Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206
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ABSTRACT |
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Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.
epithelial cell culture; keratinocyte growth factor
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INTRODUCTION |
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SECRETION OF THE PHOSPHOLIPID components of surfactant has been studied in vitro especially with type II cells soon after isolation (10, 11, 14, 17, 37, 40). However, secretion of the surfactant proteins has been much more difficult to study. This is because the secretion of phospholipids is usually studied with type II cells maintained on tissue culture plastic, a substratum on which the expression of the surfactant proteins is rapidly diminished (42, 43). To study secretion requires a culture system that maintains both expression of the surfactant proteins and access to the apical cell surfaces.
Many investigators have provided insights and culture conditions to improve the differentiated function of type II cells in vitro (9). In this report, we use the term "differentiation" to mean the expression and secretion of the surfactant proteins and not the transdifferentiation of type II cells into type I cells. Previous studies (9, 12, 43, 46) indicate that maintenance of type II cell differentiation is best achieved with a permissive substratum and a combination of differentiation factors and/or coculture with fibroblasts. The substratum on which the type II cells are cultured is critically important. Maintaining type II cells on tissue culture plastic leads to cell spreading and dedifferentiation as determined by decreased expression of surfactant protein mRNAs and altered patterns of phospholipid biosynthesis. Improvement in functional differentiation has been achieved by culturing type II cells on contracted collagen gels, Engelbreth-Holm-Swarm (EHS) tumor basement-membrane matrix, human amnion, and filters coated with extracellular matrix (9, 16, 42-44, 52). Substrata rich in laminin maintain the cells in a more cuboidal shape and are beneficial, whereas substrata rich in fibronectin cause the cells to flatten and dedifferentiate (5, 36). A cuboidal shape and thereby a permissive cytoarchitecture appears to be a prerequisite for maintaining type II cell differentiation and surfactant protein mRNA stability (45). This cell shape can be achieved by culturing type II cells on EHS gels where the cells form spherules or by having a substratum such as a floating collagen gel that allows the cells to contract the gel and assume a native cuboidal shape (16, 42-44). Neither of these two strategies requires cell growth to increase the number of cells per surface area to maintain a cuboidal shape.
Soluble factors are also important for maintaining differentiated function. Keratinocyte growth factor (KGF), fibroblast growth factor (FGF)-7, and acidic FGF (FGF-1) increase expression of surfactant proteins A, B, and D (SP-A, SP-B, and SP-D, respectively) (46, 48, 52). Both SP-A and SP-D are increased in lavage fluid of rats instilled with KGF (54). Corticosteroids are especially stimulatory for the expression of SP-B but may be inhibitory for SP-A and SP-C (2, 46). The type of serum in which the cells are maintained also alters type II cell phenotype. Fetal bovine serum (FBS) causes the cells to flatten and diminishes differentiation, whereas rat serum stimulates type II cell DNA synthesis (26) and maintains the type II cell phenotype (4, 6). In addition, rat serum and porcine serum stimulate surfactant lipid synthesis (6).
In the fetal lung, fibroblasts or fibroblast products are critical for the development of the alveolar epithelium. Recently, we demonstrated (46) that culturing type II cells on EHS gels with adult lung fibroblasts maintains type II cells in a highly differentiated state. However, in this system the type II cells form spherules and the apical surfaces face inward, so that some functions, such as secretion and lipid uptake, cannot be easily studied.
Our goal was to stimulate type II cells to proliferate and form a monolayer of differentiated cells that allows access to the apical surfaces for the study of secretion. Proliferation of adult type II cells has been difficult to sustain in vitro. A number of growth factors can stimulate thymidine incorporation and increase the thymidine labeling index, but only a few growth factors can actually increase cell numbers over a period of days to weeks. In studies of type II cell proliferation at very low-density plating (1,000 cells/cm2), significant growth required a combination of growth factors and the addition of concentrated rat bronchioalveolar lavage fluid (25). We recently reported that a combination of KGF and EHS matrix added to the medium increased proliferation and differentiation of rat type II cells, but this system was not always reproducible for unknown reasons (52). Thus we sought to determine conditions that would consistently produce type II cell proliferation and differentiation over an extended culture period.
