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Published ahead of print on March 20, 2003, doi:10.1165/rcmb.2002-0149OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 259-266, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0149OC

In Vitro Surfactant Protein B Deficiency Inhibits Lamellar Body Formation

Cherie D. Foster, Peggy X. Zhang, Linda W. Gonzales and Susan H. Guttentag

Division of Neonatology, Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Address correspondence to: Susan Guttentag, M.D., Abramson Research Center 416G, Children's Hospital of Philadelphia, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail: guttentag{at}email.chop.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein (SP) B is essential for normal pulmonary surfactant activity and lamellar body genesis in type 2 cells. However, the role of SP-B in lamellar body genesis is poorly understood. We developed an adenovirus vector expressing antisense SP-B as an alternative in vitro model of SP-B deficiency to begin to explore the role of SP-B in lamellar body genesis. RT-PCR analysis revealed that antisense SP-B expression interfered with translation of endogenous SP-B mRNA. Antisense SP-B expression resulted in reliable in vitro reproduction of many features of SP-B deficiency, including absent mature SP-B and decreased lamellar bodies and SP-C. Light and electron microscopy demonstrated significant reductions in lamellar body number. Western blotting revealed a significant reduction in mature 8-kD SP-B protein and decreased mature SP-C. Our data indicate that antisense SP-B can be effectively used to replicate the SP-B–deficient type 2 cell phenotype in vitro, and provides an attractive alternative to transgenic models for the further study of the role of SP-B in lamellar body genesis.

Abbreviations: enhanced green fluorescent protein, EGFP • glyceraldeyde-3-phosphate deydrogenase, GAPDH • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The lamellar body of the alveolar type 2 cell is a lysosome-derived organelle similar in many respects to other specialized membrane-bound organelles such as melanosomes, platelet dense granules, neutrophil azurophil granules, and the lytic granules of natural killer and cytotoxic T cells (1). These organelles possess a low lumenal pH and contain lysosome-associated membrane proteins in their limiting membranes. The lamellar body is involved in the synthesis, organization, and secretion of pulmonary surfactant from the type 2 cell, just as the melanosome is necessary for the synthesis and storage of melanin for export from the melanocyte. However, the process of lamellar body genesis is poorly understood.

Ultrastructural studies suggest that the lamellar body is derived from the maturation and fusion of multivesicular bodies into composite bodies and finally lamellar bodies (2). The only critical component of this process identified to date is the hydrophobic surfactant protein (SP)-B. Disrupted lamellar body genesis is a prominent feature of SP-B deficiency in both humans (with a variety of SP-B gene mutations [3]) and in an SP-B knockout mouse model (4). Additional ultrastructural findings include increased numbers of vesicles and multivesicular bodies, further supporting the role of the multivesicular body in lamellar body genesis. Although there is evidence of secreted surfactant phospholipids in the alveolar space of SP-B–deficient humans and transgenic mice, the surfactant lacks mature SP-B and the other hydrophobic surfactant protein, SP-C, rendering the surfactant dysfunctional (5). The combination of accumulating intermediate peptides of SP-C biosynthesis with reduced amounts of mature SP-C indicates a relative block in SP-C post-translational processing.

The mechanisms by which SP-B fosters lamellar body genesis are poorly understood. Transgenic mouse models, although useful in elucidating critical components of the SP-B proprotein, have not clarified the process of lamellar body genesis. Antisense technology provides an attractive in vitro method to study the role of SP-B in this process. Gene expression can be selectively inhibited by adenoviral-mediated introduction of complementary antisense RNA (reviewed in Refs. 6, 7) or, more recently, by the introduction of small inhibitory synthetic RNA oligonucleotides (reviewed in Ref. 8). The mechanisms by which antisense RNA silences gene expression are varied, but are postulated to involve translation disruption via physical obstruction of ribosome attachment, or increased degradation of RNA: RNA duplexes by RNases (6, 9). We hypothesized that antisense RNA expression would facilitate the study of lamellar body genesis in a cell culture model of type 2 cell differentiation. Because of the well-characterized type 2 cell phenotype in SP-B deficiency, we sought to duplicate these findings using adenoviral-mediated antisense SP-B expression during type 2 cell differentiation in vitro. We describe herein the effects of antisense SP-B mRNA on type 2 cell phenotype and SP synthesis, and demonstrate that this phenotype can be reliably reproduced without undue cellular toxicity. As such, we show that the combination of adenovirus-mediated antisense expression and our in vitro model of type 2 cell differentiation is a powerful tool for future studies of the key components of lamellar body genesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Dexamethasone, isobutyl methylxanthine, and 8-bromo-cAMP were obtained from Sigma Chemical (St. Louis, MO). Waymouth's media (GIBCO BRL) and human embryonic kidney 293 cells (HEK) were obtained from the Cell Center, University of Pennsylvania (Philadelphia, PA). All other reagents were electrophoretic grade and were purchased from Fisher Scientific (Fair Lawn, NJ), Pierce (Rockford, IL), Invitrogen (Carlsbad, CA), or Sigma Chemical.

