Introduction

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Respiratory distress syndrome (RDS) of infants is due most often to a developmental deficiency of pulmonary surfactant combined with immature lung structure. Premature infants have decreased lung content of all surfactant components, including surfactant-specific proteins (SP) A, SP-B, and SP-C, as well as the surface-active phospholipid disaturated phosphatidylcholine (DSPC). Recently, RDS has been described in full-term human infants who are homozygous for a null mutation in a single surfactant protein, specifically SP-B (1). The most common mutation described to date is a net 2 bp insertion at codon 121 in exon 4 of the SP-B gene (121ins2). This frameshift mutation introduces a downstream premature stop codon, and SP-B is undetectable in both lung tissue and lavage fluid. Other mutations resulting in total or partial SP-B deficiency have been described and mostly occur in compound heterozygous infants with the 121ins2 mutation on one allele (4, 5). Infants with inherited deficiency of SP-B develop respiratory failure at birth, requiring supplemental oxygen, assisted ventilation, and often extracorporeal membrane oxygenation. Surfactant replacement is not effective in SP-B-deficient infants and without lung transplantation all affected infants have died before 1 yr of age. The prevalence of inherited SP-B deficiency has not been established, but this condition is estimated to account for 25% of the cases of refractory RDS in term infants (6).

The 10-kb SP-B gene is located on chromosome 2 and encodes a 2.2-kb messenger RNA (mRNA) that gives rise to a 40-kD preproSP-B (5, 7). Processing of both amino and carboxy regions results in an 8-kD mature SP-B that is highly lipophilic and is packaged with surfactant phospholipids in lamellar bodies of alveolar type II cells (8, 9). The SP-B gene is also expressed in Clara cells of airway epithelium, but its function in these cells is unknown. In vitro, SP-B causes aggregation of lipids and promotes both formation and stability of the surface-active phospholipid film (5). Studies in animal models with SP-B-neutralizing antibodies (10, 11) and with SP-B supplementation of replacement surfactant (12) indicate a vital role for SP-B in surfactant function in vivo. Moreover, ablation of the SP-B gene in transgenic mice causes respiratory failure at birth and death in homozygous pups (13).

Lungs of infants homozygous for the SP-B null mutations have structural abnormalities and altered biochemical properties (1, 14). Histologically, changes in lung tissue include fibrosis and emphysema, typical of all forms of chronic lung disease (CLD), plus alveolar proteinosis, which reflects accumulation of SP-A, proSP-C, and serum proteins in airspaces. Electron microscopy has indicated abnormal ultrastructure of type II cells, including a lack of mature lamellar bodies and increased content of multivesicular bodies; alveoli contain desquamated type II cells, foamy macrophages, and secreted lipid vesicles. SP-B deficiency is associated with accumulation of a 6- to 12-kD form of SP-C, which may represent incomplete processing of the precursor protein to the mature 3.7-kD form. In the index case of homozygous 121ins2 SP-B deficiency, the content of SP-A and SP-C mRNA was normal, and SP-B mRNA was not detected (1).

The major objectives of this study were to determine whether there are alterations in SP-B gene transcription rate, processing of SP-C, and phospholipid synthesis and content in SP-B-deficient tissue obtained at lung transplantation or postmortem. We found that the mutated SP-B gene is normally transcribed, suggesting SP-B mRNA instability, and that absence of SP-B protein blocks processing of SP-C. CLD of various etiologies reduced surfactant function and altered content but not synthesis of phosphatidylglycerol (PG). A preliminary report of this study has been published as an abstract (19).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents

Express Protein Labeling Mix was obtained from New England Nuclear (Boston, MA). Protein A-agarose was obtained from Life Technologies, Inc. (Gaithersburg, MD). Dexamethasone, isobutylmethylxanthine, and 8-Bromo-cAMP were obtained from Sigma Chemical Co. (St. Louis, MO). Radioisotopes [³H]choline (81 Ci/mmol), [³H]acetate (129 Ci/mmol), and [¹⁴C]glycerol (185 mCi/mmol) were purchased from New England Nuclear, and reagents for enhanced chemiluminescence were purchased from Amersham Searle (Arlington Heights, IL). All other reagents were purchased from Bio-Rad Laboratories (Hercules, CA). Culture medium was obtained from the Cell Center Facility, University of Pennsylvania (Philadelphia, PA).

Antibodies

The production and specificity of antisera have been previously described (20). Affinity-purified, polyclonal anti- SP-A antiserum was produced in rabbits using purified rat SP-A and recognizes human SP-A but not other surfactant proteins or serum proteins. Polyclonal monospecific anti- SP-B (designated PT3) was prepared in rabbits using an organic lipid extract of bovine surfactant (Surfactant TA; Ross Laboratories, Columbus, OH) and recognizes precursor and mature human SP-B and was used for Western blot analyses; a polyclonal antiserum prepared against mature SP-B purified from human alveolar proteinosis fluid (designated hSP-B) was used for immunoprecipitation experiments. A site-specific proSP-C antiserum (NPROSP-C) was made in rabbits using a synthetic peptide (Met¹⁰-Pro²⁷ of rat proSP-C) and recognizes human proSP-C forms containing sequence immediately amino-terminal to the mature region of SP-C.

