Main

The hydrophobic surfactant protein C (SP-C) is synthesized from the 191 or 197-amino-acid precursor SP-C (proSP-C), which is encoded by SFTPC on human chromosome 8p21.3. ProSP-C is an integral membrane protein that contains four regions: a short cytoplasmic N-terminal domain (residues 1–23), a mature SP-C region (residues 24–58) mainly composed of a transmembrane domain (residues 33–58), a non-BRICHOS region (residues 59–89), and a BRICHOS domain (residues 90–197) (1). ProSP-C undergoes palmitoylation and a series of proteolytic C- and N-terminal cleavages to yield a 3.7-kDa mature peptide within subcellular compartments of the endoplasmic reticulum (ER), multivesicular bodies, lamellar bodies (LBs), and others (2). Mature SP-C is exclusively produced by type II alveolar epithelial cells (AECs), packaged into LBs, which are a subclass of lysosomal-like organelles, and subsequently secreted to the alveolar surface. Meanwhile, phospholipids and proteins are endocytosed from the alveolar surface into early endosomes and routed back either to lysosomes for degradation or LBs for recycling (3).

Interstitial lung disease (ILD) in infants and children represents a heterogeneous group of respiratory disorders that are mostly chronic and associated with higher morbidity and mortality than those reported in adults (4,5,6). SP-C is critical in reducing alveolar surface tension at the air/liquid interface to prevent alveolar collapse at the end of expiration. Since the first report of an infant and mother with ILD due to a mutation in the SFTPC gene (4), several additional mutations have been identified in ILD patients that were associated with abnormal expression of SP-C (7,8). The great majority of SFTPC mutations are located in the BRICHOS domain, and associated with chronic accumulation of misfolded proSP-C (9,10). In contrast, in vitro studies have shown that mutations located in the non-BRICHOS region, such as proSP-CI73T, misdirected proSP-C to the plasma membrane, leading to the accumulation of abnormally processed isoforms (11). Thus, mutations involving different domains of proSP-C may represent different pathogeneses of ILD. Although several mutations located within the mature domain of SFTPC have been recognized (7,8,12), the pathway and mechanism of such mutations remain unclear.

In the present study, we describe a novel heterozygous SFTPC mutation associated with ILD in a young Japanese girl. Lung biopsy samples performed using transmission electron microscopy (TEM) revealed abnormal subcellular organelles. We also investigated whether the subcellular localization of novel proSP-CL55F was altered in A549 cells.

Results

Case Summary

The patient was a young girl who had been delivered at full-term to nonconsanguineous parents without any apparent perinatal diseases. She presented with poor feeding at 2 mo of age and exhibited a failure to thrive. She was hospitalized at the age of 5 mo due to cough and tachypnea. High-resolution computed tomography at the age of 5 mo showed bilateral ground glass opacities and minimal reticulation ( Figure 1a ). Histopathological evaluation of her lung tissue by open-lung biopsy revealed type II AEC hyperplasia and alveolar interstitial fibrosis with obvious inflammation ( Figure 1d ) as compared with a control ( Figure 1c ). According to clinical features, biochemical indicators, and radiographic and pathological findings, she was given a diagnosis of ILD. She was treated with methylprednisolone pulse therapy followed by oral prednisolone in addition to cyclosporine and azathioprine. Due to her persistently elevated supplementary oxygen requirement, the patient required conventional mechanical ventilation and hydroxychloroquine was added to her regimen. These treatments were associated with a slowing of her disease progression, and she was discharged at 2 y of age with home oxygen therapy. At the age of 10 y, her pulmonary function began to deteriorate rapidly. She successfully underwent living-donor right single-lobe lung transplantation from her mother soon afterwards (13). Three months after transplantation, high-resolution computed tomography demonstrated a well-inflated lung allograft without obvious atelectasis ( Figure 1b ).

Figure 1
figure 1

Characteristics of interstitial lung disease in this patient. (a,b) High-resolution computed tomography (HRCT) images observed before (a) and after (b) right single-lobe lung transplantation. (a) HRCT image at 5 mo of age showed bilateral ground-glass pulmonary opacities, small cysts, and several thickened septae. (b) HRCT revealed normal right lung parenchyma and a round tracheal shape after lung transplantation. (c) Normal lung tissue, hematoxylin and eosin. (d) The patient’s lung tissue showed interstitial thickening, type II alveolar epithelial cell hyperplasia, and alveolar interstitial fibrosis with obvious inflammation consisting of lymphocytes, plasma cells, and macrophages, hematoxylin and eosin (scale bars = 100 μm).