Antibodies
Rabbit anti-human SP-A, a polyclonal antibody that recognizes human SP-A but not other surfactant proteins or serum proteins, and an epitope-specific proSP-C rabbit antiserum (NPROSP-C; Met10–Glu23 of rat proSP-C) were the kind gifts of Dr. Michael Beers. Mature SP-C protein was detected using rabbit polyclonal antiserum to mature SP-C substituting Phe for Cys 3 and 4 of the mature sequence (Byk Gulden, Konstanz, Germany). Human SP-B antiserum was prepared using purified human SP-B8 isolated from patients with pulmonary alveolar proteinosis as described previously (10). Mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antiserum (Chemicon, Temecula, CA) was used to assess loading of Western blots.

Construction of the Antisense Plasmid
The recombinant adenoviral vector H5010.CMV.asSPB (AdCMVasSPB) was prepared by directional cloning using the Adeno-X Expression System (Clontech Laboratories, Palo Alto, CA) first developed by Mizuguchi and Kay (11, 12). A SpeI/Bam HI segment containing the first 649 base pairs of the SP-B cDNA was cloned in antisense orientation into the pShuttle plasmid (Figure 1). Next, a fragment containing the CMV promoter, antisense SP-B cDNA, and BGH poly A was excised by PI-Sce I/I-Ceu-I digestion. This segment was then ligated into the replication-incompetent Ad5 E1/E3-deletion adenovirus genome (contained within the pAdeno-X plasmid) via an in vitro ligation reaction, followed by Swa I digestion to remove wild-type vector. Positive clones of AdCMVasSPB were identified by PCR screening. Using similar techniques, we constructed AdCMVasSPBCMV-EGFP, which expresses both antisense SP-B and enhanced green fluorescent protein (EGFP) RNA from separate promoters.



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  		Figure 1. Construction of Ad.CMVas SPB-EGFP. Schematic diagram of the orientation of bases 1–674 (gray box, capital letters) of the SP-B cDNA after directional cloning into a shuttle plasmid used to construct AdCMVasSPB-EGFP. Antisense RNA expression is controlled by a proximal CMV promoter and is terminated in a bovine growth hormone (BGH) polyadenylation signal. This is followed by a distal CMV promoter controlling the expression of EGFP. The arrows indicate the forward and reverse primers for RT-PCR of antisense mRNA; the reverse primer anneals to vector sequences (gray box, lower case letters).

 
Viral Stock Preparation
Low passage HEK 293 cells were successfully transfected with Pac I–digested recombinant adenoviral DNA using Fugene 6 (2 vols/µg DNA; Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Cells were carried for 7–10 d until cytopathic effect was noted. The resultant cell pellets were expanded and purified by the Institute for Human Gene Therapy, University of Pennsylvania Vector Core Lab. The viral titer of the resultant stock was analyzed by plaque assay and particle number was determined by measuring the A260 of purified virus (1 A260 = 1012 viral particles). The standard concentration of the prepared AdCMVasSPB was 4.5 x 1012 particles per ml. The stock solution was stored at -70°C until ready for use, and a fresh aliquot was used for each experiment. Two additional adenoviral vectors were utilized as controls in the experiments that follow: AdCMVsSPB, prepared from full length SP-B cDNA in sense orientation using the methods described above, and AdCMVEGFP, a recombinant adenovirus expressing EGFP. Both control vectors were purchased from The Institute for Human Gene Therapy Vector Core, University of Pennsylvania School of Medicine.