Tissues and Lavage Samples

Postnatal human lung tissues and/or lung lavage fluid were obtained at the time of lung transplantation or postmortem from nine medical centers in North America. Fetal lung was obtained from second trimester abortuses. Lung lavage of postnatal lung was performed on a single lobe (usually an upper lobe) within 1 h of lung removal. A cannula was anchored in a major airway and lavage carried out by five instillations and withdrawals of approximately 10 ml of sterile salt solution (1.25 mM NaCl, 5 mM KCl, 25 mM Na₂HPO₄, 17 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, pH 7.4). For off-site collections, tissues and fluid were shipped frozen to St. Louis, and tissue in culture medium was shipped to Philadelphia on ice for explant culture. Studies were performed under protocols approved by the Committees for Human Research at each of the respective institutions.

Patient Genotyping

DNA was prepared from blood leukocytes using a salt precipitation method with a commercially available kit (Puregene; Gentra Systems, Minneapolis, MN) (23). Genomic DNA was screened for the presence of the 121ins2 mutation by polymerase chain reaction (PCR) amplification and restriction analysis using Sfu I as previously described (3). When initial analyses were consistent with the 121ins2 mutation on only one allele, genomic DNA was screened for other SP-B gene mutations by use of heteroduplex analysis as previously described (4). Mutations were confirmed by direct sequence analysis of PCR products amplified from genomic DNA, by end-labeling a specific primer with [³³P]deoxyadenosine triphosphate using T4 polynucleotide kinase (New England Biolabs, Beverly, MA), and by cycle sequencing using the Circumvent kit from New England Biolabs. The two mutations in the patient heterozygous for the 121ins2 and C248X mutations were demonstrated to be derived from different alleles by sequence analysis of SP-B-specific complementary DNA (cDNA), generated from RNA, subcloned into a plasmid vector (PCR 2.1; Invitrogen, La Jolla, CA), and sequenced as previously described (3).

Explant Culture

Lung tissue was cultured as previously described (24). Briefly, 1-mm³ pieces of tissue were cultured in serum-free Waymouth's medium using a rocking platform (3 oscillations/min) and 95% air/5% CO₂. Most studies of lipid and surfactant protein synthesis were performed after overnight culture that was carried out to allow recovery of tissue metabolic activity. Some explants were maintained an additional 3 d in medium containing 10 nM dexamethasone with addition of fresh medium every 24 h.

cDNA Probes

The human cDNAs for SP-A, SP-B, SP-C, and -actin have been previously described (25). The human SP-B cDNA used for mRNA analysis was excised with BamH1 from the 5' end of a nearly full-length cDNA (~ 1.9 kb) and contained 662 bp. This cDNA is designated SP-B 5' probe and corresponds to the coding region for amino acids 1-221 of proSP-B. A second SP-B probe was prepared by digestion of the full-length cDNA with Sac1 and EcoR1 and subcloning of the fragment into Bluescript plasmid. This cDNA is designated SP-B 3' probe and contains the final 570 bp of the 3' terminus of SP-B cDNA corresponding to the 3' untranslated region of the mRNA.

mRNA Analysis

Total RNA was prepared from frozen tissue using the acidic phenol extraction method, and the content of SP-A, SP-B, SP-C, and -actin mRNAs was determined by cDNA hybridization using both Northern blot analysis and a dot blot procedure as previously described (28). The cDNA probes were labeled with [³²P]cytidine triphosphate using a random primer labeling technique (oligo labeling kit; Pharmacia LKB, Piscataway, NJ). Blots were analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) or exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) with Cronex intensifying screens (Du Pont Instruments, Wilmington, DE) for 1 to 5 d at 70°C, and the autoradiograms were scanned using a densitometer (Hoeffer GS 300; Hoeffer Scientific Instruments, San Francisco, CA). Relative densities were calculated from linear portions of the dose-response curve for each RNA sample.

Nuclear Run-On Assay

Transcription rate of surfactant protein genes was assessed by nuclear run-on assay, which has been described (29). In brief, nuclei were isolated from frozen preculture lung tissue or from tissue cultured as explants for 1 to 4 d and incubated for 30 min with [³²P]uridine triphosphate and other unlabeled nucleotides. RNA was isolated and hybridized to strips of Duralose (Stratagene, La Jolla, CA) containing 5 µg of cDNA for SP-A, SP-B (5' and/or 3' probe), SP-C, and -actin, which served as a control probe, as well as plasmid vector, which uniformly gave no signal. The strips were washed under stringent conditions, treated with RNase A, and then exposed to phosphorescence screens. The intensity of the hybridization signal was quantitated by PhosphorImager scanning. In most experiments, duplicate preparations of nuclei were used, and data were calculated as the mean of duplicate results for each sample.