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SFTPC Genetic Analysis

Polymerase chain reaction (PCR) followed by direct sequencing of SFTPC, SFTPB, and ATP-binding cassette subfamily A, member 3 (ABCA3) were performed, and three heterozygous substitutions at bases c.163, c.413, and c.557 were detected in SFTPC (Supplementary Figure S1 online). The c.163C>T in exon 2 located at codon 55 in the mature transmembrane domain resulted in a leucine to phenylalanine substitution (p.L55F). Neither 61 healthy Japanese volunteers, nor the patient’s parents, carried this c.163C>T mutation.

The patient’s mother and several volunteers also possessed substitutions of c.413C>A and c.557G>A, which have been described as common coding SNPs in previous studies (14). Paternal gene sequences were identical to those of healthy control volunteers.

Lung Histology

To evaluate whether the proSP-C mutation influenced the expression of surfactant proteins or ABCA3, immunohistochemical assessment of the girl’s lung tissue specimens was performed. In type II AECs, proSP-C exhibited eccentrically located cytoplasmic distribution adjacent to subcellular organelles ( Figure 2c , d ) in comparison with normal controls ( Figure 2a , b ). Immunostaining for SP-B, precursor SP-B (proSP-B), and ABCA3 showed granular cytoplasmic expression in type II AECs of the patient (Supplementary Figure S2a–f online) in amounts comparable to those of controls (data not shown).

Figure 2
figure 2

Immunohistochemical staining for precursor surfactant protein C (proSP-C) and electron microscopic images of type II alveolar epithelial cells (AECs). (a–d) Immunohistochemical staining of lung tissue samples for proSP-C. Immunostaining for proSP-C was detected in lung tissue specimens from a control (a) and the patient (c) at low-power magnification (scale bars = 100 μm). Solid boxes were magnified on the left for better resolution (right) (scale bars = 50 μm). (b) Normal staining for proSP-C in type II alveolar epithelial cells (AECs) of normal lung tissue. (d) Type II AECs of patient lung tissue showed abnormal focal brown staining of the cytoplasm adjacent to subcellular organelles. (e–h) Ultrastructure of type II AECs. Type II AECs of normal lung tissue (e) and the patient (g) at low-power magnification (scale bars = 2 μm). Solid boxes were magnified on the left for better resolution (right) (scale bars = 500 nm). (f) High-power magnification of normal lung type II AECs showed numerous lamellar bodies (LBs) filled with concentric lamellae having diameters of 1–2 μm. (h) High-power magnification of patient lung type II AEC showed multiple smaller condensed LBs with an electron-dense core (dark arrows).

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Congo red is a well-known marker for β-sheet amyloid aggregates. A common feature seen in BRICHOS domain mutations of proSP-C is a high tendency to misfold and form Congo red-positive aggregates (15,16). However, we did not detect any amyloid fibrils as identified by Congo red-positivity as green birefringent materials in the explanted lung tissue of the patient (Supplementary Figure S2g,h online).

TEM of Lung Tissue

Patient lung tissue samples were examined by TEM to characterize the alterations in subcellular organelles related to ILD. Smaller, denser, and abnormally shaped LBs were observed in type II AECs as 0.5 μm organelles ( Figure 2g , h ), which was in contrast to normal LBs having regular concentric “onion skin” lamellae and a diameter of 1–2 μm ( Figure 2e , f ).

Processing of ProSP-CL55F

To identify potential processing differences between proSP-CL55F and previously reported mutations, we analyzed proSP-CI73T (17) and proSP-CA116D (18) in the non-BRICHOS and BRICHOS domains of proSP-C, respectively. Semiquantitative PCR showed that A549 cells stably expressing green fluorescent protein (GFP)-tagged proSP-C isoforms expressed comparable levels of proSP-C mRNA (data not shown). Lysates of A549 cells stably expressing GFP-tagged proSP-C isoforms were also analyzed by western blotting using anti-GFP to assess relative protein expression among proSP-CL55F, proSP-CI73T, proSP-CA116D, and proSP-CWT.

As shown in Figure 3 , a 27 kDa product was expressed in cells transfected with the pAcGFP-C1 vector alone. We observed doublet bands for proSP-CWT and proSP-CL55F that likely corresponded to the primary translation product of the fusion protein (48 kDa) and its known palmitoylated isoform (50 kDa) (19).