Although much is known about secretion of the surfactant phospholipids, much less is known about secretion of the surfactant proteins. SP-B and SP-C are very hydrophobic and are thought to be integral components of lamellar bodies and hence are secreted with the phospholipid components of surfactant. SP-D is much more hydrophobic, is not found in lamellar bodies, and is thought to be secreted by a nonlamellar body route. The relationship of the secretion of SP-A to lamellar body phospholipids is more complex and controversial. The current hypothesis is that newly synthesized SP-A is secreted independent of lamellar bodies, and then the extracellular SP-A is endocytosed, routed to lamellar bodies, and secreted with lamellar bodies (15, 32, 39). However, studies directly measuring the secretion of SP-A and phospholipids have been difficult to perform, because phospholipid secretion by type II cells is usually done under culture conditions (e.g., on tissue-culture plastic) that do not support surfactant protein gene expression. Direct studies of SP-A and phospholipid secretion require a steady-state culture system with high levels of SP-A expression, lamellar body formation (required for metabolic labeling), and access to cell apices. Such a system should allow us to determine whether SP-A is secreted independent of the lipid components of surfactant and whether SP-A and SP-D secretion is regulated or constitutive.
In this study, we describe a culture system for maintaining the synthesis and secretion of surfactant proteins and phospholipids by rat type II cells in vitro. With this system, we have found that both SP-A and SP-D are secreted independently of regulated phospholipid secretion.
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MATERIALS AND METHODS |
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The source of most reagents is stated in the description of the individual methods. KGF was purchased from Promega Biotech (Madison, WI) or R&D Systems (Minneapolis, MN). FGF-10, heparin-binding epidermal-like growth factor (HB-EGF), and hepatocyte growth factor (HGF) were purchased from R&D Systems. LysoTracker Green, MitoTracker Green, and BODIPY phosphatidylcholine were purchased from Molecular Probes (Eugene, OR).
Isolation and culture of alveolar type II cells. Alveolar type II cells were isolated from specific pathogen-free adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) by dissociation with porcine pancreatic elastase (Worthington Biochemical, Freehold, NJ) and partial purification on discontinuous metrizamide gradients (10). Type II cells were plated on a filter insert (30-mm-diameter Millicell-CM; Millipore, Bedford, MA) that had been coated with 0.4 ml of a 4:1 (vol/vol) mixture of rat-tail collagen and EHS tumor matrix (Matrigel; Collaborative Biomedical Products, Bedford, MA). The mixture was prepared at 4°C and allowed to gel at room temperature and contained ~0.8 mg of rat-tail collagen and 2 mg of EHS protein per milliliter. Viable cells (2.5 × 106) were plated in 2 ml of DMEM containing 10% FBS (Irvine Scientific, Santa Ana, CA) [or in later experiments 5% rat serum (Pel Freez Biologicals, Rogers, AK)] plus 2 mM glutamine, 2.5 µg/ml amphotericin B, 100 µg/ml streptomycin, and 100 µg/ml penicillin G; and 2.5 ml of the same media was added to each well outside the insert. After attachment for 20 h, the monolayers were rinsed; 0.4 ml of the specified medium was added to the apical surface, and 2.5 ml of the medium was placed outside the insert. In different experiments, the medium contained combinations of 1% charcoal-stripped FBS (CS-FBS), 5% rat serum, 10 ng/ml KGF, or dexamethasone. The wells were then placed on a rocking platform inside an incubator gassed with 10% CO2. The medium was changed every 48 h.
