Exogenous gene transfer of Rab³8 small GTPase ameliorates aberrant lung surfactant homeostasis in Ruby rats

Unless otherwise specified, chemicals were purchased from Sigma (St. Louis, MO) or Wako Chemicals (Osaka, Japan). Restriction enzymes were obtained from Nippongene (Tokyo, Japan). Rabbit anti-rat Rab³8 polyclonal antibody was produced in our laboratory [8]. Mouse anti-pig surfactant protein B (SP-B) monoclonal antibody (8B5E) was a generous gift from Dr. Yasuhiro Suzuki (Department of Molecular Pathology, Chest Disease Research Institute, Kyoto University), and mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Chemicon (Temecula, CA). Mouse anti-rat surfactant protein A (SP-A) monoclonal antibody (1D6) was a generous gift from Dr. Dennis R. Voelker, the National Jewish Health (Denver, CO). Horseradish peroxidase-conjugated goat anti-rabbit IgG antibody, anti-mouse IgG antibody, and a broad-range prestained SDS-PAGE molecular marker were purchased from Bio-Rad (Hercules, CA). A chemiluminescent detection kit, a stripping buffer, and a micro bicinchoninic acid (BCA) protein assay kit were purchased from Pierce (Rockford, IL). A thin-layer chromatography apparatus was purchased from Advantec (Tokyo, Japan), and Silica gel G plates were purchased from Analtec (Uniplate, Newark, DE).

An adenovector construction kit (AdMax) and HEK293 cells were purchased from Microbix Biosystems Inc. (Toronto, Ontario, Canada) [12] (Fig. 1). We digested a shuttle plasmid pDC315 from the kit with BamH1 and Sal1 enzymes to construct a recombinant plasmid containing lacZ-cDNA or Rab³8-cDNA. The lacZ-cDNA was excised from the pRSET/lacZ plasmid (Invitrogen, Carlsbad, CA) using BamH1 and Hind3 enzymes, inserted into a pBlueBacHis2A (Invitrogen) and then re-digested with BamH1 and Sal1 enzymes. Rab³8-cDNA was excised from pBlueBacHis2A-Rab³8 [8] using BamH1 and Sal1 enzymes. All plasmids, including an adenovirus genomic plasmid (pBHGloxΔE1,3Cre) which harbor cytomegalovirus (CMV) promoter and Cre/loxP recombination sites, were purified by CsCl-density gradient ultracentrifugation. HEK293 cells were co-transfected with either of the recombinant shuttle plasmids (lacZ or Rab³8) and the adenovirus genomic plasmid which lacked the early-transcribed regions (E1 and E3) according to the manufacturer’s protocol. The 293 cells harbor the early-transcribed regions (E1 and E3) for replication of the recombinant adenovirus. Recombinant viral plaques generated from Cre-lox recombination appeared within 2-3 weeks, some of which were isolated and propagated. The expression of lacZ and Rab³8 were confirmed by β-galactosidase enzyme (lacZ) assay on live 293 cells and Western blot analysis of 293 cell lysates with a rabbit anti-Rab³8 polyclonal antibody [8], respectively. The viruses were amplified using 293 cells, and purified high-titer adenovirus stocks were prepared with CsCl₂-density gradient ultracentrifugation, subsequently dialyzed against 10 mM Tris-HCl, pH 8.0, aliquoted in presence of 10% glycerol, and stored at –80 °C.

All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Kanazawa Medical University. Specific-pathogen-free (SPF) male Sprague-Dawley (SD), Long Evans/Kwl (LE), and Long Evans Cinnamon/Crj (LEC) rats were purchased from Japan Charles River Inc. (Yokohama, Japan) and housed in the SPF rat room of Kanazawa Medical University Laboratory Animal Center. The genotypes of the rats were determined by DNA sequencing of PCR products of Rab³8 exon 1 as previously reported [3]. Eight to twelve-week-old male rats, weighing 148–249 g, were used. For adenovector administration experiments, LE or LEC rats were anesthetized by intraperitoneal administration of pentobarbital sodium (~15 mg) and intubated with a 16-G plastic venous catheter. Through the catheter, an extra-thin soft polypropylene catheter connected to an adenovector-containing 1-ml syringe was inserted into the left lung, and purified high-titer adenovirus (10⁹ pfu in 0.5 ml PBS/1% glycerol) was slowly delivered into the left lung at the left decubitus position (inclined head-up), followed by several air injections to flush the airway tract [13]. At 2-weeks post administration, the rats were anesthetized with intraperitoneal injection of pentobarbital sodium (~ 15 mg) and sacrificed by cutting abdominal aorta, and used for further investigation.

