Exosurf enhances adenovirus-mediated gene transfer to alveolar type II cells

S. Machelle Manuel1, Yi Guo1, and Sadis Matalon1,2,3

Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35233-6810

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We assessed the role of surfactant replacement mixtures in the enhancement of adenovirus-mediated gene transfer to pulmonary epithelial cells both in vitro and in vivo. A549 cells, a pulmonary epithelium-derived adenocarcinoma cell line, were incubated with either media alone or media containing 10 µg phospholipid/ml Exosurf or Infasurf for 50 min followed by addition of a replication-deficient adenovirus (E1-deleted) expressing the luciferase reporter gene [AdCMV-Luc; 10 plaque-forming units (PFU)/cell] for 4 h. Pretreatment with Exosurf, but not Infasurf, at 37°C, but not at 4°C, enhanced luciferase activity in A549 cells 24 h later by 156% (P < 0.01). Intratracheal instillation of AdCMV-Luc (2 × 109 PFU) into rats resulted in luciferase expression mainly in alveolar macrophages and to a smaller extent in alveolar type II (ATII) cells 24 h later. However, when the AdCMV-Luc instillation was preceded by Exosurf (250 µl; 25 mg/ml), a 10-fold increase in ATII cell luciferase activity was noted. Preincubation of cultured ATII cells with Exosurf also enhanced their transfection by AdCMV-Luc by 515% (P < 0.001). The results of these studies provide a new strategy for targeting ATII cells for gene delivery.

calf lung surfactant extract; macrophages; luciferase; alveolar epithelium; A549 cells

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE TRANSFECTION of epithelial cells using adenoviral and plasmid vectors is an expanding field of study. Advances in this area are important to the development of gene therapy protocols to treat inherited diseases with severe pulmonary manifestations such as cystic fibrosis and alpha 1-antitrypsin deficiency. In addition, other noninherited pulmonary diseases, including the acute respiratory distress syndrome (ARDS) and inflammatory lung disease (1), may be abrogated by the use of gene therapy.

To date, two different types of gene delivery systems have been used for the delivery of transgenes to mammalian airway and alveolar epithelial cells in vivo: plasmid vectors and replication-deficient viral vectors. The latter have been used extensively and successfully for gene transfer in a variety of organs, including lung airway and epithelial cells (2, 22, 29). The advantages of adenoviral vectors include the potential of obtaining high titers of these vectors relative to retroviral vectors and the ability to incorporate foreign genes as large as 7.5 kb into their genome (2, 33). It is also possible to generate recombinant adenoviruses that are replication deficient using cell lines (human 293 cells) that provide the E1a and E1b early gene regions in trans. These vectors retain the capacity to infect both replicating and nonreplicating terminally differentiated cells in vivo, and the DNA functions in an extrachromosomal fashion that eliminates the risk of insertional mutagenesis.

On the other hand, instillation of replication-deficient adenoviruses into the trachea of live animals results in marked pulmonary inflammation, due to the production of viral proteins that generate neutralizing antibodies of the immunoglobulin (Ig) A subtype directed against the virus capsid (hexon and fiber proteins), limiting the ability of the adenoviral vectors to effectively transfect cells on readministration (39). Currently, a number of approaches are being tested to overcome the problem of recombinant adenovirus-mediated immunogenicity, including more extensive deletions of the adenovirus genome (E2 and E3 regions; see Ref. 40) and modulation of the cellular and humoral immune systems.

One strategy to optimize gene transfer to alveolar epithelial cells is to coinstill replication-deficient adenoviruses with pulmonary surfactants that enhance both the intrapulmonary distribution of substances instilled into air-filled lungs and their uptake by alveolar epithelial cells (32, 38). Intratracheal instillation of superoxide dismutase and catalase, two water-soluble antioxidant enzymes encapsulated into emulsions or surfactant liposomes, led to increased antioxidant activities in lung tissue (4, 31, 37). Furthermore, intratracheal instillation of adenoviral vectors suspended in Survanta, a surfactant replacement mixture, resulted in increased transgene expression in lung tissues with preferential expression in peripheral lung tissues (24). However, these studies did not identify the type of lung cells targeted by the adenovirus-surfactant mixtures and did not identify which components of surfactant mixtures were responsible for increased efficacy of gene transfer.

Herein, we demonstrate that Exosurf, a surfactant mixture shown to be effective in repleting surfactant stores in infants with neonatal respiratory distress syndrome (NRDS; Ref. 6), increased the transfection efficacy of both A549 and cultured alveolar type II (ATII) cells by replication-deficient adenoviral vectors containing firefly luciferase (AdCMV-Luc). In a subsequent series of experiments, we demonstrated that a single intratracheal instillation of Exosurf into rats before AdCMV-Luc instillation resulted in a considerably higher fraction of AdCMV-Luc taken up by ATII cells rather than by alveolar macrophages (AM). The results of these studies provide a new strategy for targeting ATII cells for gene delivery.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. A549 pulmonary epithelial cells (American Type Culture Collection, Rockville, MD; an adenocarcinoma cell line derived from alveolar epithelial cells; see Ref. 28) were maintained in Dulbecco's modified Eagle's medium-F-12 (1:1) media with 10% (vol/vol) fetal bovine serum (FBS), 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 28 mM sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin in 75-cm2 tissue culture flasks at 37°C in 5% CO2-95% air. A549 cell monolayers were lifted from the flasks by addition of 2 ml of 0.05% trypsin and were seeded on Falcon Primaria-coated six-well tissue culture plates at a density of 1.2 × 105 cells/well.