Measurement of SP-A and SP-D. SP-A and SP-D were measured by ELISA as described previously (30). Initially, rat SP-A and SP-D, which were purified from lavage fluid from rats 28 days after silica instillation (19), were used as standards for the assay. In later studies, recombinant rat SP-D produced in Chinese hamster ovary cells was used as the SP-D standard. The IgG was purified on protein A sepharose. The standards and antibodies were generous gifts of Dennis R. Voelker (Denver, CO). Polyclonal anti-rat SP-A or anti-rat SP-D rabbit IgG (10 µg/ml in 0.1 M sodium bicarbonate) was bound to wells in microtiter plates (Immulon 1 plates; Dynatech Laboratories, Alexandria, VA) overnight at room temperature. The wells were then incubated with a 5% (wt/vol) solution of nonfat dry milk in phosphate-buffered saline (PBS; pH 7.2) to block nonspecific binding (blocking buffer). After the wells were washed with the blocking buffer, 100 µl of purified rat SP-A or SP-D (0-20 ng) for standards or appropriately diluted lavage samples were added to each well. Plates were incubated for 90 min at 37°C and then washed with 20% blocking buffer and 1% Triton X-100 (vol/vol) in PBS (antibody buffer). Anti-SP-A or SP-D antibody-conjugated horseradish peroxidase (200 µl; 2 µl/ml and 30 µl/ml in antibody buffer for SP-A and SP-D, respectively) was added to the wells, and the plates were incubated for 90 min at 37°C. After additional washing with 1% Triton X-100 in PBS, 200 µl of the color-developing agent (0.1% o-phenylenediamine and 0.015% hydrogen peroxide in 0.1 M citrate buffer, pH 4.6) were added. The reaction was carried out for 5 min at room temperature in a darkened room and was stopped by the addition of 100 µl of 1 M sulfuric acid. The absorbance at optical density of 490 nm was recorded with Microplate Autoreader EL-311s (Bio-Tek Instruments, Winooski, VT).
Measurement of DNA.
To harvest type II cells for DNA assay, the collagen-EHS gel was teased
off the insert and placed in a polypropylene tube. The matrix was
digested by incubation with 1 ml of a 4:1 (vol/vol) mixture of 5 mg/ml
type I collagenase (Worthington Biochemical, Lakewood, NJ) and dispase
(BD Biosciences, Bedford, MA). The cells were collected in saline,
sedimented, resuspended, and washed once before being resuspended in
phosphate buffer for the DNA analysis. The suspension was frozen and
stored at 20°C. After being thawed, the cells were sonicated and
the DNA content was determined fluorometrically (23).
Immunocytochemistry and electromicroscopy. Immunocytochemistry was performed as previously described (52, 53), and electromicroscopy was done by standard techniques (51). Cells were immunostained for SP-D in acid alcohol-fixed cultures, embedded in paraffin, and localized with polyclonal rabbit anti-rat SP-D IgG and FITC-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) (52). Staining with biotinylated Maclura promifera lectin (Vector Laboratories, Burlingame, CA) was performed as previously described (13, 21).
Ribonuclease protection assay for the surfactant proteins. Type II cells on the gels were directly lysed into 4 M guanidinium isothiocyanate, 0.5% N-laurylsarcosine, and 0.1 M 2-mercaptoethanol in 25 mM sodium citrate buffer. Total cellular RNA was isolated by ultracentrifugation for 18 h at 150,000 g through a 5.7 M CsCl cushion. Total RNA isolated from type II cells was analyzed for surfactant protein mRNA expression with a ribonuclease protection assay (RPA) that allowed simultaneous measurement of mRNAs for SP-A, -B, -C, and -D as described previously (46, 54). The primers used to generate the riboprobes and their sizes have been reported (54).
For the RPA, 3 µg of total cellular RNA was hybridized at 45°C for 16-18 h with 2.4 × 104 counts/min (cpm) of the antisense probe. The mRNA not protected by hybridization with a target probe was digested with a mixture of RNases A and T1. The protected RNA duplex fragments were precipitated, resuspended, and separated on an 8% polyacrylamide-7 M urea gel. The gel was dried and exposed to Hyper film (Amersham Life Science Products, Arlington Heights, IL) atRegulated phosphatidylcholine and protein secretion. On day 6 under air-liquid conditions (day 7 of culture), the cells were labeled overnight with [3H]choline. Secretion experiments were performed 24 h later. The monolayers were washed and agonists were added to the apical (0.4 ml) and basolateral solutions. Secretion was allowed to proceed for 3 h. The medium was centrifuged at 300 g for 10 min, and the supernatant was extracted and processed as described previously (41). Secretion was calculated as the percent of total phosphatidylcholine recovered in cells plus medium as described previously (40). SP-A and SP-D were measured via ELISA.