Cell Culture
Human fetal lung was obtained from second-trimester therapeutic abortions (14–18 wk estimated gestational age) under protocols approved by the Committee for Human Research, Children's Hospital of Philadelphia. Fetal lung parenchyma was dissected free of large airways, chopped into 1 mm3 explants, and cultured in Waymouth media on a rocking platform as previously described (13). After overnight explant culture, epithelial cells were isolated with trypsin, collagenase, and DNase digestion, followed by panning on plastic culture dishes to remove contaminating fibroblasts as described previously (14). Ten nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutyl methylxanthine (DCI) were added to the culture media to induce type 2-cell differentiation. These conditions have been shown to induce the type 2 cell phenotype, including: SP-A, SP-B, SP-C, and SP-D RNA and protein expression, SP-B and SP-C post-translational processing, phospholipid enzyme expression, and formation of lamellar bodies and microvilli (14). Cells were treated with adenovirus at 1 x 102 to 5 x 104 particles/cell, simultaneously with DCI. Control groups included cells treated with DCI alone, the adenovirus containing "sense" SPB or AdCMVEGFP. Viral exposure occurred for 24 h. Subsequently, fresh Waymouth media with DCI was replaced daily until the cells were harvested 48–120 h after viral treatment.

Toxicity Studies
Cytotoxicity was assessed by trypan blue exclusion assay. Cultured cells were washed, incubated with 0.4% trypan blue for 5 min, and counted at a total magnification of x400 (four fields in each of two dishes per treatment group). Live cells excluding trypan blue were expressed as a percentage of total cells. Cell viability was assessed by labeling live cells with Carboxy SNARF-1, a long-wavelength fluorescent pH indicator (Molecular Probes, Eugene, OR). Cells cultured on glass coverslips were washed, then incubated with 2 µM Carboxy SNARF-1 (1,000x stock solution in high-quality anhydrous dimethylsulfoxide) for 30 min (15), counterstained with the nuclear stain DAPI, fixed with 1% paraformaldehyde in phosphate-buffered saline, then washed and mounted onto glass slides using the Prolong Antifade Kit (Molecular Probes). Fluorescence was examined with an Olympus 1X70 microscope and Metamorph imaging system (Universal Imaging Corporation, West Chester, PA), with viable cells exhibiting fluorescence emission between 580 and 640 nanometers, detectable using a Texas Red filter. Live cells (DAPI-positive, SNARF-positive) were expressed as a percentage of total cells.

Morphology
Cells were fixed in 2.5% glutaraldehyde and 0.1M Na Cacodylate buffer (pH 7.2) for 1 h, followed by overnight post-fixation in 0.1 M Na Cacodylate at 4°C. Preparation of all samples for morphologic analysis was performed at the Biomedical Imaging Core Facility at the University of Pennsylvania.

For examination by light microscopy, cells were embedded in epon, and thick sections were stained with toluidine blue by methods previously described (13). Toluidine blue sections were assessed by field counting of lamellar bodies and type 2 cells under oil immersion light microscopy at a total magnification of x600. Lamellar bodies and numbers of cells exhibiting microvilli were counted in four fields per slide/treatment group.

For transmission electron microscopy, ultrathin sections were cut with a diamond knife, and contrasted with uranyl acetate and lead citrate, as previously described (16). Ultrastructure was examined in a JEOL CX100II transmission electron microscope operated at 80 kV, in the Biomedical Imaging Core of the University of Pennsylvania School of Medicine. Two to three blocks were prepared and examined from each experimental group. Electron micrographs of 6–8 grid subdivisions per section were taken at x2,500 and x7,500–x10,000 magnification. The total number of cells, percentage of cells with microvilli, and number of lamellar bodies/microvilli-positive cell were determined within each grid.