Isolation and culture of ATII cells. ATII cells were isolated from the lungs of young adult male Sprague-Dawley rats (150-250 g) as previously described (20). Briefly, the rats were anesthetized and killed by an intraperitoneal pentobarbital sodium injection (260 mg/kg body wt). Their lungs were perfused with buffered saline, removed en bloc from the thoracic cavity, and lavaged with balanced salt solution and buffered saline alternately to remove macrophages. Subsequently, the lungs were filled with Joklik's modified minimum essential medium (JMEM; 37°C) containing elastase (Worthington; 2.5 U/ml in JMEM) and deoxyribonuclease (DNase). Proteolytic digestion was stopped after 30 min by addition of cold (4°C) JMEM containing 10% (vol/vol) heat-inactivated FBS, DNase, and trypsin inhibitor. The lungs were minced with scissors into small pieces, and ATII cells were separated from the crude cell suspension by discontinuous density-gradient centrifugation consisting of 10 ml of Percoll (Sigma) with specific density (rho ) = 1.035 and 2 ml of Percoll with rho  = 1.08. Cells on the rho  = 1.035 Percoll interface were layered on sterile disposable petri dishes (Labcraft) precoated with rat IgG, and the dishes were incubated at 37°C in 5% CO2-95% air for 1 h. Nonadhering cells were removed by carefully panning the plates and were pelleted by centrifugation. Cell viability and purity were >90% as checked by trypan blue exclusion and Papanicolaou staining, respectively. When appropriate, the purified ATII cells were seeded onto 4 µm pore size 0.33-cm2 Transwell tissue culture filters at a density of 1.5 × 106 cells/cm2 and were maintained in minimum essential medium with 10% FBS plus 0.1 mM dexamethasone (3). Monolayer formation was confirmed by the measurement of the resistance of the cell monolayer with chopstick electrodes (Millipore). Cells were considered to be confluent and to demonstrate tight junction formation when their resistance values exceeded 400 Omega /cm2.

Surfactant preparations. Exosurf, a generous gift of Glaxo/Wellcome (Research Triangle Park, NC) is an artificial surfactant replacement product consisting of 81 mg/ml dipalmitoylphosphatidylcholine (DPPC), 9 mg/ml cetyl alcohol, 6 mg/ml tyloxapol, and 5.845 mg/ml sodium chloride, without surfactant apoproteins. Calf lung surfactant extract (Infasurf; a generous gift of ONY, Buffalo, NY) was obtained by chloroform/methanol extraction of the cell-free bronchoalveolar lavage of freshly killed calves as previously described (25). Infasurf contains 98% lipid and 2% protein by weight, consisting exclusively of the hydrophobic surfactant proteins-B and -C.

Adenoviral vector preparation. AdCMV-Luc (15) or recombinant adenoviral vector containing beta -galactosidase (AdCMV-LacZ) (39) reporter genes were propagated in 293 cells and were purified by cesium chloride gradient centrifugation as previously described (10). Both recombinant vectors are replication-deficient adenovirus type 5, with deletion of the E1 region required for virus replication. The reporter proteins were expressed under the control of the cytomegalovirus promoter. Virus stocks were typically 1010 plaque-forming units (PFU)/ml for AdCMV-Luc and 5 × 108 PFU/ml for AdCMV-LacZ.

Luciferase activity. Cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), lysed with 200 µl of distilled water, and scraped from tissue culture wells, if appropriate. The cell suspension was then centrifuged at 1,000 revolutions/min (500 g) for 10 min. Fifty microliters of the supernatant were added to an assay reagent containing (in mM) 20 tricine, 1.07 magnesium carbonate, 2.67 magnesium sulfate, 0.1 EDTA, and 33.3 dithiothreitol, as well as 530 µM ATP, 270 µM coenzyme A, and 470 µM luciferin and were assayed on a monolight 20-10 luminometer (Analytical Luminescence Labs). Results were expressed in relative light units (RLU) per microgram of cell protein and were measured by the bicinchoninic acid method using bovine serum albumin as the standard.