In some experiments, we compared the signal using SP-B 5' probe and SP-B 3' probe. In the run-on assay, transcription continues in vitro for RNA molecules already initiated in vivo, but there is no new initiation of transcription. Treatment of the Duralose strips with RNase A, which degrades single-stranded RNA, before exposure to phosphorescence screens results in detection of only the labeled RNA, which is hybridized with cDNA probe. Using the SP-B 5' probe therefore provides an estimate for transcription rate of the 5' region of the gene, whereas the 3' probe detects only newly synthesized mRNA from the SP-B 3' untranslated region (i.e., distal to exon 4 containing codon 121, which is the site of the most common mutation in SP-B deficiency). The ratio of signal with SP-B 3' probe versus 5' probe was calculated from results obtained on the same Duralose strip.

Western Blot Analysis

Explant tissue was sonicated and total protein was quantified by the method of Bradford (30). One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 16.5% polyacrylamide gels using a Tris-tricine buffer system, and samples were transferred to Duralose or polyvinylidene difluoride membrane (Bio-Rad) for subsequent immunoblotting or autoradiography as previously described (21).

Pulse/Chase Labeling

Explants in culture were starved by replacing Waymouth's medium with Met/Cys-free Dulbecco's modified Eagle's medium (DMEM; 2 ml/60 mm plate) for 2 h while incubating in 95% air/5% CO₂ on a rocking platform. This medium was then replaced with Met/Cys-free DMEM supplemented with 200 µCi/ml of Express Protein Labeling Mix (2 ml/60 mm plate), which is composed of 70% Met and 15% Cys (New England Nuclear). After 1 h, the tissue was washed and placed in complete Waymouth's medium. Duplicate samples were harvested at 0 h and single samples at 1, 2, and 4 h of chase unless otherwise specified. Samples were washed in phosphate-buffered saline with protease inhibitors (10 mM N-ethyl maleimide, 2 mM benzamidine HCl, and 80 mM phenylmethylsulfonyl fluoride) and then sonicated in the same solution with addition of 1% SDS for studies of SP-B.

Immunoprecipitation

Radiolabeled lung homogenates were immunoprecipitated as previously described (9, 22). Total protein and total trichloroacetic acid (TCA) precipitable counts were determined from duplicate 10-µl samples of labeled homogenate. Immunoprecipitation was performed on samples containing 10⁶ incorporated counts using human SP-B antibody (hSP-B), anti-NPROSP-C antiserum, or preimmune rabbit serum. After immunoprecipitation using protein A- agarose beads, proteins were solubilized in gel sample buffer (62.5 mM Tris-HCl, pH 6.8/2% SDS/0.72 M 2-mercaptoethanol/10% glycerol/0.0075% bromophenol blue). An aliquot was taken for scintillation counting and the remainder was subjected to Tris-tricine SDS-PAGE as described above. After transfer to membranes, blots were analyzed by either PhosphorImager or with an Ambis 4000 radioanalyzer (Scanolytics, San Diego, CA).

Labeling and Analysis of Lipids

In experiments for lipid studies, explants were cultured overnight and then [³H]choline (5 µCi/ml), [³H]acetate (20 µCi/ml), or [¹⁴C]glycerol (2 µCi/ml) were added and explants were harvested after 6 h. Unlabeled precursors (1 mM for acetate and glycerol or 1.8 mM for choline) were added to ensure rapid equilibration of the endogenous precursor pools in all tissues and linear incorporation with time. Tissue was sonicated in saline and lipids were extracted by the method of Bligh and Dyer (31). For explants labeled with [³H]choline, phosphatidylcholine (PC) was isolated from the total lipid extract by thin layer chromatography in chloroform:methanol:7 M NH₄OH (60:35:5, vol/vol/vol) as described (24). To separate all phospholipids ([³H]acetate- and [¹⁴C]glycerol-labeled tissue), extracts were spotted on Whatman LK5D thin layer chromatography (TLC) plates and developed twice in a neutral lipid solvent (hexane:ethyl ether:acetic acid (60:40:1, vol/vol/vol) with drying between runs. In preliminary experiments, these preruns were shown to markedly increase migratory distance between the free fatty acid and PG spots, thus ensuring no cross-contamination between these components. The plates were subsequently developed in the solvent system created by Touchstone and colleagues (32), and the phospholipids were visualized with 8-anilino-naphthalene-sulfonic acid, scraped, and counted in a scintillation counter. In most experiments, either control (normal lung) or CLD extracts were separated on the same TLC plate simultaneously with the SP-B deficient sample extracts.

Surface Tension and Phospholipid Analysis

Lavage fluid was centrifuged at 300 × g for 15 min to remove cells, and the supernatant was then centrifuged at 48,000 × g to obtain a crude surfactant pellet (33). The pellet was resuspended in 0.15 M NaCl/5 mM CaCl₂, and aliquots were taken for determination of protein content and extraction of phospholipid for phosphorus assay (34). The lipid extract was also subjected to two-dimensional TLC (35), and the phospholipid composition was determined. DSPC was determined by extracting the PC spot and determining lipid phosphorus after treatment with OsO₄ and TLC (36). The remaining surfactant material was adjusted to a phospholipid concentration of 1.5 mM, and minimal surface tension was determined after 5 min pulsation in a pulsating bubble surfactometer (Electronetics, Buffalo, NY) (37).