Western Blot Analysis
Cell pellets were sonicated and total protein was quantified by the method of Bradford (17). For SP-A, SP-B, ProSP-C, and GAPDH analysis, the cell pellets were solubilized in 20 µl of gel sample buffer, separated using a 12% NuPAGE Bis-Tris gel with MES SDS Running Buffer, and transferred to Duralose (Stratagene, Cedar Creek, TX), as per the manufacturer's protocol (Invitrogen). For SP-C analysis, SDS-PAGE was performed in 12.5% polyacrylamide gels using a Tris-Tricine buffer system as described previously (18). Electrophoresed samples were transferred to Duralose at 20 mA/cm2 for 13–16 h for subsequent immunoblotting. Immunoblotting was performed using a horseradish peroxidase system (Bio-Rad Laboratories, Hercules, CA), and bands were visualized by enhanced chemiluminescence using the SuperSignal West Pico enhanced chemiluminescence kit (Pierce) as previously described (19). Primary antibody concentrations were as follows: SP-A, SP-B, and ß-actin at 1:5,000; NProSP-C at 1:4,000; and GAPDH at 1:10,000. Secondary antibody was used at a dilution of 1:10,000 for all primary antibodies used. Blots were stripped free of antibody by using the Reblot Western Blot Recycling Kit (Chemicon) for 20 min at 50°C and then reprobed with an additional primary antibody.

mRNA Analysis
Total RNA was isolated from cell pellets with RNA STAT-60 Reagent (Tel-Test, Friarswood, TX) according to the manufacturer's instructions. Purity was verified by OD 260:280 ratio and by visualization of ribosomal RNA bands on a formaldehyde agarose gel. Samples were then treated with RQ1 RNase-free DNase (Promega, Madison, WI) and ethanol precipitated after phenolchloroform extraction. cDNA was synthesized from RNA samples using the SuperScript First-Strand RT-PCR kit (Invitrogen). The following primers were used for RT-PCR: sense-oriented SP-B, forward: 5'-AGGACATCGTCCACATCCTT-3', sense-oriented SP-B, reverse: 5-'GAGCAGGATGACGGAGTAGC-3' with an amplicon length of 556 corresponding to bases 218–774 of the human SP-B mRNA sequence (GenBank reference NM_000542); antisense SP-B, forward: 5'-CACAGGGAGGACGAGCTT-3', antisense SP-B reverse: 5'-GCTTGAGCTCGAGATCTGATA-3', with an amplicon length of 571 (see Figure 1); GAPDH, forward: 5'-ACCACAGTCCATGCCATCAC-3', GAPDH, reverse: 5'-TCCACCACCCTGTTGCTGTA-3', with an amplicon length of 451 corresponding to bases 601–1052 of the human GAPDH mRNA sequence (GenBank reference NM_002046). Each PCR product was sequenced by the Nucleic Acid and Protein Core Facility of the Children's Hospital of Philadelphia and the sequence confirmed by BLAST search. PCR was performed on 1 µg cDNA with PCR conditions of 94°C x 5 min (94°C x 30 s, Annealing Temp [57°C for SP-B/GAPDH and 55°C for antisense SP-B/GAPDH] x 30 s, 72°C x 30 s) x 30 cycles and 72°C x 10 min. Cycle number was determined to be in the linear response range for GAPDH amplification. Samples of the final reaction (5 µl) were run on 2% agarose gels with ethidium Br, and UV images were obtained using a Kodak Digital Science Imaging System 1D 2.0.2 (New Haven, CT). Images were scanned and quantified using MacBas v2.4 software (Fuji Photo Film, Tokyo, Japan) and analyzed with Statview 4.1 software (Abacus Concepts, Berkeley, CA).

Statistical Analysis
Results are given as mean ± SE unless otherwise specified. Data were compared for significance using Students' t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antisense SP-B Selectively Decreases SP-B Expression in a Dose-Dependent Fashion without Significant Toxicity
Undifferentiated epithelial cells expressed proSP-B with no processing to mature SP-B and, after 72 h of hormone treatment alone, exhibited a ~ 11-fold increase in 8-kD mature SP-B (10.7 ± 3.1-fold, n = 3), as previously noted (14, 18). Uniformity of transduction by viral vectors was monitored using recombinant adenovirus-expressing EGFP, and was 65 ± 2% at viral doses between 1,000–10,000 particles/cell (n = 7). Viral treatment using a vector expressing EGFP did not alter SP-B8 expression compared with DCI alone (EGFP vector, 2.69 ± 0.63 densitometry units; no virus, 2.22 ± 0.59 densitometry units, n = 3, P = NS).