In vitro transfection studies. All procedures were carried out at 37°C unless otherwise noted. A549 or ATII cells seeded on culture plates or filters were incubated in complete tissue culture medium for ~18 h, as described above. At this time, the medium was removed and was replaced by CellGro tissue culture medium containing Exosurf or Infasurf (A549 cells only) in concentrations ranging from 0 to 200 µg phospholipid/ml. Fifty minutes later, AdCMV-Luc, diluted to 10 PFU/cell in a final volume 0.5 ml of CellGro, was added into the medium, and the cells were returned to the incubator for 4 h (pretreatment). In a number of experiments, cells were incubated with medium containing 10 mg/ml Exosurf and AdCMV-Luc (10 PFU/cell; Exosurf as vehicle) for 4 h. The media was then aspirated and was replaced with fresh CellGro medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and the cells were returned to the incubator for 24 h, at which time their luciferase activity was determined as described above. To investigate the dependence of transfection efficiency on temperature, A549 cells were maintained at 4°C during the preincubation and transfection intervals by immersing the culture plates in ice. All other procedures were carried out as described above.

In vivo transfection studies. Adult Sprague-Dawley rats (175-250 g) were anesthetized using 1-2% Metofane inhalation, and the trachea was exposed using a midline incision. Rats were randomly assigned to one of three groups. Rats in the first group (control) received an intratracheal instillation of 2 × 109 PFU AdCMV-Luc or 107 PFU AdCMV-LacZ suspended in 500 µl of 10 mM sterile PBS. Rats in the second group (pretreatment) received an instillation of 250 µl of 25 mg phospholipid/ml Exosurf followed 10 min later by a 250-µl instillation of 2 × 109 PFU AdCMV-Luc suspended in 10 mM PBS. Rats in the third group (Exosurf as vehicle) were instilled with 2 × 109 PFU AdCMV-Luc suspended in a 25 mg/ml Exosurf solution (total volume was 500 µl in sterile 10 mM PBS). All solutions were instilled via a 27-gauge needle inserted into the trachea over an ~5-min period, during which the rats were slowly rotated along the horizontal plane to enhance delivery of instilled solutions to all lung fields. The incision was then sutured, and the rats were returned to the animal care facility. Twenty-four hours later, the rats were killed by a 260 mg/kg pentobarbital sodium injection. AM and ATII cells were isolated and were processed for measurement of luciferase activity as described above. Surgical mortality was <5%.

In vivo detection of galactosidase activity. Twenty-four hours after AdCMV-LacZ instillation, rats were killed, the thorax was opened, and lungs were removed from the chest cavity, filled with 10 ml of Tissue-Tek II optimum cutting temperature compound (Miles Diagnostics Division), and frozen immediately at -20°C. Cryosections (6 µm) were cut using a Tissue Tek II cryostat (model 4553; Miles) at -20°C, placed onto glass slides, fixed for 10 min in 0.2% glutaraldehyde and 2% paraformaldehyde, and stained with a solution containing (in mM) 80 disodium phosphate, 20 sodium phosphate, 3 potassium ferricyanide, and 1.3 magnesium chloride in PBS with 1 mg/ml 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-Gal). Slides were examined by light microscopy 12 h after staining, and galactosidase-positive fields were identified by their blue color.

Statistical analysis. Results are expressed as means ± SE. Statistical differences among group means were determined using one-way analysis of variance. Data points that fell outside two standard deviations from their means were omitted, and statistics were recalculated using the remaining data.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Transfection of A549 cells by AdCMV-Luc. Addition of 10 µg/ml Exosurf into the incubation medium of A549 cells 50 min before addition of the recombinant vector AdCMV-Luc significantly enhanced their luciferase activity, as evidenced by a 156% increase in RLU per microgram protein over control values 24 h later (Fig. 1). No further augmentation of luciferase activity was noted when the concentration of Exosurf was increased up to 200 µg/ml (data not shown). Coinstillation of Exosurf with AdCMV-Luc resulted in a similar augmentation of luciferase activity. In contrast to these findings, preincubation of A549 cells with Infasurf, under identical conditions, did not enhance A549 luciferase activity above that seen when these cells were transfected by AdCMV-Luc alone (Table 1).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of Exosurf on transfection of A549 cells by recombinant adenoviral vector containing firefly luciferase (AdCMV-Luc). Luciferase activity is expressed as mean relative light units (RLU) per microgram of protein (µg pr) and represents the mean value (+SE) of 3 different wells. Figure shows results of a representative experiment that was repeated 6 times. Control cells (plated on tissue culture plates at a density of 1.2 × 105 cells/well) were incubated in media only for 50 min, after which AdCMV-Luc [10 plaque-forming units (PFU)/cell], suspended in medium, was added to each well. Cells in the pretreatment group were incubated with 10 µg/ml Exosurf for 50 min, after which AdCMV-Luc (10 PFU/cell) was added to each well. Cells in the Exosurf as vehicle group were incubated with medium plus 10 µg/ml Exosurf and AdCMV-Luc (10 PFU/cell) for 50 min. Four hours after addition of AdCMV-Luc, supernatant was aspirated, cells were washed, and media was replaced with medium containing 10% fetal bovine serum (FBS) and antibiotics. After 24 h, the cells were harvested, and luciferase activity was determined as described in the text. * Significantly different from control (P < 0.05).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of Infasurf on AdCMV-Luc transfection of A549 cells

