beta-D-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels. Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin. There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production. These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination.

Retinoic acid has both early or immediate (within hours) and late (after days) effects on gene expression. We studied the early effects of retinoic acid on the surfactant protein (SP) genes. Exposure of fetal rat lung explants to all trans-retinoic acid for 4 h resulted in a significant dose-dependent increase in SP-A, -B, and -C mRNA with markedly different dose-response characteristics. The maximal (2.5x) increase in SP-A mRNA was observed with 10(-10) M retinoic acid, whereas treatment with 10(-5) M resulted in a tendency to decreased levels. In contrast, maximal stimulation of SP-C (6x) was noted at 10(-5) M retinoic acid and that of SP-B (2x) at 10(-7) to 10(-5) M retinoic acid. Similar differences in the dose-response characteristics of SP-A and SP-C were observed with 9-cis-retinoic acid. A retinoic acid response element consensus sequence was identified in the rat SP-A gene; we hypothesize that retinoic acid-receptor complexes act directly on the SP-A gene via this response element.

Secretion of surfactant phosphatidylcholine has been extensively studied and there is evidence that it is a regulated process that can be influenced by a variety of physiological factors and pharmacological agents. In contrast, secretion of the major surfactant protein, surfactant protein A (SP-A), has been investigated to much lesser extent. It is not known whether SP-A secretion is constitutive or regulated and, if regulated, whether its regulation is similar to that of phosphatidylcholine. To address those questions we measured SP-A secretion in primary cultures of type II pneumocytes under conditions identical to those used to study phosphatidylcholine secretion. Freshly isolated cells from adult rats were cultured overnight, washed, and then incubated in fresh medium in the presence and absence of surfactant phospholipid secretagogues. As previously reported for phosphatidylcholine, SP-A secretion was linear with time for up to 4 h. However, the rate of SP-A secretion, approximately 6% of total SP-A (cells+medium) released into the medium per hour, was more than sixfold greater than that of the lipid. Although freshly isolated cells contained 70% more SP-A than cells that were cultured overnight, the rate of SP-A secretion was not significantly different. Secretion of SP-A by freshly isolated or cultured type II cells was not increased by a combination of ATP, terbutaline, the adenosine A2 receptor agonist 5'(N-ethylcarboxyamido)adenosine, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin at concentrations that optimally stimulated phosphatidylcholine secretion. We conclude that secretion of the major lipid and protein components of surfactant are independently regulated.

Activated alveolar macrophages secrete both nitric oxide and superoxide in the alveolar lining fluid which combine rapidly to form peroxynitrite, a potent oxidizing agent capable of damaging lipids and proteins in biological membranes. Peroxynitrite (1 mM) plus 100 microM Fe3+EDTA inhibited calf lung surfactant extract (CLSE) from reaching a minimum surface tension below 10 mN/m on dynamic compression. Peroxynitrite and its by-products reacted with the unsaturated lipid components of CLSE, as evidenced by the appearance of conjugated dienes and thiobarbituric acid products, and damaged all surfactant proteins. A mixture of the hydrophobic proteins [surfactant protein B (SP-B) and surfactant protein C (SP-C)] exposed to peroxynitrite became incapable of lowering phospholipid minimum surface tension on dynamic compression. Exposure of SP-A to peroxynitrite decreased its ability to cause lipid aggregation and to act synergistically with SP-B and SP-C in lowering surface tension of surfactant lipids. Western blot analysis of SP-A exposed to peroxynitrite was consistent with fragmentation and polymerization of the 28- to 36-kDa triplet band, and amino acid analysis revealed the presence of significant levels of 3-nitro-L-tyrosine. We conclude that peroxynitrite and its reactive intermediates inhibit pulmonary surfactant function by lipid peroxidation and damaging surfactant proteins.