Lung tissue was homogenized and a surfactant-enriched pellet was prepared as described previously (38). An aliquot of resuspended material was extracted, and total phospholipid and protein content, lipid composition, and DSPC content were determined as described.

Statistics

Mean ± standard error (SE) data are presented for replicate determinations (n  three specimens). Comparisons between study groups were made by analysis of variance (ANOVA) and Fisher t test with significance of P < 0.05.

	Results

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Patient Population

Over a 4-yr period, lung tissue and/or lavage fluid were acquired from ten infants with severe lung disease secondary to inherited deficiency of SP-B (Table 1). Six infants received a lung transplant, two infants died awaiting transplantation, and support was withdrawn from two infants after diagnosis. The genotype was determined for all infants; eight were homozygous 121ins2 (the most commonly described mutation) and two infants were compound heterozygotes for 121ins2 and separate mutations (C248X and R236C). All infants lacked detectable immunoreactive SP-B in samples of tracheal aspirate taken for the initial diagnosis and on histochemical evaluation of lung tissue obtained at transplantation or death. The age at transplantation or death ranged from 3 wk to 10 mo.

Lung tissue and/or lavage fluid were obtained from 15 other infants with other irreversible lung diseases who underwent lung transplantation (13 infants) or died (two infants) (Table 1). These lung diseases included idiopathic alveolar proteinosis, interstitial lung diseases of unknown etiology, structural abnormalities, and bronchopulmonary dysplasia. All of these infants required mechanical ventilation from birth until transplantation or death at 2 wk to 24 mo of age. These infants all had normal levels of SP-B and they comprised a comparison group, designated CLD.

Lung tissue (n = 1) and lavage fluid (n = 3) were obtained from an additional four infants with pulmonary vascular abnormalities and refractory pulmonary hypertension, all of whom required lung transplantation. These infants did not require long-term mechanical ventilation but did require supplemental oxygen until the time of transplantation. Normal postnatal lung tissue was obtained from three donor lungs when reduction of lung size was required at the time of transplantation, and lavage fluid was available from one donor lung.

The histologic appearance of the lungs from SP-B-deficient infants has been described elsewhere (1, 2, 4, 14, 18). Type II pneumocyte hyperplasia and desquamation together with variable amounts of eosinophilic proteinaceous material within the airspaces were the prominent features, prompting the histopathologic descriptions of desquamative interstitial pneumonitis or alveolar proteinosis. Except for the absence of SP-B by immunohistochemistry, the microscopic appearance of the lungs of the group of infants with other forms of CLD was indistinguishable from that of the SP-B-deficient infants (data not shown). Specifically, the type II pneumocytes were similarly abundant. Microscopic examination of the lungs from the infants with pulmonary vascular disease showed vascular proliferation and medial and intimal hypertrophy of the vessels but no abnormalities of the alveolar epithelium. For the purposes of this study, lungs of infants with vascular disease were considered to be normal. The donor lungs were histologically normal.

As an additional control for precursor incorporation experiments, data are presented for second trimester human lung tissue, which was cultured as explants for 5 d in the presence of dexamethasone. Under these conditions, lung epithelial cells differentiate into type II alveolar cells containing lamellar bodies and produce both lipid and protein components of surfactant (24, 39). The explant culture system has been extensively characterized for human fetal lung; the fetal tissue remains viable and hormone responsive for at least 5 d (24).

Lung tissue was received in cold culture medium from the referring hospital the day after transplantation or death and placed in explant culture using serum-free medium. Most of the studies described subsequently were carried out on tissue cultured for 1 d, which allowed time for the tissue to recover from storage but avoided possible changes in metabolic activity associated with more prolonged explant culture.

Content and Synthesis of SPs

As previously described (1, 4, 16), Western blot analysis of tissue from SP-B-deficient infants confirmed the absence of any immunoreactive SP-B forms, abundant SP-A, and a prominent band at approximately 6 kD on immunostaining for SP-C, which was not observed in control samples (data not shown). It has been proposed that this form of SP-C results from incomplete processing of proSP-C (1, 16), but this possibility has not been addressed previously in metabolic labeling studies.

To examine synthesis and processing of SP-B and SP-C, we performed pulse/chase studies using [³⁵S]Met/Cys as previously described for fetal lung. For SP-B, CLD lungs demonstrated initial labeling of proSP-B 40- and 42-kD forms and SP-B₂₅ intermediate; with increasing time of chase, the proSP-B signal decreased and SP-B₂₅ increased, and a faint band of SP-B₈ appeared at 4 and 8 h (Figure 1). In parallel experiments with tissue from three homozygous 121ins2 infants, no labeled protein was immunoprecipitated with the hSP-B antibody (not shown).