Figure 3
figure 3

Processing features of GFP/proSP-CWT GFP/proSP-CL55F, GFP/proSP-CI73T, or GFP/proSP-CA116D. (a) Total protein from A549 cells stably transfected with pAcGFP-C1 vector, GFP/proSP-CWT, GFP/proSP-CL55F, GFP/proSP-CI73T, or GFP/proSP-CA116D was analyzed for expression of proSP-C by western blotting using antibodies directed against GFP or β-actin. (b) Densitometric quantitation of band intensity normalized to β-actin from three separate experiments. Data of proSP-CL55F (black column), proSP-CI73T (gray column), and proSP-CA116D (hatched column) were expressed as fold change over proSP-CWT (white column) values in each experiment. ProSP-CL55F yielded Band 1 similar to those of proSP-CWT. Band 2 of proSP-CL55F was observed at a reduced expression level as compared with proSP-CI73T. The lowest processing intermediate of proSP-CL55F (Band 3) indicated a significant decrease as compared with those of proSP-CWT, proSP-CI73T, and proSP-CA116D. Significant differences were indicated by asterisks. *P < 0.05, **P < 0.001.

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ProSP-CL55F yielded bands similar to those of proSP-CWT for the primary translation product of the fusion protein (48 kDa). Two lower molecular weight intermediates were observed at a reduced expression level, and the lowest processing intermediate (Band 3) displayed a significant decrease as compared with those of proSP-CWT, proSP-CI73T, and proSP-CA116D. In proSP-CI73T cells, a band around 37 kDa migrated to a higher molecular weight compared with those in proSP-CWT, proSP-CL55F, and proSP-CA116D.

Abnormal Subcellular Localization of ProSP-CL55F

Immunofluorescence analysis was performed to investigate the subcellular localization of proSP-CL55F in A549 cells. ProSP-CL55F poorly colocalized with CD63/LAMP3 as compared with proSP-CWT ( Figure 4a ). According to quantitative analysis, the correlation of proSP-CL55F was significantly lower than those of proSP-CWT and proSP-CA116D, but was similar to that of proSP-CI73T ( Figure 4b ). Similarly to proSP-CI73T, which was reported to localize at the plasma membrane, proSP-CL55F also demonstrated signs of partially trafficking toward the plasma membrane ( Figure 4a ). Increased plasma membrane localization was observed for proSP-CL55F as compared with proSP-CWT at low magnification (Supplementary Figure S3 online). On the other hand, proSP-CL55F was detected in early endosome antigen (EEA1)-positive vesicles at a level similar to that of proSP-CWT. Statistical analysis using Pearson’s correlation coefficient showed no significant differences among proSP-CL55F, proSP-CWT, proSP-CI73T, and proSP-CA116D (Supplemental Figure S4 online). Likewise, no remarkable difference was seen between proSP-CWT and mutant proSP-C when we analyzed colocalization with ER-Tracker-Red (data not shown).

Figure 4
figure 4

Intracellular localization of proSP-CWT and mutant proSP-C forms cultured for 12 h in stably expressing A549 cells. (a) Immunofluorescence analysis with a CD63/LAMP3 antibody showed complete colocalization of proSP-CWT and proSP-CA116D and partial colocalization of proSP-CL55F and proSP-CI73T (scale bars = 5 μm). Green channel: proSP-C visualized by GFP fluorescence; Red channel: LAMP-positive structures used to denote lamellar bodies; Merge: overlapped green and red channels for visualization of the colocalization between different SP-C forms and CD63/LAMP3 (yellow). Nuclei were visualized by DAPI (blue). Regions delineated by white boxes were enlarged to illustrate colocalization with the plasma membrane of proSP-CL55F and proSP-CI73T (white arrows). (b) Pearson’s correlation coefficients for proSP-CWT, proSP-CL55F, proSP-CI73T, and proSP-CA116D colocalization with CD63/LAMP3. **P < 0.001.

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Altered Subcellular Organelles in ProSP-CL55F

To further characterize the structure of subcellular organelles, A549 cells transfected with either proSP-CWT or mutant proSP-C isoforms were evaluated by TEM.

At the ultrastructural level, nontransfected A549 cells contained various cellular components, such as early endosomes and late endosomes, within the cytoplasm (data not shown). Cells transfected with proSP-CWT showed an increased number of these organelles, which featured regular “onion skin” electron-dense and concentric lamellae with phospholipid membranes ( Figure 5a , b ). Numerous disorganized lysosomal-like organelles with a double membrane (white arrows) and tightly packed electron-dense dark bodies (black arrow) were observed in A549 cells stably expressing proSP-CL55F or proSP-CI73T. A549 cells transfected with proSP-CA116D displayed several lysosomal-like organelles with irregularly arranged and eccentrically packed lamellae ( Figure 5 ).