Previously, it was found that lung explants from mid-trimester human abortuses differentiate spontaneously in organ culture in serum-free defined medium in an atmosphere of 95% air-5% CO2. Dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) treatment of human fetal lung in culture increases the rate of morphological differentiation and enhances expression of the surfactant protein A (SP-A) gene. To begin to define the factors responsible for this accelerated in vitro differentiation, we analyzed the effects of atmospheric oxygen on the morphological and biochemical development of human fetal lung in culture and on responsiveness of the cultured tissue to DBcAMP. We found that when lung explants were maintained in an atmosphere containing 1% oxygen they failed to differentiate spontaneously and no induction of SP-A gene expression was apparent. Furthermore, at 1% oxygen, DBcAMP had no effect to stimulate morphological differentiation or SP-A gene expression. When lung tissues that had been maintained for 5 days in 1% oxygen were transferred to an environment containing 20% oxygen, there was rapid morphological development and induction of SP-A gene expression. The effects on morphological development were manifest within 24 h of transfer to the 20% oxygen environment; within 72 h, a marked stimulatory effect of DBcAMP on SP-A gene expression also was observed. Our findings further suggest that the effects of oxygen on the levels of SP-A and SP-A mRNA are concentration dependent. Interestingly, the inductive effects of DBcAMP on SP-A gene expression were apparent only at oxygen concentrations > or = 10%. Morphological differentiation of the cultured human fetal lung tissue also was influenced by oxygen in a concentration-dependent manner. These findings suggest that oxygen plays an important permissive role in the spontaneous differentiation of human fetal lung in vitro.

Synthesis and secretion of surfactant protein A (SP-A) were studied in the isolated perfused rat lung using Trans35S-label (approximately 85% methionine, 15% cysteine) in the perfusate with or without 1 mM ATP or 0.1 mM 8-bromoadenosine 3',5',-cyclic monophosphate (8-BrcAMP) for up to 6 h of perfusion. By enzyme-linked immunosorbent assay, the SP-A content was 36 +/- 0.3% of total protein in extracellular surfactant and 10.8 +/- 1.9% of total protein in lamellar bodies of control lungs; these relativr proportions were maintained in the presence of ATP or 8-BrcAMP. Incorporation of [35S]methionine (cysteine) into the surfactant and lamellar body protein fraction could be detected at 4 h of perfusion. At 6 h, specific activity of total protein [disintegrations per minute (dpm)/micrograms)] was significantly increased in both the surfactant (54%) and lamellar body fractions (30%) under the influence of either secretagogue compared with control conditions. In the presence of ATP, there was a significant increase in the SP-A immunoprecipitable counts of 61 and 72% in extra- and intracellular compartments, respectively. However, no significant change was observed in the relative abundance of SP-A mRNA between control and secretagogue-treated lungs. This dissociation of SP-A mRNA abundance and label incorporation into protein indicates that alteration in translational efficiency or posttranslational factors may be involved in the secretagogue-induced stimulation of SP-A synthesis.

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.

Surfactant protein A (SP-A) metabolism was studied in vivo in 33 preterm ventilated lambs at 138 +/- 1 days gestational age by measuring recoveries of exogenously administered surfactant containing both radiolabeled SP-A and labeled saturated phosphatidylcholine (Sat PC) given via the trachea at birth. Endogenously secreted SP-A was also labeled with [35S]methionine and followed over 24 h. The exogenously labeled SP-A left the alveolar pool more rapidly than did Sat PC over the first 5 h of life (P less than 0.05), and both exogenously labeled SP-A and Sat PC were detected within lamellar bodies by 2 h, indicating uptake from the airspaces. The quantity of SP-A in alveolar washes increased about twofold from birth to 5 h of age, whereas alveolar Sat PC pools were constant over 24 h. The SP-A endogenously labeled with [35S]methionine was recovered at highest specific activities in the alveolar washes at 10 and 45 min after birth with no labeled SP-A detectable in lamellar body fractions until 2 h. The curve for endogenous SP-A labeling of lamellar bodies was similar to that for exogenous labeling, indicating that SP-A was initially secreted by a pathway independent of lamellar bodies with subsequent SP-A labeling of lamellar bodies. The kinetics of SP-A metabolism were very different than for Sat PC in preterm lambs.

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27-36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.

The distribution of the major surfactant apoprotein (SP-A) in adult rat lung was determined in order to gain insight into its metabolism, including packaging of SP-A into lamellar bodies. The effect of glucocorticoid treatment on surfactant apoprotein was studied to test whether regulation of surfactant apoprotein genes, which has been described for the fetal lung, can be demonstrated in the adult animal. We measured the amounts of immunoreactive SP-A in several lung tissue compartments and lavage fractions from control animals and from the lungs of rats given dexamethasone for 1 wk. Protein and phospholipids were measured, SP-A was quantitated with a noncompetitive enzyme-linked immunoabsorbent assay (ELISA) and SP-A, SP-B, and SP-C mRNAs were estimated by Northern blotting. We found an 85-fold concentration of SP-A in a lamellar body-rich fraction compared with lung tissue homogenate and we calculated that as much as one-half of all the tissue SP-A might be accounted for by a lamellar body pool. After 1 wk of dexamethasone treatment, there was an increase in adult rat lung SP-A, SP-B, and SP-C mRNA and a substantial increase in tissue and lavage fluid immunoreactive SP-A pools. Lamellar body fraction SP-A content per lung was 1.4-fold higher after dexamethasone, and there was a fivefold increase in the lavage SP-A pool, much of which was inseparable from the alveolar macrophages. We conclude that SP-A is concentrated in the lamellar bodies of type II cells, that dexamethasone treatment increased all surfactant mRNAs, and that it increased SP-A content in adult rat lung.