View larger version (86K): [in this window] [in a new window]   Figure 1.   Kinetics of SP-B processing in CLD tissue. Cultured tissue from an infant with idiopathic alveolar proteinosis was labeled with [35S]Met/Cys and chased for 2 to 20 h with Met/Cys replete medium. SP-B was immunoprecipitated and resolved by electrophoresis. After the pulse labeling (time 0 of chase), immunoprecipitated radioactivity is observed in ~ 40-kD proSP-B and ~ 25-kD intermediate. During the chase period, the signal decreases in 40- and 42-kD forms and is maximal in the 25-kD band at 4 h. Faint bands at ~ 8 kD (mature SP-B) are present at 4 and 8 h of chase. Results are representative of four experiments. NIS = nonimmune serum.

Representative results for processing of SP-C are shown in Figure 2. In CLD tissue, a 1-h pulse with [³⁵S]Met/Cys produced strong signals for proSP-C₂₁ and SP-C₂₄ and weaker bands for processing intermediates at 16 and approximately 6 kD. The antibody used in these experiments (NPROSP-C) does not recognize mature SP-C_3.7. The intensity of the SP-C₆ signal was maximal at 1 h of the chase and reproducibly undetectable after 4 h for CLD lung. In SP-B-deficient tissue, labeling of precursor 21- and 24-kD and intermediate 16-kD forms of SP-C occurred with a similar time course as for CLD infants; however, the SP-C₆ band was consistently present after 4 h of chase. These findings indicate active synthesis of proSP-C but incomplete processing in SP-B-deficient tissue.

View larger version (83K): [in this window] [in a new window]   Figure 2.   SP-C synthesis and processing in tissue from CLD (A) and SP-B-deficient patients (B). The CLD infant had interstitial lung disease and received a lung transplant at 5 mo, and the SP-B-deficient infant was transplanted at age 2 mo. Cultured tissue was labeled with [35S]Met/Cys and chased for 1 to 4 h with Met/ Cys replete medium. SP-C was immunoprecipitated and resolved by electrophoresis. In the CLD tissue, proSP-C21 appears and diminishes with time and there is transient appearance of intermediates at 24, 16, and ~ 6 kD. In the SP-B-deficient tissue, SP-C6 remains after 4 h of chase. Results are representative of three experiments. The apparent delay in appearance of SP-C6 in the SP-B-deficient tissue was not a consistent finding. NIS = nonimmune serum; MW = molecular weight markers.

Labeling experiments were also carried out with lung tissue from normal (donor) infants; however, only low amounts of radioactivity were recovered after immunoprecipitation with both anti-SP-B and anti-proSP-C antibodies (data not shown). We attribute these findings to the relative paucity of type II cells in normal lung tissue compared with both diseased lung, which has type II cell hyperplasia (40), and hormone-treated cultured fetal lung, in which most of the respiratory epithelium is comprised of type II cells (24, 39).

The overall rate of protein synthesis in cultured tissue was assessed by TCA precipitation after labeling with [³⁵S]Met/Cys. Rates of incorporation (cpm × 10⁶/µg protein) after overnight explant culture were similar for all groups: SP-B-deficient, 5.0 ± 0.6; CLD, 2.8 ± 1.6; normal, 3.2 ± 0.8; and fetal, 4.9 ± 1.6 (mean ± SE, n = three to five per group; nonsignificant [NS] by ANOVA).

Content and Synthesis of SP mRNAs

Content of SP mRNAs in lung tissue was initially examined by dot blot hybridization under stringent conditions. This approach detects partially degraded as well as intact message and provides more quantitative data than Northern blot analysis. As shown in the representative blot of Figure 3, a low concentration of SP-B mRNA was observed in tissue from a 121ins2 homozygous infant compared with the donor tissue that was processed in parallel; signals for SP-A and SP-C were comparable for the two tissues. Normalized to the -actin value, a low but detectable SP-B mRNA hybridization signal was seen for each of five SP-B-deficient patients (7.6 ± 2.1% of mean value for CLD [n = 7]; range, 2.3-13.1%). Corresponding data for SP-A and SP-C mRNA content were 180 ± 50% and 148 ± 41% of CLD (NS), respectively.

View larger version (44K): [in this window] [in a new window]   Figure 3.   Representative dot blot hybridization for quantitation of surfactant protein mRNAs. Total RNA was prepared from donor and recipient (121ins2) lung tissues and hybridized with cDNA probes for the surfactant proteins and -actin. The hybridization signal for donor and recipient tissue is comparable for SP-A and SP-C, whereas SP-B is markedly reduced in the recipient.

By Northern blot analysis, using RNA isolated after 24 h of explant culture, lung of 121ins2 infants contained primarily degraded SP-B mRNA (Figure 4B, lanes 1 and 3), whereas a strong band was seen at 2 kb for cultured fetal lung (Figure 4B, lane 5). In both donor and CLD lungs, a variable mixture of intact and degraded SP-B message was seen for Day 1 RNA (not shown). After explant culture for 4 d, however, 121ins2 tissue demonstrated increased SP-B mRNA signal, including some intact mRNA (Figure 4B, lanes 2 and 4). Some degradation of SP-A and SP-C mRNAs was also observed for 121ins2 tissues on Day 1 of culture (Figures 4A and 4C, lanes 1 and 3) compared with Day 4 of culture (lanes 2 and 4), suggesting that some autolysis occurred after surgical removal.