When A549 cells were transfected at 4°C instead of 37°C with the same amount of AdCMV-Luc, their luciferase activity was decreased by ~87% (Fig. 2). Preincubation with Exosurf at 4°C enhanced luciferase activity, albeit to a much lesser extent than at 37°C.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of temperature and Exosurf on A549 cell transfection efficiency. Luciferase activity is expressed as mean RLU per microgram of protein and represents the mean value (+SE) of at least 3 different wells. Figure shows results of a representative experiment that was repeated 2 times. A549 cells (plated on tissue culture plates at a density of 1.2 × 105 cells/well) were incubated with medium (control) or 10 µg/ml Exosurf suspended in medium (Exosurf) for 50 min at either 37 or 4°C. Four hours after the addition of AdCMV-Luc, supernatant was aspirated, cells were washed, and media was replaced with medium containing 10% FBS and antibiotics. After 24 h, the cells were harvested, and luciferase activity was determined as described in the text. * Significantly different from 37°C control (P < 0.001). ** Significantly different from 37°C Exosurf and 4°C control (P < 0.001).

To determine which component of Exosurf was responsible for the increased transfection efficiency, A549 cells were preincubated for 50 min with 0.72 µg/ml tyloxapol, 1.08 µg/ml cetyl alcohol, and 10 µg/ml DPPC, the concentrations found in 10 µg/ml Exosurf. Tyloxapol increased luciferase activity in A549 cells by 45%. None of the other agents altered luciferase activity (Fig. 3).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of Exosurf components on A549 transfection efficiency. Luciferase activity is expressed as mean RLU per microgram of protein and represents the mean value (+SE) of at least 3 wells. Figure shows results of a representative experiment that was repeated 3 different times. A549 cells (plated on tissue culture plates at a density of 1.2 × 105 cells/well) were incubated with medium (CON), tyloxapol (TYL; 0.72 µg/ml), cetyl alcohol (CA; 1.08 µg/ml), dipalmitoylphosphatidylcholine (DPPC; 10 µg/ml), or a mixture of all three components (TYL/CA/DPPC) at 37°C for 50 min. At this time, AdCMV-Luc (10 PFU/cell) was added. Four hours later, supernatant was aspirated, cells were washed, and media was replaced with medium containing 10% FBS and antibiotics. After 24 h, the cells were harvested, and luciferase activity was determined as described in the text.

Transfection of lung cells with AdCMV-Luc in vivo. In rats instilled with AdCMV-Luc (2 × 109 PFU) alone, AM luciferase activity 24 h postinstillation was much higher than in ATII cells (4.36 × 106 ± 4 × 106 RLU/µg protein vs. 1.385 × 106 ± 3.42 × 105 RLU/µg). In contrast, instillation of Exosurf 10 min before AdCMV-Luc resulted in significant redistribution of the instilled AdCMV-Luc to ATII cells (Fig. 4). Coinstillation of Exosurf and AdCMV-Luc also increased luciferase activity in ATII cells, albeit to a smaller extent (Fig. 4). Data shown in Fig. 5 also demonstrate significantly higher X-Gal staining of lung sections taken from rats that received Exosurf and AdCMV-LacZ vs. AdCMV-LacZ alone.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of Exosurf on in vivo transfection of alveolar macrophages (AM) and alveolar type II (ATII) cells by AdCMV-Luc. Luciferase activity of AM and ATII cells is expressed as mean RLU per microgram of protein and represents the mean value (+SE) of cells harvested from 6 different rats per group. Anesthetized rats received an intratracheal instillation of 2 × 109 PFU AdCMV-Luc alone (Control), 25 mg/ml Exosurf followed 10 min later by 2 × 109 PFU AdCMV-Luc (pretreatment), or 2 × 109 PFU AdCMV-Luc suspended in 25 mg/ml Exosurf (Exosurf as vehicle). After 24 h, rats were killed, AM and ATII cells were harvested, and their luciferase activity was determined as described in the text. * Significantly different from control ATII (P < 0.05). ** Significantly different from pretreatment ATII (P < 0.05).


View larger version (143K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of Exosurf on in vivo transfection of lung tissue by recombinant adenoviral vectors containing beta -galactosidase. Anesthetized rats received an intratracheal instillation of AdCMV-LacZ (2 × 109 PFU) alone (Control) or 25 mg/ml Exosurf followed 10 min later by AdCMV-Luc (2 × 109 PFU; pretreatment). After 24 h, rat lungs were excised en bloc, and frozen sections were prepared and stained with a 1 mg/ml 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside solution. beta -Galactosidase-positive fields were identified by their blue color. A: control lung sections. B: lung sections from a rat in the pretreatment group. Magnification is ×400. Bar, 10 µm.