The amino acid sequence of surfactant protein B (SP-B), derived from human genomic and cDNA sequences, indicates that the active polypeptide is contained within the sequence of a preproprotein of 381 residues. Synthesis of mature SP-B, which requires proteolytic processing at both the NH2- and COOH-termini of the proprotein, was studied in primary cultures of rat alveolar type II epithelial cells. Type II cells were pulse labeled with [35S]methionine-cysteine for 15-30 min and chased for 0-18 h. SP-B proprotein (Mr = 42,000) accumulated in the medium at early time points but declined at later time points suggesting extracellular proteolysis of the proprotein. In contrast, surfactant protein A (SP-A), another surfactant protein, continued to accumulate extracellularly during this time period. A proteolytic fragment of SP-B (Mr = 25,000) accumulated in the medium with a slightly slower time course, consistent with extracellular proteolysis of the proprotein. Intracellular processing of SP-B was also detected. SP-B polypeptides of Mr 8,000 and 12,000 were detected intracellularly and in the medium at late time points. These forms of SP-B (Mr = 8,000 and 12,000) comigrated in two-dimensional isoelectric-focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mature SP-B isolated from rat alveolar lavage fluid. The mature form of SP-B (Mr = 8,000), but not the Mr 42,000 and 25,000 SP-B precursors, was also detected in lamellar bodies isolated from rat lung homogenates. These experiments demonstrate complete proteolytic processing of prepro-SP-B to the alveolar Mr 8,000 form by type II epithelial cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

SP-A, a glycoprotein of pulmonary surfactant, consists of an NH2-terminal domain containing a collagen-like sequence and a COOH-terminal domain with sequence homology to several Ca2(+)-dependent lectins. We have compared the size, thermal stability, and secondary structure of recombinant SP-A, the product of a fibroblast line transfected with a single human gene encoding SP-A, with natural SP-A isolated from canine and human lungs. Our results suggest both recombinant and natural SP-A are assembled as large oligomers. More variability in the degree of oligomerization was observed with recombinant human SP-A than with natural canine SP-A. As shown by collagenase digestion, the full assembly of protein subunits was dependent on an intact collagen-like domain. The cysteines in the noncollagen domain of SP-A form intrachain bonds between residues 135-226 and 204-218. The circular dichroism spectra of both recombinant and natural SP-A were consistent with the presence of a collagen-like triple helix. As determined by the change in ellipticity at 205 nm, the thermal transition temperatures of canine, natural human, and recombinant SP-A were 51.5, 52.3, and 42.0 degrees C, respectively. These results suggest differences in the assembly and stability of the natural and recombinant proteins.

The effects of postnatal dexamethasone treatment in vivo on the synthesis of surfactant protein A (SP-A) were examined at the protein and RNA levels. Rats ranging from 1 day old to adult were injected with 200 micrograms of dexamethasone/kg body wt or with vehicle alone and were killed 24 h after injection. One portion of the lung was metabolically labeled with [35S]methionine, the proteins immunoprecipitated using an antiserum to SP-A, and analyzed electrophoretically. Both newly synthesized intracellular and secreted SP-A levels were increased by dexamethasone, reaching averages of 2.3 and 4.5 times control values, respectively. Another portion of the lung tissue was used for RNA analysis. SP-A mRNA levels were also elevated an average of 1.4 times control values by hormone treatment. Dose-response experiments using 16-day-old pups showed that both total SP-A, as measured by enzyme-linked immunosorbent assay, and total SP-A mRNA levels were elevated with dexamethasone treatment, reaching maximal stimulation at 2 mg. We conclude that postnatal dexamethasone treatment in vivo results in increased levels of both newly synthesized SP-A and SP-A mRNA, suggesting that pretranslational events may in part contribute to this process.