Treatment with AdCMVasSPB or AdCMVasSPB-EGFP during differentiation of type 2 cells resulted in a significant dose-dependent decrease in both 42-kD proSP-B and mature 8-kD SP-B (Figure 2) at >= 1,000 particles/cell. At 1,000–10,000 particles/cell, there was an average decrease of 48 ± 12% (P < 0.01, n = 7) in mature SP-B8 compared with hormone-treated controls. In contrast, SP-A, which is not dependent upon lamellar bodies for de novo synthesis (20), and proSP-C, the unmodified SP-C primary translation product, were unchanged.




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  		Figure 2. Dose dependency of SP-B expression in response to antisense SP-B. (A) Representative (n = 7) Western blot analysis for human SP-A, SP-B, and proSP-C in AdCMVasSPB-treated cells at 72 h. Forty-two–kilodalton proSP-B and mature 8-kD SP-B were decreased in cells treated with AdCMVasSPB at doses >= 1,000 particles/cell. SP-A at 34–36 kD and proSP-C at 21 kD were not affected by antisense treatment compared with an adenovirus vector containing full-length SP-B cDNA and untreated control cells. (B) Densitometry of immunoblots from dose–response experiments (n = 3–7) demonstrated a statistically significant decrease in mature 8 kD SP-B protein in antisense-treated versus control cells (*P < 0.05 versus control/no virus) at doses >= 1,000 particles/cell.

 
Cell viability was assessed by trypan blue exclusion and Carboxy-SNARF-1 staining (Table 1). Carboxy SNARF-1, a long wavelength fluorescent pH indicator, passively crosses cell membranes as a polar ester, and then is hydrolyzed by intracellular esterases into a polar compound that cannot leave intact cells. At physiologic intracytoplasmic pH, viable cells emit fluorescence at 580–640 nanometers. Treatment with AdCMVasSPB up to 10,000 particles/cell (n = 3–5 experiments) resulted in no significant difference in cell viability using either assay compared with control.


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  		TABLE 1 Cell viability after viral treatment

 
Antisense SP-B Inhibits Lamellar Body Genesis
Type 2 cell morphology was examined first by light microscopy, using Toluidine blue to detect lamellar body inclusions in those cells with obvious microvilli. Undifferentiated lung epithelial cells exhibited no lamellar bodies, as previously noted in both cells (14) and lung explants (13). As expected, cells treated with DCI demonstrated abundant microvilli and Toluidine blue inclusions/high power field (Toluidine blue positive lamellar bodies/cell: 9.3 ± 0.38). By contrast, cells treated with AdCMVasSPB or AdCMVasSPB-EGFP at 5,000–10,000 particles/cell exhibited significantly decreased numbers of lamellar bodies (5,000 particles/cell: 2.17 ± 0.12, 10,000 particles/cell: 2.19 ± 0.14, P < 0.01, n = 3) compared with control.

Morphologic differences were more obvious by electron microscopy. Figure 3 shows composite EM images from a representative experiment. As expected, undifferentiated lung epithelial cells had scant microvilli and large amounts of glycogen. Cell cultures treated with DCI demonstrated numerous differentiated type 2 cells, with abundant microvilli. In comparison, whereas 99 ± 0.9% of DCI-treated cells had microvilli, only 5.0 ± 1.6% of preculture cells exhibited microvilli (P < 0.001, n = 15).



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  		Figure 3. Differentiating type 2 cells cultured with antisense SP-B (AdCMVasSPB) demonstrate a paucity of lamellar bodies. Representative electron micrographs at x5,000 magnification. Freshly isolated undifferentiated epithelial cells (A) demonstrated none of the characteristics of differentiating type 2 cells (B), specifically microvilli and large, abundant lamellar bodies. In contrast, cells treated with antisense SP-B at 10,000 particles/cell (C) demonstrated decreased numbers of lamellar bodies.