Eight- to ten-week-old male SD or LEC rats were used. Type II cells were isolated from these rats by tissue dissociation using elastase and metrizamide density-gradient centrifugal separation as previously described [14‐16]. Isolated cells were immediately suspended in DMEM/10% FBS with the recombinant adenovirus (Ad-lacZ or Ad-Rab³8) at a multiplicity of infection (MOI) (13) of 5 and incubated for 1 h in a 37 °C water bath with gentle shaking. Next, the cells were seeded at 2 × 10⁶ cells in 35-mm plastic culture dishes. The next day, adherent cells were washed, and fresh DMEM/10% FBS was replenished every 2 days.

Freshly isolated type II cells were plated on a plastic chamber slide (Lab-Tek II chamber slides, Nalge Nunc International, Naperville, IL). After overnight culture, the cells were infected with adenovirus (Ad-lacZ or Ad-Rab³8) at MOI = 2 to reduce expression level of Rab³8 protein and facilitate identification of co-localization with SP-B. The cells were further cultured for 24 h and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 10 min and followed with 100% acetone for 30 s. The fixed cells were blocked with 25% normal goat serum, incubated with a primary antibody (anti-Rab³8 and anti-SP-B), followed by a secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG antibody and Alexa 594-conjugated goat anti-mouse IgG antibody) (Abcam, Tokyo, Japan). The slides were mounted with an antifade solution containing DAPI (S7113, Millipore, Temecula, CA). The immunofluorescence images were acquired using a fluorescence microscope (Keyence Biorevo BX-9000, Osaka, Japan).

Male SD rats were used as controls, and male LEC rats were used as Rab³8-deficient rats. Type II cells were isolated as described above [14‐16]. Isolated cells were infected with adenovirus (Ad-lacZ or Ad-Rab³8) at MOI = 5 for 1 h and plated in 35-mm plastic culture dishes at 2 × 10⁶ cells in 2 ml of DMEM containing 10% FBS and incubated in a 10% CO₂ incubator at 37 °C for 22 − 24 h. The adherent cells were replenished with 2 ml of DMEM containing 10% FBS and 0.5 μCi/ml of [³H]choline chloride (ARC, St. Louis, MO) and further cultured for 22 − 24 h. After wash out of the [³H]-containing medium, 1.8 ml DMEM at 37 °C was added to the adherent cells and incubated for 15 min, followed by addition of the following agonist: 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 100 nM), ATP (10 μM), and terbutaline (100 μM). Phospholipid secretion was allowed to proceed for 3 h. The supernatant media and cells were separated, and the lipids were extracted according to the Bligh-Dyer method [17], and [³H]phosphatidylcholine (PC) was counted using a liquid scintillator. Basal secretion values (%) were expressed as 100 × [³H]PC in supernatant/(supernatant + cell). Agonist-induced secretion value was normalized by the basal secretion value within each experimental group, i.e. fold of the basal secretion value. LDH release was evaluated using a LDH Cytotoxicity Detection Kit (Clontech, Mountain View, CA); less than 3% of LDH released into the media from total cellular LDH was considered as insignificant cellular toxicity.

Eight- to twelve-week-old male LE or LEC rats were used. The left lungs were excised and weighed. Next, a 2.5 ml of saline/10 mM Hepes (pH 7.4) was infused into the left lung and gently recovered. This procedure was repeated 4 times, yielding approximately 12 ml of lavage fluid. The half volume of cell-free bronchoalveolar lavage (BAL) fluids were concentrated to a 0.5-ml volume with a centrifugal filter (MWCO 5000, Millipore). The remaining lung was weighed and cut into two parts of equal weight. One part was cut into small pieces and homogenized using a Potter-Elvehjem-type homogenizer. One-tenth volume was reserved as a lung homogenate, and the remaining nine-tenths were further used for lamellar body purification [15, 16]. Discontinuous sucrose density gradients were centrifuged at 100,000 × g for 3 h. The 0.4–0.6 M layers were recovered as the lamellar body fraction, diluted, and centrifuged at 100,000 × g for 1 h to pellet the lamellar bodies. The pellet was divided into two portions, which were used for Western blot and lipid extraction/phosphorus assay. The other half of the left lung was cut into small pieces and used for lipid extraction with Bligh-Dyer lipid extraction and subsequent phosphorus assay [17, 18].