View larger version (29K): [in this window] [in a new window]   Figure 4.   Northern blot analysis. Equal amounts of total RNA from two SP-B-deficient infants and a fetal lung specimen (cultured 5 d with dexamethasone) were subjected to electrophoresis and blotting with cDNAs for SP-A (A), 5' SP-B (B), SP-C (C), and -actin (D). Lanes 1 and 3: SP-B-deficient tissue on Day 1 of culture; lanes 2 and 4: SP-B-deficient tissue on Day 4 of culture; lane 5: fetal lung. With SP-B probe, only a broad band < 2 kb is seen with the SP-B-deficient tissues on Day 1 (lanes 1 and 3), representing degraded mRNA, whereas some intact SP-B mRNA is present in the Day 4 samples (lanes 2 and 4, arrow). Largely intact SP-A mRNA (2.2 kb), SP-C mRNA (0.9 kb), and -actin mRNA (2.0 kb) are present in both Day 1 and Day 3 samples. A single actin mRNA band was clearly observed in lane 3 on longer autoradiographic exposure. Staining of the gel with ethidium bromide indicated intact ribosomal RNA bands of similar intensity except for decreased staining of lane 3.

Transcription rates for SP genes were assessed by nuclear RNA run-on assay as illustrated for an SP-B-deficient lung and the corresponding donor tissue (Figure 5A). The signals for each SP were comparable in the two lungs relative to that of -actin, which is expressed in most cell types and serves as an internal standard. Quantitative results for transcription rate in 11 different lungs, assayed on Day 1 of explant culture, are summarized in Table 2. The rates for SP-A, SP-B, and SP-C in three SP-B-deficient lungs were similar to the values for both CLD and normal samples. The relative rates for the three SP genes, normalized to the rate for SP-A, were also similar among the three groups.

View larger version (77K): [in this window] [in a new window]   Figure 5.   Nuclear run-on assay for transcription rates. (A) Representative results for surfactant proteins. Nuclei were prepared from donor tissue and SP-B-deficient (121ins2) tissue after 1 d (left) and 3 d (right) of explant culture. The run-on assay was performed as described in MATERIALS AND METHODS, and autoradiographic results are shown for duplicate samples (except for Day 3 donor). Compared with the signal for -actin, donor and 121ins2 data are comparable for each of the surfactant proteins. Quantitative results for all samples studied are shown in Table 2. (B) SP-B transcription rate using cDNA probes for the 5' and 3' regions. These probes detect SP-B mRNA that is labeled during the run-on assay in the 5' region, including sequence before codon 121, and in the 3' untranslated region (beyond codon 121), respectively, as described in MATERALS AND METHODS. Autoradiographic results are shown for nuclei prepared from lungs of nine patients as indicated. In each case, the intensity of the signal is greater with the 3' probe compared with the 5' probe. Quantitative results are presented in Table 2.

Comparable results for transcription rates were obtained on Day 3 of culture (Figure 5A, right panel) compared with day 1 (Figure 5A, left panel). In experiments with four lungs (one SP-B deficient, one CLD, and two normal), transcription rates for SP-A, SP-B, SP-C, and actin on Days 3-4 of culture were 79 to 89% of the Day 1 value. Overall RNA synthetic activity of the tissues on Days 3-4 of culture, as assessed by rate of precursor incorporation, was 61 to 109% of the initial level after overnight culture (Day 1).

The experiments of Figure 5A and Table 2, using a 5' SP-B cDNA probe in conjunction with RNase A treatment after hybridization, detected only those SP-B mRNA molecules that were labeled in the 5' region during the in vitro run-on reaction (i.e., growing mRNA molecules recently initiated in vivo before freezing tissue). To investigate whether the rate of transcription of the 3' region of SP-B gene (i.e., distal to both codon 121 and the premature stop codon) was affected by the presence of the 121ins2 mutation, we used an additional SP-B cDNA probe corresponding to the 3' untranslated region of the gene. In a comparison of nine samples, a stronger signal was consistently observed with the SP-B 3' probe compared with the 5' probe (Figure 5B). The ratio of signal intensity with the 3' probe versus 5' probe was not different for three SP-B- deficient tissues compared with both CLD and normal specimens (Table 2). These findings establish that the mutated SP-B gene is transcribed in vitro at a normal rate, under the conditions used, and that SP-A and SP-C gene expression is not altered in SP-B-deficient lungs.

Phospholipid Studies

We examined phospholipid content, surface activity, and composition in SP-B-deficient and control infants, and data are summarized in Table 3. The ratio of total phospholipid to protein was significantly lower in both lavage fluid and tissue of SP-B-deficient and CLD infants compared with normal tissue. This change could reflect decreased lipid content and/or increased protein content as a result of fibrosis and pulmonary edema. The minimal surface tension of a surfactant fraction isolated from lavage fluid was similarly elevated in both SP-B-deficient and CLD infants compared with normal infants. Whereas this likely reflects absence of SP-B (and mature SP-C) in the deficient infants, the cause in CLD infants may be multifactorial.