 
Because microvilli are a reliable marker of type 2 cells, we examined mean lamellar body number in all microvilli-positive cells. DCI-treated control cells demonstrated 6.3 ± 0.25 lamellar bodies/cell, whereas no lamellar bodies were seen in preculture cells (n = 3 experiments, P < 0.01). Cells treated with AdCMVasSPB at 5,000–10,000 particles/cell had significantly decreased numbers of lamellar bodies (2.4 ± 0.1 lamellar bodies/cell) compared with DCI-treated controls (n = 3 experiments, P < 0.01).

Antisense SP-B Alters SP-C Processing
Because of the known association of abnormal SP-C processing with SP-B deficiency (5, 21), we examined the effects of antisense SP-B on the expression of SP-C by Western immunoblotting. As mentioned earlier, proSP-C was not altered by antisense treatment (Figure 2A). We have previously observed in this culture model that although SP-B expression is robust by 72 h, mature SP-C3.7 expression is not reliably detected until 5 d in culture (14). Figure 4 shows a representative time course of SP-C expression compared with SP-B in differentiating, hormone-treated human alveolar type 2 cells in the presence or absence of AdCMVasSPB. Mature SP-B was decreased throughout the 120-h time course in antisense-treated cells. Mature SP-C, which was not detectable before 96 h in control cells, was also decreased in antisense-treated cells. There was no detectable accumulation of SP-C intermediates over this period.



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  		Figure 4. Time course of SP-B and SP-C expression in antisense SP-B–treated type 2 cells. Left: time course of SP-B expression in cells treated with DCI ± AdCMVasSPB at 7,500 particles/cell. In the absence of antisense SP-B, mature 8-kD SP-B appeared at 72 h in culture, with robust expression after 120 h. In contrast, cells treated with antisense SP-B demonstrated decreased mature SP-B. Right: time course of mature SP-C expression. Stable amounts of proSP-C were detected throughout the time course in both untreated and antisense-treated cells. Mature SP-C was not detected in cells cultured without virus until 120 h (upper right panel; previously published in Ref. 14). In contrast, there was a decrease in the expression of mature SP-C in cells exposed to the antisense vector (lower right panel). *Nonspecific band.

 
The Mechanism of Action of Antisense SP-B Does Not Involve RNA Degradation
Antisense RNA interference with endogenous gene expression results from duplex formation followed by either RNA degradation or interference with translation. To determine the mechanism of action of antisense SP-B inhibition of endogenous SP-B protein expression, we developed RT-PCR to examine expression of both antisense and endogenous SP-B mRNA. The antisense SP-B primers were chosen to eliminate the possibility of amplification off of endogenous SP-B message. This was accomplished by choosing the reverse primer from bacterial vector sequence lying between the antisense sequence and the bovine growth hormone polyA sequence (Figure 1). No homology of this primer sequence with the human SP-B mRNA sequence was detected by BLAST searching. In addition, primers for endogenous SP-B were chosen to eliminate the possibility of amplification off antisense SP-B mRNA. The reverse sense SP-B primer consists of bases 755–774 of the human SP-B mRNA, and no homology of this primer sequence with the antisense SP-B adenovirus construct was detected by BLAST searching. Thus, the PCR reactions were specific for detecting sense or antisense products.

Over the range of virus doses tested, endogenous SP-B mRNA expression remained constant and there was no indication of RNA degradation (Figure 5). Furthermore, Northern blot analysis confirmed stable, intact SP-B message (not shown). By contrast, antisense SP-B mRNA increased in a dose-dependent fashion, and corresponded to the observed dose-dependent decrease in SP-B protein levels at 72 h (Figure 2). The constant expression of intact, endogenous SP-B mRNA in association with decreasing protein levels indicates antisense interference with message translation.




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  		Figure 5. Endogenous SP-B mRNA expression is stable with increasing AdCMVasSP-B/EGFP dose. (A) Representative RT-PCR from a dose–response experiment showing expression of antisense SP-B, endogenous SP-B, and GAPDH at 72 h. (B) Graphic representation of RT-PCR results from three experiments for antisense (hashed bars) and endogenous SP-B mRNA (solid bars). Results were corrected for loading based on GAPDH expression. Expression of antisense SP-B mRNA was dose-dependent from 100–10,000 particles/cell. *P < 0.05 versus 100 particles/cell, n = 3.