Samples containing fixed amount of protein were subjected to 4–12% Bis-Tris SDS-PAGE under reducing condition and transferred to nitrocellulose membranes. The membranes were immunoblotted with a rabbit anti-rat Rab³8 antibody, or a rabbit (or mouse) anti-surfactant protein antibody, followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit (or anti-mouse) IgG antibody. Chemiluminescent detection assay was performed, and the bands were developed using an autofluorography film. Exposure times were adjusted to between 15 s and 5 min (usually 1 min) depending on the signal intensities obtained. When indicated, after stripping off the antigen-antibody complex, the same membrane was reused for the second antigen-antibody reaction.

Total lipids were extracted according to the Bligh and Dyer method [17] from one of the two cell-free BAL fluids, one of the two lamellar body fractions, and half of the left lung. In all cases, 20% of each sample was subjected to phospholipid phosphorus content assessment according to the method described by Bartlett [18]. The remaining 80% of each sample was subjected to two-dimensional thin-layer chromatography [19]. After development, the silica gel spots corresponding to each phosphatidylcholine species were scraped off and recovered quantitatively. The phosphorus content was then quantified by the Bartlett method [18].

Two weeks after the administration of adenovirus, the LE or LEC rats were sacrificed, and freshly excised lungs were cut into small pieces and fixed for 2 h with fresh fixative containing 2.5% glutaraldehyde/0.1% picric acid/2% osmium tetroxide/4% sucrose/0.1 M cacodylate buffer (pH 7.4). The blocks were post-fixed with 1% aqueous uranyl acetate solution for 1 h followed by dehydration in a graded series of ethanol and subsequent propylene oxide. The samples were finally embedded in Epon. Semi-thin sections (0.8 μm-thick) were prepared and stained with 1% toluidine blue with warming. Ultrathin sections (60 nm-thick) were then counterstained with 2% uranyl acetate, followed by 2.6% lead nitrate/3.5% sodium citrate (pH 12). The sections were examined using a Hitachi H-7100 transmission electron microscope. Using ~25 electron microscopic photographs per experimental group, the areas of cells and lamellar bodies were quantified by an area-calculating software (Area Manager Lite, Visionary Co. Ltd., Kobe, Japan), and the numbers of lamellar bodies per single cell were counted.

Data were expressed as mean ± SEM. Single-factor ANOVA was used for data analysis among the three experimental groups and followed by Student-Newman-Keuls post hoc test for multiple comparisons. P values <0.05 was considered as statistically significant.

The adenovirus carrying lacZ-cDNA (Ad-lacZ) at MOI of 5 infected ~ 100% of cultured type II cells in vitro and expressed functional β-galactosidase (Fig. 2b). The Ad-lacZ-infected and -non-infected (control) cells were stained with 0.5 mg/ml X-gal. Western blot (Fig. 2a) confirmed exogenously expressed Rab³8 in Ad-Rab³8- infected LEC type II cells in primary culture. Freshly isolated cells (day 0) were infected with Ad-Rab³8 at MOI = 5 for 1 h and cultured for the indicated time. The post nuclear supernatant equivalent of 0.5 × 10⁶ cells was used for the analysis. As previously reported [3], intact LEC type II cells completely lacked Rab³8 protein. In contrast, Ad-Rab³8-infected cells expressed Rab³8 protein (molecular weight ~ 26 kDa) by 24 h after infection. This expression persisted for more than 21 days with a peak in expression at approximately 7 days. Immunofluorescence cell staining of cultured LEC type II cells with anti-Rab³8 antibody revealed no protein expression in intact LEC type II cells (Fig. 2c; a), but significant exogenous Rab³8 protein in Ad-Rab³8-infected LEC type II cells (Fig. 2c; e, i). The exogenous Rab³8 appeared to at least partially co-localize with granularly distributed SP-B in the cells (Fig. 2c; l, arrow heads).