Phospholipid composition was determined for surfactant fractions prepared from both lung tissue and lavage fluid. Compared with normal infants, SP-B-deficient infants had elevated phosphatidylinositol (PI) and percent DSPC (tissue) and decreased content of PG in both lavage and tissue. CLD infants had a more marked alteration in composition compared with normal infants, with elevated content of sphingomyelin, phosphatidylserine, and phosphatidylethanolamine, and decreased PG, PG/PI, PC, and DSPC (lavage only). All of the differences in composition between SP-B-deficient and CLD infants reflected the abnormalities of CLD infants relative to normal infants, perhaps reflecting longer and/or more severe disease of CLD infants, with greater sloughing of epithelial cells and therefore increased content of membrane phospholipids.

To examine synthesis of phospholipids, we incubated explanted lung tissue with labeled lipid precursors and determined the rate of incorporation into phospholipid (Table 4). Compared with the tissue of CLD infants, SP-B- deficient tissue demonstrated elevated rates of choline incorporation into PC (approximately 2-fold) and acetate incorporation into total phospholipids (approximately 14-fold); the rates of glycerol incorporation into phospholipid were not significantly different between the two groups. The rates for the two available normal lungs were variable. Rates of choline and acetate incorporation for hormone-stimulated fetal lung, which has a high proportion of type II cells, had higher mean values.

The composition of newly synthesized phospholipid, assessed after incorporation of labeled acetate, is shown in Table 5 for SP-B-deficient and CLD groups together with data from fetal lung for comparison; tissue from only one normal control lung was available for these studies. Compared with CLD tissue, the tissue of SP-B-deficient infants had decreased amounts of phosphatidylethanolamine, PI, and sphingomyelin (P < 0.05). Thus, different synthetic rates between SP-B-deficient and CLD infants may in part be responsible for observed differences in content (Table 3) of sphingomyelin and phosphatidylethanolamine but not phosphatidylserine or PC. The relatively high level of PG synthesis in both SP-B-deficient and CLD tissue (8 to 12% of total phospholipid) suggests that the low content of PG in tissue and lavage of both groups (Table 3) is related to increased turnover.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, we describe the first studies on surfactant protein gene expression and phospholipid biosynthesis in lungs of human infants with SP-B deficiency. We included two comparison groups: infants with refractory lung disease from a variety of causes other than inherited SP-B deficiency (CLD) and infants with normal alveolar epithelium. The CLD infants were a heterogeneous group, and it is possible that some of the infants have, as yet uncharacterized, genetic diseases as a cause of their lung disease. The importance of the CLD comparison group in this study of SP-B deficiency is underscored by findings of surfactant abnormalities common to both groups of infants (decreased phospholipid/protein ratio, increased surface tension, decreased PG content). The lungs of both CLD and SP-B-deficient infants had been subjected from birth to the same therapeutic interventions (oxygen, assisted ventilation, and in some cases extracorporeal membrane oxygenation), and these treatments may have affected type II cell biosynthetic activity independent of the underlying disease process. The normal, donor infants received short-term ventilatory support but their lungs were not exposed to significant volutrauma and oxygen toxicity, and infants with pulmonary vascular disease received only supplemental oxygen. The proportion of type II cells in these lungs was normal in contrast to the type II cell hyperplasia typical of all forms of CLD. As a result, studies of SP-B and SP-C biosynthesis were not technically possible in normal tissue owing to the low levels of label incorporated into immunoprecipitable products.

As expected, we could not demonstrate any primary translation product of the mutated SP-B mRNA. The 121ins2 mutation alters subsequent amino composition and introduces a premature stop codon at amino acid 214. Any translation product formed would be expected to migrate at approximately 20 kD on PAGE and would not be recognized by the antiserum to mature 8 kD SP-B (Phe₂₀₁ through Met₂₇₉). In view of the very low content of SP-B mRNA, however, appreciable levels of the abnormal primary translation product are unlikely.

The time course of labeling and initial processing of proSP-C was similar in SP-B-deficient lung tissue and CLD. Based on gel migration and epitope recognition, an identical, approximately 6-kD intermediate occurred in both types of tissue. The delayed disappearance of the SP-C₆ band provides direct evidence for a block in SP-C processing in SP-B-deficient tissue. This probably reflects the absence of normal lamellar bodies where the latter events in SP-C processing have been localized (8, 41), whereas processing of proSP-C to the 16-kD intermediate appears not to be specific for type II cells (42).

In the initial description of the 121ins2 mutation (1), SP-B mRNA was not detected by Northern blot analysis of total RNA, and content was approximately half the expected level in a compound heterozygous SP-B-deficient infant with a point mutation on one allele (121ins2/R236C) (4). Our current studies establish that most if not all affected infants have a low but detectable amount of degraded SP-B mRNA. By contrast, mRNAs for other surfactant proteins and -actin were largely intact. The findings of a normal transcription rate and low mRNA content are consistent with an accelerated rate of degradation of mutated SP-B mRNA in vivo. Attempts to determine SP-B mRNA t_1/2 using RNA synthesis inhibitors were unsuccessful owing to the low level of message.