Transfection of ATII cells by AdCMV-Luc. Pretreatment of cultured rat ATII cells with Exosurf increased transfection by AdCMV-Luc as demonstrated by an almost 6-fold increase in their luciferase activities (12,772 ± 2,727 RLU/µg protein vs. 2,075 ± 806 RLU/µg protein for AdCMV-Luc alone; Fig. 6).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of Exosurf on transfection of cultured rat ATII cells by AdCMV-Luc. Luciferase activity is expressed as mean RLU per microgram protein and represents the mean value (+SE) of 5 wells derived from 2 different rats. Control ATII cells were incubated in media only for 50 min, after which AdCMV-Luc (10 PFU/cell), suspended in medium, was added to each well. Cells in the Exosurf group were incubated with 10 µg/ml Exosurf for 50 min, after which AdCMV-Luc (10 PFU/cell) was added to each well. Four hours after the addition of AdCMV-Luc, supernatant was aspirated, cells were washed, and media was replaced with medium containing 10% FBS and antibiotics. After 24 h, the cells were harvested, and luciferase activity was determined as described in the text. * Significantly different from control (P < 0.001).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

At present, recombinant adenoviruses are considered among the most promising vectors used for gene therapy, and several clinical phase I trials utilizing these vectors are in progress (27). Two major limitations of recombinant adenovirus-mediated gene transfer in the lung are the inefficiency of in vivo transgene expression and vector-induced tissue inflammation. In fact, widespread inflammation associated with pulmonary diseases may hinder transfection. Zsengeller et al. (41) demonstrated that administration of either cyclosporin A or dexamethasone to cotton rats before their transfection with an E1a-E3-deleted adenoviral vector expressing the luciferase reporter gene resulted in decreased inflammation and increased luciferase expression in the lungs of these rats. Furthermore, this same group demonstrated that, in immune-deficient mice, lung luciferase activity could be maintained longer than in wild-type mice after adenovirus-mediated transfection presumably due, in part, to lymphocytic infiltration and cellular proliferation in wild-type mice.

The development of protocols that circumvent the drawbacks of gene therapy, as detailed above, are essential if adenoviral gene transfer is to be useful clinically. Of particular concern is the reduction of perivascular inflammation associated with adenoviral delivery that causes reduced recombinant gene transfer. Studies in our laboratory have demonstrated that increased levels of nitric oxide, expected to be present during inflammation, cause a decrease in efficiency of gene expression (14). Already, investigators have manipulated adenoviral vectors by deleting the E1 and E2a genes responsible for encoding viral replication proteins to provide increased recombinant gene expression while reducing levels of perivascular inflammation (8). Clearly, methods that improve adenovirus-mediated transfection efficiency allowing a reduction in adenoviral titer delivered to the lung are of particular importance when devising gene transfer strategies for the lung.

In the last few years, surfactant replacement therapy has become a routine procedure in the prevention and treatment of NRDS characterized by the lack of surfactant in the premature lung. This treatment is known to be highly effective in improving lung mechanics and gas exchange, leading to improved clinical outcome and decreased mortality rates (23). A surfactant-deficient state, due to injury of the various components of the pulmonary surfactant system by inflammatory mediators and highly reactive O2 and N2 species (13), coupled with the presence of increased amounts of plasma proteins in the alveolar hypophase, is also present in the lungs of patients with ARDS and contributes to its complex pathophysiology (11, 34).

On the basis of these observations, surfactant replacement has been proposed as a way to decrease the pulmonary pathophysiology in ARDS. In mechanically ventilated baboons rendered surfactant deficient by exposure to 100% O2 for 96 h, aerosolized surfactant administration potentiated the beneficial effects of positive end-expiratory pressure (7, 21). Instillation of porcine surfactant into the lungs of patients with ARDS resulted in a transient improvement of gas exchange (35). Exosurf inhibited endotoxin-stimulated cytokine secretion from AM in vitro and thus may reduce inflammatory cytokine production in ARDS (36). Thus there is potential for the use of pulmonary surfactants as vehicles for the delivery of adenoviruses in the lungs of patients with inflammatory diseases. Pulmonary epithelial cells are known to recycle the lipid components of the alveolar lining fluid in a specific way, therefore, it stands to reason that if adenoviral vectors were suspended in these lipids, the efficiency of viral uptake and subsequent gene expression might also increase; however, there is considerable controversy as to which surfactant forms the most effective delivery vehicle.

In a recent study, Jobe et al. (24) showed that the suspension of an adenoviral vector in 10 and 25 mg/ml Survanta, a surfactant replacement product used clinically, resulted in increased luciferase gene expression in the lungs of rabbits and preferential expression into peripheral lung tissues without changing lobar distribution. The period of transgene expression was not lengthened by surfactant and, in fact, was absent 7 days postinstillation. Incubation of the virus with surfactant did not result in inactivation of the virus, as evidenced by retention of adenoviral plaque-forming capability in 293 cells.

Data presented herein indicate that preincubation of A549 cells with Exosurf, but not Infasurf, significantly enhanced their transfection by adenoviral vectors (Fig. 1). Recently, Pataki et al. (32) showed that intratracheally instilled Exosurf was taken up more efficiently by AM and ATII cells than an equivalent amount of Infasurf. These observations are consistent with the higher efficiency of Exosurf in augmenting adenoviral-mediated gene transfer in A549 cells, as shown herein. It should be pointed out that our studies with Infasurf involved identical conditions to that used for Exosurf studies in A549 cells. We did not directly assay the impact of Infasurf on adenoviral activity, so we cannot rule out the possibility that proteases or nucleases present in the Infasurf inactivated the adenovirus. However, we have repeated our experiments a number of times using different batches of Infasurf with identical results. Whereas Infasurf may prove to have an impact on adenoviral transfection in pulmonary epithelial cells at higher concentrations or different experimental conditions, it was outside the scope of the present study to pursue this possibility.