 
We also performed a time-course analysis of antisense SP-B RNA and endogenous SP-B mRNA expression (Figure 6), which demonstrated stable expression of endogenous SP-B mRNA over 168 h after viral treatment. Unexpectedly, antisense SP-B mRNA decreased beyond 72 h of viral treatment (n = 4). This corresponded with our observations of mature SP-B protein in antisense-treated cells at 96 and 120 h by immunoblotting (Figure 5, lower left panel). These data suggest that transduced cells are silencing the antisense message produced over time in culture, allowing the stable endogenous SP-B mRNA to be translated and processed to mature SP-B.



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  		Figure 6. Time course of antisense SP-B/EGFP mRNA expression. Representative RT-PCR from a time course experiment showing expression of antisense SP-B, endogenous SP-B, and GAPDH of the at 7,500 particles/cell of AdCMVasSPB/EGFP. In virus-treated cells, antisense mRNA expression peaked at 72 h after viral exposure, decreasing over the next 96 h, respectively (A). Endogenous SP-B mRNA expression was stable throughout the time course in both control and antisense SP-B/EGFP-treated cells (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lamellar body genesis is a vital but poorly understood process in alveolar type 2 cells. The lamellar body lies at the intersection of the secretory pathway for surfactant proteins, the synthetic pathway for surfactant specific phospholipids, and the endocytic pathway, through which recycled surfactant components return for re-export from the type 2 cell. We have now shown that the combination of antisense technology and an in vitro model of type 2 cell differentiation are powerful tools for the study of lamellar body genesis. We have duplicated many features of the SP-B–deficient type 2 cell phenotype, most importantly disruption of lamellar body formation. Antisense SP-B expression reliably and predictably perturbed type 2 cell phenotype in a dose-dependent fashion without significant cytotoxicity through interference with protein translation, rather than RNA:RNA duplex degradation. These studies provide a valuable foundation for future studies directed at perturbing proteins and processes essential for lamellar body formation.

Antisense strategies have been used successfully to selectively inhibit gene expression in many different in vitro systems (reviewed in Refs. 6, 7, 22). We selected antisense RNA methodology for superior intracellular stability, as compared with antisense DNA, which improves the duration of expression (reviewed in Ref. 7). In addition, the superior transfection efficiency by recombinant adenovirus allowed for detectable reductions in SP-B protein expression in our primary cell cultures. Little is known about the fate of RNA:RNA duplexes. The mechanisms examined to date include enzymatic degradation of duplexes and nonenzymatic structural changes compromising protein translation (6, 23). Our findings strongly suggest that the complementary antisense SP-B message from our recombinant adenovirus forms stable RNA:RNA complexes within the type 2 cell, because both RNAs were readily detected by RT-PCR. Furthermore, the presence of decreased SP-B protein concomitant with stable antisense SP-B and endogenous SP-B messages suggest that the duplexes interfere with translation of proSP-B.

SP-B deficiency results in neonatal lethality in humans and transgenic mice (4, 24). However, the mechanism of pulmonary insufficiency extends beyond the simple absence of SP-B from alveolar surfactant. Both humans and mice with SP-B deficiency exhibit aberrant type 2 cell ultrastructure, most significantly showing a complete lack of lamellar bodies (4, 25). In the absence of lamellar body formation, SP-B–deficient type 2 cells accumulate precursor organelles, including multivesicular bodies and an abundance of intracytoplasmic vesicles. SP-B antisense expression during in vitro differentiation recapitulates many of the characteristics of the well-described human phenotype, including reduced numbers of lamellar bodies. We did not observe accumulation of multivesicular bodies over the 72 h during which antisense expression was stable. This is in contrast to our recent studies of inhibition of SP-B processing by a cysteine protease inhibitor in which cells accumulated large numbers of vesicles and multivesicular bodies in addition to exhibiting absent lamellar bodies (26). We speculate that the paucity of multivesicular bodies in our current study is due primarily to decreased production of proSP-B and intermediates of SP-B processing, whereas multivesicular bodies predominated in protease inhibitor studies due to the accumulation of SP-B intermediates resulting from the normal induction of SP-B expression.