Figure 5
figure 5

Characterization of cellular components in A549 cells by transmission electron microscopy (TEM). Representative TEM images of A549 cells stably expressing proSP-CWT, proSP-CL55F, proSP-CI73T, or proSP-CA116D (left) (scale bars = 5 μm). Solid boxes were magnified on the left for better resolution (right) (scale bars = 500 nm). (a,b) In A549 cells transfected with proSP-CWT, normal lysosomal-like organelles were observed. (c,d) Tightly packed electron-dense dark granules (dark arrow) and numerous disorganized lysosomal-like organelles with a double membrane (white arrow) were evident in mutant proSP-CL55F cells. Abnormal lysosomal-like organelles (white arrow) with a double membrane were detected for proSP-CI73T (e,f) and proSP-CA116D (g,h).

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Discussion

In the present study, we described the radiological findings and histopathological and ultrastructural characteristics of a child associated with ILD at initial diagnosis. A novel mutation in one SFTPC allele (c.163C>T) in the region encoding the mature peptide of proSP-C was identified in the infant, who eventually required lung transplantation due to chronic respiratory symptoms. This mutation was not detected in her parents, healthy control volunteers, or the literature, nor has it been listed in large publicly available databases, such as the 1000 Genomes Project (http://browser.1000genomes.org/index.html), or Exome Variant Server (http://evs.gs.washington.edu/EVS/), suggesting that it was a de novo variant.

The clinical presentations of ILD depending on patient age and outcome in infants and children have been associated with disorders of SP-C, SP-B, and ABCA3 expression. According to previous reports, recessive SP-B gene mutations were often found as a cause of lethal respiratory failure in the newborn period; most infants with an SP-B deficiency presented within hours of birth with respiratory failure that required mechanical ventilation (20). ABCA3 gene mutations have also been described in fatal respiratory diseases in newborns and severe chronic lung disease in infancy (21,22). In contrast, SFTPC mutations found in autosomal dominant diseases were often associated with ILD severities ranging from mild to severe and affected patients from the neonatal period (4) to adulthood (6). In the current study, a child presented with the symptoms, signs, and radiographic findings typical of ILD at the age of 5 mo and exhibited a history of persistent respiratory insufficiency for 9 y, indicating that SFTPC mutations were a possible contributor to her condition.

Immunohistochemical staining for proSP-C displayed abnormal distribution within secretory compartments in type II AECs of patient lung tissue specimens as compared with a control, indicating a potential association between proSP-CL55F and the alteration of subcellular proSP-C localization. This was supported by morphological alteration of type II AECs detected by TEM evaluation of lung tissue sections, which provided distinctive characterization by small and densely packed LBs with electron dense inclusions in type II AECs.

To investigate the pathogenesis of proSP-CL55F associated with ILD, we constructed a model of A549 cells stably transfected with proSP-CWT, proSP-CL55F, proSP-CI73T, or proSP-CA116D. TEM examination of A549 cells stably transfected with proSP-CL55F uncovered abnormal lysosomal-like organelles and increased dense core bodies of lysosomal-like organelles, suggesting that proSP-CL55F might have disrupted normal intracellular storage and secretion of SP-C in lysosomal-like organelles.

Western blotting analysis showed similar patterns of intermediate products among proSP-CWT, proSP-CL55F, and proSP-CA116D, suggesting that proSP-CL55F and proSP-CA116D did not interfere with the cleavage step of proSP-C at the C-terminus. An aberrant 37 kDa band was identified for proSP-CI73T and supported the findings of abnormal protein processing. ProSP-CL55F showed a significant reduction in an intermediate processing band as compared with proSP-CWT, proSP-CI73T, and proSP-CA116D, which indicated a loss in proSP-CL55F expression in the processing pathway of A549 cells.