Exosurf may potentiate recombinant adenovirus-mediated gene transfer by direct effects on viral entry into cells. Adenoviruses contain a heterodimeric protein complex consisting of a 186-kDa fiber protein that mediates high-affinity virus attachment to cells and a 400-kDa pentavalent subunit that contains five Arg-Gly-Asp (RGD) sequences. The entry of adenoviruses into cells can be divided into two stages. During the binding stage, fiber protein on the adenoviral surface plays a crucial role in adenovirus transfection by attaching the virus to an as yet unidentified cellular receptor at neutral pH and begins virus transfection. In the second stage of transfection, the virion particle is internalized via an interaction between an RGD sequence and a specific subset of vitronectin binding integrins on the cell membrane (9). After internalization, the adenovirus efficiently disrupts cell endosomes, allowing the virus genome and transgene to be rapidly transported to the cell nucleus where, under the influence of the inserted promoter, transgene expression is initiated. Potential mechanisms by which Exosurf may directly enhance recombinant adenovirus gene transfer include enhanced endocytosis, fiber receptor interaction with cells, endosome disruption, or transgene expression. The fact that transfection efficiency was decreased significantly at 4°C compared with 37°C strongly suggests that Exosurf enhances the rate of adenovirus fluid phase endocytosis into epithelial cells. Furthermore, data shown in Fig. 3 implicate tyloxapol as the component of Exosurf most likely responsible for enhancing transfection. However, as shown in Fig. 3, the enhancement of transfection efficiency by tyloxapol alone (45% increase above control) was much smaller than that of Exosurf (156% increase), indicating synergism among the various components present in Exosurf.

Our initial efforts to transfect cultured rat ATII cells on plastic dishes were unsuccessful. When we employed Transwell filters in our culture system for rat ATII cells, we were able to demonstrate transfection by AdCMV-Luc that was augmented by Exosurf. Pretreatment of rat ATII cells with Exosurf resulted in a significant enhancement of luciferase activity (Fig. 6). To our knowledge, this is the first successful demonstration of adenovirally mediated transfection of a primary culture of rat ATII cell monolayers. It is interesting to note that the luciferase activity in ATII cells transfected in vivo was much higher than those transfected in vitro using similar doses of adenovirus.

An essential question to be answered is whether gene expression increases because a larger number of ATII cells are transfected in the presence of surfactants or because the efficiency by which individual ATII cells are transfected is enhanced. It is well established that surfactant can augment the rate of spreading and improve the distribution of saline suspensions instilled into the lungs (26). In the previously described studies, Jobe et al. (24) suggested that surfactant-induced spreading would allow an adenoviral vector to reach more of the lung surface and result in increased gene expression. However, Infasurf, which contains hydrophobic surfactant apoproteins responsible for lipid monolayer spreading and absorption, failed to enhance transfection of A549 cells by AdCMV-Luc, indicating that spreading may not be the dominant mechanism by which surfactants enhance uptake into the pulmonary epithelium.

An exciting aspect of this study is the finding that preinstillation of Exosurf resulted in significant enhancement of luciferase activity in alveolar epithelial cells instead of macrophages. ATII cells synthesize and secrete surfactant in the alveolar hypophase (38), transport sodium ions in a vectorial fashion from the alveolar hypophase into the lung interstitial space (30), a process linked to increased fluid reabsorption and reduction of alveolar edema in both patients and animals with ARDS type injury, and serve as progenitors to the squamous alveolar type I cells during normal lung development as well as in the reparative response of the alveolar epithelium to injury (5, 12). ATII cells isolated from lungs of rabbits exposed to hyperoxia had significantly lower levels of surfactant synthesis (18), which resulted in lower levels of lavageable phospholipids in the alveolar space of these rabbits and the development of ARDS-type injury due to surfactant deficiency (17, 19). Furthermore, enhancement of ATII cell antioxidant enzyme content in vitro prevented oxidant-induced decreases in surfactant synthesis (16). Thus our data provide the rationale for the use of surfactant replacement mixtures for the delivery of adenoviral vectors containing antioxidant enzymes or other anti-inflammatory agents to ATII cells to mitigate oxidant injury.

    ACKNOWLEDGEMENTS

We acknowledge the technical assistance of Carpantato Myles and Lynne Addington.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-31197, HL-51173, T32 HL-07553, and HL-09491 and by Grant N00014-97-1-0309 from the Office of Naval Research.

Address for reprint requests: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 S. 19th St., 940 THT, Birmingham, AL 35233-6810.

Received 4 February 1997; accepted in final form 17 June 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Brigham, K. L., A. E. Canonico, B. O. Meyrick, H. Schreier, A. A. Stecenko, and J. T. Conary. Gene therapy for inflammatory diseases. Prog. Clin. Biol. Res. 388: 361-365, 1994[Medline].