Type II cells are unique cells that specifically synthesize large amount of [³H]PC as a predominant surfactant constituent using [³H]choline chloride precursor that is added to the culture medium. [³H]PC is hydrophobic and is extracted into organic phase, whereas [³H]choline chloride is hydrophilic and extracted into hydrophilic phase using the Bligh-Dyer lipid extraction method [17]. Figure 3 shows the secretion of newly synthesized [³H]PC from cultured type II cells. Compared with wild-type (SD) cells, basal secretion is significantly reduced in Ad-lacZ-infected LEC cells (Fig. 3a). Basal secretion in Ad-Rab³8 LEC cells was significantly higher than Ad-lacZ-infected LEC cells, which exhibited no significant difference compared with the wild-type cells. TPA, ATP, and terbutaline are known as an agonist for PC secretion from cultured type II cells [14, 20]. TPA- and ATP-induced [³H]PC secretion was remarkably increased in LEC cells as previously reported [3] (Fig. 3b). However, TPA- and ATP-induced [³H]PC secretion from Ad-Rab³8-infected LEC cells was significantly decreased compared to that from Ad-lacZ-infected LEC cells. Basal, TPA-, ATP-, and terbutaline-induced [³H]PC secretion from Ad-Rab³8-infected cells were not significantly different from that of wild-type cells.

The efficiency of adenovector (Ad-lacZ) spreading into the left lung by a single endobronchial instillation was evaluated by X-gal staining of a whole left lung. Two-week post-administration of Ad-lacZ or vehicle (0.85% NaCl/1.0% glycerol), the left lungs were excised, fixed, and stained for 4 h with an intratracheal administration of a 0.5 mg/ml of X-gal/2 mM MgCl₂/5 mM K₄Fe(CN)₆/5 mM K₃Fe(CN)₆/20 mM Tris/PBS (pH 8.3) [13]. The Ad-lacZ-administered lung (Fig. 4a, right) exhibited extensive staining by a lacZ assay, suggesting the effectiveness of the administration. The stained lung was embedded in paraffin, sectioned, and counter-stained with nuclear fast red (Kernechtrot) stain. The lower respiratory tract including alveolar tissues also exhibited effective staining by the lacZ assay (Fig. 4c). These results indicate that a single endobronchial administration of adenovector can effectively transduce lower respiratory tract cells.

Two-week post-administration, the left lungs were weighed and lavaged with a saline/10 mM Hepes (pH 7.4). The remaining lungs were homogenized, and lamellar body fractions were isolated by sucrose density-gradient ultra-centrifugation. Total lipid contents were extracted from cell-free bronchoalveolar lavage fluids, lamellar body fractions, and lung homogenates. Phosphatidylcholine was analyzed by two-dimensional thin layer chromatography. Phosphatidylcholine levels in the lamellar body fractions and lung homogenates in Ad-Rab³8-infected lungs were significantly lower than those in Ad-lacZ-infected lungs (Fig. 5). However, phosphatidylcholine levels in the bronchoalveolar lavage fluids did not differ.

The SP-A and SP-B amounts in the lamellar body fractions were evaluated by Western blot (Fig. 6a). Fixed amounts of the resuspended lamellar body fraction (5 μg protein) were used (n = 3 rats for each group). Densitometry indicated that SP-B was significantly decreased in Ad-Rab³8-infected lungs compared to Ad-lacZ-infected lungs (Fig. 6c), whereas SP-A did not differ (Fig. 6b).

Compared with Ad-lacZ-infected wild-type (LE) lungs, type II cells in Ad-lacZ-infected LEC lung exhibited strikingly large lamellar bodies similar to intact LEC lungs as previously reported (Fig. 7a) [3]. In contrast, type II cells in Ad-Rab³8-infected LEC lung exhibited smaller lamellar bodies compared with Ad-lacZ-infected LEC lungs. Quantitative area measurement revealed that type II cells and lamellar bodies in Ad-Rab³8-infected LEC lungs were smaller than those of Ad-lacZ-infected LEC lungs, although they were still significantly larger than those of Ad-lacZ-infected wild-type lungs (Fig. 7b).