Immunofluorescence analysis of proSP-CWT, proSP-CL55F, proSP-CI73T, and proSP-CA116D in transfected A549 cells was performed to observe subcellular localization. The A549 cell line has been widely studied as a model for type II AECs to characterize the early steps of proSP-C maturation in vitro. Although A549 is a nonlamellar body cell line, when the wild-type isoform of proSP-C is transfected into A549 cells, proSP-C localizes to acidic, lysosomal-like organelles (23). CD63/LAMP3 is used to identify LBs, late endosomes, and lysosomes in type II AECs. Similarly to proSP-CI73T, proSP-CL55F also trafficked partially to CD63/LAMP3-positive vesicles, presumably for routing to the plasma membrane. Mutations located in the non-BRICHOS domain of proSP-C have been described as mistargeting proteins. Previous studies showed that proSP-CI73T was directed to plasma membrane and resulted in abnormal accumulation of the protein in endosome/lysosome compartments in vitro (11). These data suggested that proSP-CL55F might be related to some extent to alterations in the subcellular localization of proSP-CI73T in A549 cells, although we could not confirm this result by western blotting. Ultrastructural images of proSP-CI73T with increased numbers of large, electron-dense vesicles containing organellar debris have demonstrated the disruption of degradation pathways through autophagy (24). Therefore, the western blotting results, alterations in subcellular localization, and abnormally large electron-dense bodies in proSP-CL55F in our system might have been due in part to degradation pathway disruption by autophagic processes.

Consisting of a transmembrane and short extramembrane domain, the mature peptide of proSP-C is known to form a very rigid and stable α-helix (25). Valine is typically over-represented (>50% of amino acids) in the transmembrane domain of proSP-C. The hydrophobic peptides of SP-C and amyloid β-peptide are derived from the transmembrane region of their precursor protein with higher propensities for the β strand than helix conformation (26). However, Congo red staining in this patient revealed no formation of amyloid-like deposits, which indicated that proSP-CL55F might not induce proSP-C misfolding and aggregation.

The main limitation of this study was that our A549 system corresponded to a homozygous rather than a heterozygous SFTPC mutation. To date, all known ILD-related SP-C mutations have been heterozygous, and thus a normal allele is also expressed in vivo situation (27). Therefore, in previous multiple heterozygous expressions of mutant proteins associated with autosomal dominant diseases performed in vitro, cells coexpressing combinations of wild-type and mutant proteins were adopted to analyze the mechanism of genetic dominance of interfering mutations (9,28). Additional evaluations are required to establish the precise in vivo pathogenic mechanism of proSP-CL55F.

In summary, we uncovered a new pathogenic mutation in SFTPC located in the mature domain of proSP-C in a Japanese girl with ILD. Functional analyses of transfected A549 cells expressing this mutant proSP-CL55F protein revealed that it may have caused altered subcellular localization. Further studies are needed for a more detailed understanding of the pathogenic mechanisms of genetic onset of this mutation.

Methods

This study was approved by the institutional review board of the Shinshu University School of Medicine. Written informed consent was obtained from the patient’s guardians and Japanese healthy volunteers.

Genetic Analyses

Genetic analyses were conducted on the patient from the age of 12 mo. Genomic DNA was extracted from peripheral blood samples of the patient, her parents, and 61 healthy control subjects using the QIAamp DNA blood mini kit (Qiagen GmbH, Hilden, Germany). The coding exons of SFTPC were amplified by PCR using previously described primers (29). PCR products were confirmed by sequencing.

Immunohistochemistry of Lung Tissue

Lung tissue sections from the patient obtained by open lung biopsy at 5 mo of age were prepared from formalin-fixed paraffin-embedded samples. Control lung tissue was resected for malignancy. Immunohistochemistry was performed with antibodies against proSP-C, SP-B, ABCA3 (Abcam, Cambridge, UK), or proSP-B (Novocastra, Newcastle, UK), according to standard protocols (30).

Congo Red Staining of Lung Tissue

Formalin-fixed paraffin-embedded lung tissue sections obtained by open chest biopsy at 5 mo of age were treated by Congo red solution and dehydrated using increasing concentrations of ethanol. The fluorescence signals were observed by polarization microscopy, and images were collected.

GFP/SP-C cDNA Constructs

To obtain wild-type human proSP-C (Met1 to Ile197), mRNA was extracted from sputum samples of healthy volunteers with the QIAamp RNA blood mini kit (Qiagen GmbH). cDNA fragments of SFTPC were amplified by the primer pairs A and D ( Table 1 ) at the enzyme sites of BglII and EcoRI, respectively.