2.   Brody, S. L., and R. G. Crystal. Adenovirus-mediated in vivo gene transfer. Ann. NY Acad. Sci. 716: 90-101, 1994[Abstract].

3.   Cheek, J. M., K. J. Kim, and E. D. Crandall. Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am. J. Physiol. 256 (Cell Physiol. 25): C688-C693, 1989[Abstract/Free Full Text].

4.   Davis, J. M., W. N. Rosenfeld, H. C. Koo, and A. Gonenne. Pharmacologic interactions of exogenous lung surfactant and recombinant human Cu/Zn superoxide dismutase. Pediatr. Res. 35: 37-40, 1994[Abstract].

5.   Evans, M. J., L. J. Cabral, R. J. Stephens, and G. Freeman. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp. Mol. Pathol. 22: 142-150, 1975[Medline].

6.   Farstad, T., and D. Bratlid. Pulmonary effects after surfactant treatment in premature infants with severe respiratory distress syndrome. Biol. Neonate 68: 246-253, 1995[Medline].

7.   Fracica, P. J., S. P. Caminiti, C. A. Piantadosi, F. G. Duhaylongsod, J. D. Crapo, and S. L. Young. Natural surfactant and hyperoxic lung injury in primates. II. Morphometric analyses. J. Appl. Physiol. 76: 1002-1010, 1994[Abstract/Free Full Text].

8.   Goldman, M. J., L. A. Litzky, J. F. Engelhardt, and J. M. Wilson. Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study. Hum. Gene Ther. 6: 839-851, 1995[Medline].

9.   Goldman, M. J., and J. M. Wilson. Expression of alpha vbeta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J. Virol. 69: 5951-5958, 1995[Abstract].

10.   Goldsmith, K. T., D. T. Curiel, J. A. Engler, and R. I. Garver, Jr. Trans complementation of an E1A-deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells. Hum. Gene Ther. 5: 1341-1348, 1994[Medline].

11.   Gregory, T. J., W. J. Longmore, M. A. Moxley, J. A. Whitsett, C. R. Reed, A. A. Fowler, L. D. Hudson, R. J. Maunder, C. Crim, and T. M. Hyers. Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome. J. Clin. Invest. 88: 1976-1981, 1991[Medline].

12.   Hackney, J. D., C. E. Spier, U. T. Anzar, K. W. Clark, and M. J. Evans. Effect of high concentrations of oxygen on reparative regeneration of damaged alveolar epithelium in mice. Exp. Mol. Pathol. 34: 338-344, 1981[Medline].

13.   Haddad, I. Y., H. Ischiropoulos, B. A. Holm, J. S. Beckman, J. R. Baker, and S. Matalon. Mechanisms of peroxynitrite-induced injury to pulmonary surfactants. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L555-L564, 1993[Abstract/Free Full Text].

14.   Haddad, I. Y., E. J. Sorscher, R. I. Garver, J. Hong, E. Tzeng, and S. Matalon. Modulation of adenovirus-mediated gene transfer by nitric oxide. Am. J. Respir. Cell Mol. Biol. 16: 501-509, 1997[Abstract].

15.   Herz, J., and R. D. Gerard. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc. Natl. Acad. Sci. USA 90: 2812-2816, 1993[Abstract].

16.   Holm, B., A., B. B. Hudak, L. Keicher, C. Cavanaugh, R. R. Baker, P. Hu, and S. Matalon. Mechanisms of H2O2-mediated injury to type II cell surfactant metabolism and protection with PEG-catalase. Am. J. Physiol. 261 (Cell Physiol. 30): C751-C757, 1991[Abstract/Free Full Text].

17.   Holm, B. A., and S. Matalon. Role of pulmonary surfactant in the development and treatment of adult respiratory distress syndrome. Anesth. Analg. 69: 805-818, 1989[Abstract].

18.   Holm, B. A., S. Matalon, J. N. Finkelstein, and R. H. Notter. Type II pneumocyte changes during hyperoxic lung injury and recovery. J. Appl. Physiol. 65: 2672-2678, 1988[Abstract/Free Full Text].

19.   Holm, B. A., R. H. Notter, J. Siegle, and S. Matalon. Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia. J. Appl. Physiol. 59: 1402-1409, 1985[Abstract/Free Full Text].

20.   Hu, P., H. Ischiropoulos, J. S. Beckman, and S. Matalon. Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L628-L634, 1994[Abstract/Free Full Text].

21.   Huang, Y. C., S. P. Caminiti, T. A. Fawcett, R. E. Moon, P. J. Fracica, F. J. Miller, S. L. Young, and C. A. Piantadosi. Natural surfactant and hyperoxic lung injury in primates. I. Physiology and biochemistry. J. Appl. Physiol. 76: 991-1001, 1994[Abstract/Free Full Text].