Another finding in SP-B deficiency is the aberrant processing of SP-C. SP-C, like SP-B, is synthesized as a large proprotein, and undergoes sequential proteolysis of N- and C-termini to liberate the 3.7-kD mature SP-C protein. An aberrant 6- to 10-kD SP-C precursor accumulates in humans (21) and transgenic mice (4) deficient in SP-B due to incomplete proteolysis of proSP-C intermediates in addition to reduced mature SP-C (27). We observed decreased mature SP-C production in antisense-treated cells consistent with aberrant SP-C processing, but did not observe accumulation of SP-C precursors. Despite the relatively poor identification of SP-C precursors by the antibody to mature SP-C, the time course of expression of both SP-B and SP-C in our cell culture model is not conducive to detecting accumulation of intermediates. We have shown previously that despite induction of both SP-B and -C mRNA, expression of mature SP-C protein lags behind expression of mature SP-B during in vitro differentiation of type 2 cells (14). This is due to the requirement of mature SP-B for lamellar body genesis, and the requirement of lamellar bodies for the final steps in SP-C processing. This temporal sequence has also been observed in developing human fetal lung, as well as in explant models of type 2 cell maturation (18, 28, 29). The difference in the onset of expression of mature SP-B versus SP-C, combined with silencing of the antisense SP-B RNA beyond 72 h in culture (Figure 6), limits our ability to detect accumulation of proSP-C intermediates. This is in agreement with our previous observations using cysteine protease inhibitors to disrupt SP-B processing, in which cells accumulated SP-C precursors only after 5 d in culture (26). The more dramatic accumulations of SP-C precursors in SP-B–deficient humans and mice are likely attributable to more prolonged expression and an expanding number of fully differentiated type 2 cells.

There are many advantages of this model system over transgenic models for studying lamellar body genesis. Despite the lack of accumulating SP-C precursor, antisense SP-B achieves many of the characteristics of the SP-B–deficient type 2 cell phenotype, with decreased SP-B, aberrant type 2 cell ultrastructure, and decreased mature SP-C. The early events in the process of lamellar body genesis have not been studied extensively. Theories have been proposed that include activation of the fusogenic properties of SP-B resulting in both lysis of multivesicular body vesicles with subsequent exposure of SP-C intermediates to proteolytic enzymes to complete protein processing (reviewed in Ref. 3). Validation of these theories has not been possible to date in knockout mouse models due to the neonatal lethality of SP-B deficiency and difficulties in isolating differentiating type 2 cells from the transgenic fetal mice. In vitro differentiation of type 2 cells faithfully replicates the process of lamellar body genesis, as well as lamellar body secretion (14). We are thus able to examine the cell biology of this unusual organelle by direct manipulation of genes and proteins instrumental in its biogenesis. As other critical factors in lamellar body genesis are uncovered, this method will be an important in vitro strategy for loss-of-function studies.

In conclusion, we have demonstrated the usefulness of antisense technology in reliably reproducing many of the features of SP-B deficiency in vitro. Antisense SP-B, introduced into differentiating human fetal lung type 2 cells via a replication-deficient adenoviral vector, significantly decreased lamellar bodies, and achieved a marked reduction in mature SP-B protein and mature SP-C. SP-B deficiency is likely resulting from the generation of stable RNA:RNA duplexes, with interruption of translation, rather than enzymatic degradation of RNA. The low toxicity and reproducibility of the model will enable further studies to examine the role of SP-B in lamellar body genesis, and provides a means for determining the role of other genes also involved in this process.


   Acknowledgments
 
The authors acknowledge the assistance of Sree Angampalli and Ping Wang in the preparation of type 2 cells, and Neelima Shah and the Biomedical Imaging Core of the University of Pennsylvania School of Medicine and Philip Ballard for editorial assistance. They also thank Wolfram Steinhilber (Byk-Gulden) and Michael Beers for their kind gifts of specific SP-C antisera. These studies were supported by NIH HL56401 (S.H.G., L.G.), HL59959 (S.H.G.), and by a Fellow's Grant from Forest Pharmaceuticals, Inc. (C.D.F.).

Received in original form August 8, 2002

Received in final form March 14, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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