Table 1 Primer sets used in PCR reactions for generation of proSP-CL55F insert constructs
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For mutant GFP/proSP-CL55F, the cDNA of wild-type human proSP-C served as the template using the primers by overlap extension PCR with a two-round, four-primer technique ( Table 1 ). For construction of GFP/proSP-CI73T and GFP/proSP-CA116D, mutagenesis was also performed by overlap extension PCR, as described previously (9,18,31). Following digestion with BglII at the 5’ end and EcoRI at the 3’ end, PCR products were purified for cloning into pAcGFP-C1 vectors (Clontech, Mountain View, CA). These constructs were confirmed by sequencing.

Generation of Stably Transfected A549 Cells

A549 lung epithelial cells were nucleofected with GFP/proSP-CWT, GFP/proSP-CL55F, GFP/proSP-CI73T, or GFP/proSP-CA116D using the Amaxa SF Cell line 4D-Nucleofector X kit (Lonza, Cologne, Germany).

Transfected A549 cells were treated with 600 μg/ml G418 (Roche, Indianapolis, IN) as a neomycin selection marker in a 5% CO2 incubator at 37 °C. Control cells not carrying the resistance marker were used to verify cell death by G418. After 4 wk, several neomycin-resistant clones were selected. Neomycin-resistant cells were assessed and sorted by a BD FACS Aria II cell sorter (Becton-Dickinson, Mountain View, CA) using a 488-nm laser. A549 cells stably transfected with GFP/proSP-CWT, GFP/proSP-CL55F, GFP/proSP-CI73T, or GFP/proSP-CA116D were generated for further analysis.

RNA Extraction and PCR of Stably Transfected A549 Cells

Total RNA was extracted from stably transfected A549 cells and reverse transcription to cDNA was performed using the PrimeScript RT reagent kit (TaKaRa Bio, Shiga, Japan). For semiquantitative RT-PCR, four 10-fold serial dilutions of template cDNAs from GFP/proSP-CWT, GFP/proSP-CL55F, GFP/proSP-CI73T, or GFP/proSP-CA116D were amplified using primer pairs A and D ( Table 1 ) and confirmed by sequencing. The amplification conditions of SFTPC and GAPDH were 1 cycle at 94 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min.

Western Blotting

Stably transfected A549 cells were cultured for 12 h, harvested, and subjected to protein extraction. Western blotting was performed using primary antibodies, as follows: GFP (Clontech) at a 1:5,000 dilution and β-actin (MBL, Nagoya, Japan) at a 1:1,000 dilution. Peroxidase-labeled antibodies to mouse IgG and rabbit IgG (H+L; KPL, Gaithersburg, MD), respectively, were used as secondary antibodies. β-actin was used as the standard for normalizing protein samples. Detection was performed using an ECL system and target proteins were quantified using a densitometric image analyzer with NIH Image version 1.61 software (Bethesda, MD).

Fluorescence Microscopy

Stably transfectedA549 cells were cultured for 12 h on Lab-Tek II CC2 chamber slides (Thermo Fisher Scientific, Rochester, NY). The cells were fixed and incubated with primary antibodies for CD63/LAMP3 (BioLegend, San Diego, CA) or EEA1 (Cell Signaling Technology, Danvers, MA) at 1:100 dilutions. Species-specific Alexa Fluor 555- and 568-conjugated secondary antibodies (Invitrogen, Eugene, OR), respectively, were used at 1:500 dilutions. Samples were mounted using ProLong Gold Antifade Reagent with DAPI and examined using an LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Cells were also stained with ER-Tracker-Red (BODIPY TR, Glibenclamide, Invitrogen, Eugene, OR).

Images of single costained cells were captured for colocalization analysis. Colocalizations between GFP/proSP-C and CD63/LAMP3 or EEA1 were quantified using ZEN2009 software (Carl Zeiss) based on Pearson’s correlation coefficient.

TEM Examination

Small lung tissue specimens obtained from the patient at 5 mo of age and stably transfected A549 cells were cultured for 12 h and fixed. After dehydration through a series of ethanol gradients, the samples were embedded in EPON resin. Ultrathin sections (70–80 nm) were prepared and double-stained with uranyl acetate and lead citrate. Ultrastructural investigation was carried out using a JEOL JEM-1230 TEM device.

Statistical Analysis

Statistical analyses were performed by one-way ANOVA using SPSS version 17.0 software (SPSS, Chicago, IL). All values are expressed as the mean ± SE. The level of significance was defined as a P value of <0.05.

Statement of Financial Support

No financial assistance was received to support this study.

Disclosure

The authors have no financial disclosures indicating any financial ties to products in the study or potential/perceived conflicts of interest.