22.   Hubbard, R. C., M. A. Casolaro, M. Mitchell, S. E. Sellers, F. Arabia, M. A. Matthay, and R. G. Crystal. Fate of aerosolized recombinant DNA-produced alpha 1-antitrypsin: use of the epithelial surface of the lower respiratory tract to administer proteins of therapeutic importance. Proc. Natl. Acad. Sci. USA 86: 680-684, 1989[Abstract].

23.   Jobe, A. H., and M. Ikegami. Surfactant metabolism. Clin. Perinatol. 20: 683-696, 1993[Medline].

24.   Jobe, A. H., T. Ueda, J. A. Whitsett, B. C. Trapnell, and M. Ikegami. Surfactant enhances adenovirus-mediated gene expression in rabbit lungs. Gene Ther. 3: 775-779, 1996[Medline].

25.   Kendig, J. W., R. H. Notter, C. Cox, L. J. Reubens, J. M. Davis, W. M. Maniscalco, R. A. Sinkin, A. Bartoletti, H. S. Dweck, and M. J. Horgan. A comparison of surfactant as immediate prophylaxis and as rescue therapy in newborns of less than 30 weeks gestation. N. Engl. J. Med. 324: 865-871, 1991[Abstract].

26.   Kharasch, V. S., T. D. Sweeney, J. Fredberg, J. Lehr, A. I. Damokosh, M. E. Avery, and J. D. Brain. Pulmonary surfactant as a vehicle for intratracheal delivery of technetium sulfur colloid and pentamidine in hamster lungs. Am. Rev. Respir. Dis. 144: 909-913, 1991[Medline].

27.   Knowles, M. R., K. W. Hohneker, Z. Zhou, J. C. Olsen, T. L. Noah, P. C. Hu, M. W. Leigh, J. F. Engelhardt, L. J. Edwards, and K. R. Jones. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N. Engl. J. Med. 333: 823-831, 1995[Abstract/Free Full Text].

28.   Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees, and G. Todaro. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17: 62-70, 1976[Medline].

29.   Mastrangeli, A., C. Danel, M. A. Rosenfeld, L. Stratford-Perricaudet, M. Perricaudet, A. Pavirani, J. P. Lecocq, and R. G. Crystal. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J. Clin. Invest. 91: 225-234, 1993[Medline].

30.   Matalon, S., D. J. Benos, and R. M. Jackson. Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L1-L22, 1996[Abstract/Free Full Text].

31.   Nieves-Cruz, B., A. Rivera, J. Cifuentes, G. Pataki, S. Matalon, W. A. Carlo, A. K. Tanswell, and B. Freeman. Clinical surfactant preparations mediate SOD and catalase uptake by type II cells and lung tissue. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L659-L667, 1996[Abstract/Free Full Text].

32.   Pataki, G., L. Czopf, B. A. Holm, and S. Matalon. Quantitation of the alveolar distribution of surfactant mixtures in normal and injured lungs. Am. J. Respir. Cell Mol. Biol. 15: 451-459, 1996[Abstract].

33.   Rosenfeld, M. A., G. Ronald, and R. G. Crystal. Gene therapy for pulmonary diseases. Pathol. Biol. (Paris) 41: 677-680, 1993[Medline].

34.   Seeger, W., A. Gunther, H. D. Walmrath, F. Grimminger, and H. G. Lasch. Alveolar surfactant and adult respiratory distress syndrome. Pathogenetic role and therapeutic prospects. Clin. Invest. 71: 177-190, 1993.

35.   Spragg, R. G., N. Gilliard, P. Richman, R. M. Smith, R. D. Hite, D. Pappert, B. Robertson, T. Curstedt, and D. Strayer. Acute effects of a single dose of porcine surfactant on patients with the adult respiratory distress syndrome. Chest 105: 195-202, 1994[Abstract].

36.   Thomassen, M. J., J. M. Antal, M. J. Connors, D. P. Meeker, and H. P. Wiedemann. Characterization of Exosurf (surfactant)-mediated suppression of stimulated human alveolar macrophage cytokine responses. Am. J. Respir. Cell Mol. Biol. 10: 399-404, 1994[Abstract].

37.   Walther, F. J., R. David-Cu, and S. L. Lopez. Antioxidant-surfactant liposomes mitigate hyperoxic lung injury in premature rabbits. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L613-L617, 1995[Abstract/Free Full Text].

38.   Wright, J. R., and L. G. Dobbs. Regulation of pulmonary surfactant secretion and clearance. Annu. Rev. Physiol. 53: 395-414, 1991[Medline].

39.   Yang, Y., Q. Li, H. C. Ertl, and J. M. Wilson. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69: 2004-2015, 1995[Abstract].

40.   Yang, Y., F. A. Nunes, K. Berencsi, E. Gonczol, J. F. Engelhardt, and J. M. Wilson. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7: 362-369, 1994[Medline].

41.   Zsengeller, Z. K., S. E. Wert, W. M. Hull, X. Hu, S. Yei, B. C. Trapnell, and J. A. Whitsett. Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum. Gene Ther. 6: 457-467, 1995[Medline].


AJP Lung Cell Mol Physiol 273(4):L741-L748
1040-0605/97 $5.00 Copyright © 1997 the American Physiological Society