The decreased content of SP-B mRNA associated with the 121ins2 mutation represents an example of nonsense-mediated mRNA decay that has been described for a variety of human genes (43). The magnitude of decrease in specific transcript level is variable with different nonsense mutations in the same gene (47, 51), and a reduction of mRNA is generally not found with nonsense mutations occurring in the 3' exons (47, 52). Reduced mRNA content is not a feature of missense mutations, and elimination of the premature stop codon by conversion of a nonsense mutation to a missense mutation restores normal mRNA content in transfected cells (52).

There is little previous information regarding the possible role of decreased initiation of transcription in mutated genes with premature stop codons. In a study of transfected cells, Cheng and Maquat (48) found the same rate of transcription for mutated and wild-type human triosephosphate isomerase cDNAs; however, Baserga and Benz (53) reported a decreased transcription rate for transfected human -globin cDNA with a nonsense mutation. Using probes for both 5' and 3' regions of the gene, we found normal rates of SP-B gene transcription for alleles containing the 121ins2 mutation. This finding suggests that the decreased content of nuclear, as well as cytoplasmic, mRNAs found for other genes with nonsense mutations reflects post-transcriptional events (48, 53). It is likely that SP-B mRNA stability is affected by the mutations, but this issue had not been addressed experimentally. Current models for nonsense-mediated mRNA decay involve degradation of mRNA attached to ribosomes resulting from the premature stop in translation, and studies in yeast implicate both cis-acting instability elements and trans-acting factors in the degradation process (54, 55).

Functional activity of surfactant from SP-B-deficient patients, as assessed by the pulsating surfactometer technique, was abnormal as expected, based on the known role of SP-B and SP-C in formation and stability of the surfactant phospholipid layer (5). This finding is consistent with the clinical course of these infants who present shortly after birth with respiratory failure secondary to atelectasis. Surfactant isolated from CLD infants also had abnormal surface tension lowering ability. This abnormality has been described previously for older patients with acute respiratory distress syndrome (ARDS) and could result from the alterations in surfactant content and composition and/or through inactivation by serum proteins (56).

Previously, Hamvas and coworkers (18) reported low PG and normal PC in lavage fluid of two SP-B-deficient patients. The current studies confirm this finding and establish that deficient PG is a feature of ongoing infant lung disease from a variety of causes. This was the only abnormality in phospholipid composition of lavage surfactant of SP-B-deficient infants compared with that of normal infnats, and PG was also decreased in surfactant isolated from lung tissue (Table 3). CLD infants demonstrated even lower concentrations of PG in both lavage and tissue. These findings are in agreement with previous observations of reduced PG content in surfactant isolated from lungs of adults with ARDS (56). To our knowledge, however, the synthesis of PG has not been previously studied in injured human lung tissue. We found normal to elevated amounts of PG in newly synthesized phospholipids, suggesting that the decreased PG content reflects increased degradation. PG turnover is controlled by the action of acylhydrolases and other phospholipases, although to our knowledge these pathways have not been extensively studied for PG in the lung. It is possible that the reduced PG content associated with CLD contributes to the abnormal surface tension lowering properties of lavage surfactant from these infants.

The overall rate of phospholipid synthesis was greater in SP-B-deficient lung than in other CLD tissue using both choline and acetate as precursors. Because tissue in explant culture contains all pulmonary cell types, this finding cannot be directly attributed to surfactant production in type II cells. Nevertheless, it is unlikely that synthesis of surfactant phospholipid is impaired in postnatal SP-B-deficient lungs, which is in agreement with data for synthesis and content of DSPC in lungs of SP-B-deficient fetal mice (57).

SP-B deficiency is associated with several alterations in type II cell function and ultrastructure, although at present there is limited understanding of the mechanisms mediating these changes. It has been proposed that there is a critical role for SP-B in formation of lamellar bodies within type II cells, perhaps mediating fusion of multivesicular bodies and ordering the lamellar structure of lipids (17). With SP-B absent, multivesicular bodies would accumulate, and lamellar structures that might form would be disorganized and incomplete, producing the membranous vesicles that are observed. Because cleavage of precursor SP-C to the mature form appears to occur in lamellar bodies (8, 41), processing would be disrupted and the 6-kD proSP-C intermediate would accumulate. If recycling of SP-A were disrupted because of abnormal formation of lamellar bodies in SP-B deficiency, this could account for the increased content of SP-A in airspaces and decreased amount within type II cells. Defective formation and secretion of lamellar bodies in the absence of SP-B could also delay the normal accumulation of phospholipid and surfactant proteins in amniotic fluid as reported in one case (15). The lack of efficacy of replacement surfactant in inherited SP-B deficiency (15) emphasizes the importance of normal type II cell ultrastructure and function in establishing a surface active film in the alveolus.