Statistics. A one-way ANOVA was used to determine whether the means of continuous outcomes were different by treatment. Fisher's protected least difference multiple-comparison procedure was then used to determine which pairs of means differed. Statistical significance was defined as P < 0.05 by Tukey's HSD test for multiple comparisons. Values are presented as means ± SE.
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RESULTS |
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Initial feasibility studies. Our goal was to improve type II cell culture conditions for the study of SP-A and SP-D secretion. Cells were maintained in a Millicell system where the apical medium could be sampled (12, 52). We rocked the cultures so that the epithelial cells would be exposed briefly to the gas phase as occurs in the lung in vivo. The substratum on which the type II cells were cultured was a mixture of 20% (by volume) EHS matrix (Matrigel) and 80% rat-tail collagen that we prepared (16). On gels containing a very high percentage of EHS, type II cells formed spherules that trapped the apical secretions, and secretion of SP-A and SP-D into the medium decreased substantially. We performed a variety of preliminary experiments with different growth factors, various concentrations of dexamethasone, the addition of rat serum, and the importance of a coculture with fibroblasts to discover conditions suitable for measurement of SP-A, SP-D, and phospholipid secretion.
Effect of KGF without fibroblasts on SP-A and SP-D secretion.
We knew that KGF improved the differentiation of type II cells
(46, 48, 52), and we sought to determine whether other growth factors were as effective as KGF. The growth factors and the
concentrations used were chosen based on the ability to stimulate thymidine incorporation by adult rat type II cells (24, 27, 33). As shown in Fig. 1, KGF was
the only growth factor that stimulated the production and secretion of
significant amounts of SP-A or SP-D. This was surprising, because
FGF-10 is very similar to KGF, binds the same FGF receptor splice
variant (FGFR-2IIIb) (20, 29), and stimulates type II cell
proliferation in vivo. The morphology of type II cell cultures
without KGF was that of a flat monolayer, whereas the cultures with KGF
had areas of refractile cuboidal cells. However, a uniform monolayer of
cuboidal epithelial cells was not produced with KGF alone.
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Effect of KGF with fibroblasts on SP-A and SP-D secretion. Because our previous studies that showed that coculture with fibroblasts helped maintain the phenotype of differentiated type II cells, studies were performed with KGF in the presence of fibroblasts (12, 46, 52). In other studies, induction of differentiation of fetal lung epithelium in a transwell system by fetal lung mesenchyme was maximal when the epithelial explants were placed directly above the mesenchyme (J. M. Shannon, unpublished observations). Therefore, the fibroblasts were plated in a collagen gel on the underside of the matrix-coated filter. To prevent fibroblast proliferation and gel contraction, the fibroblasts were treated with 10 µg/ml of mitomycin C for 2 h. Early-passage human lung fibroblasts were used because these were a known source of HGF and KGF and have been used in our previous studies (33). Type II cells were plated in medium containing 10% FBS for 24 h to promote attachment, and then the medium was changed to DMEM with 1% CS-FBS. The coculture with fibroblasts clearly improved the monolayer of differentiated type II cells compared with the effect of KGF alone. Type II cells cocultured with fibroblasts secreted relatively high levels of SP-A (1,149 ± 61 ng/ml) and SP-D (3,177 ± 319 ng/ml) into the apical medium (n = 4). The addition of KGF slightly increased the secretion of SP-A (1,418 ± 68 ng/ml) and SP-D (4,469 ± 347 ng/ml) in the presence of fibroblasts. By immunostaining, SP-A and SP-D could be readily seen in the epithelial cells. However, by electron microscopy these cells had relatively few lamellar inclusions (data not shown), and by immunostaining, the content of pro-SP-C was significantly less than that observed in type II cells in whole lung (data not shown). Therefore, we sought to further improve this culture system.
Effect of addition of rat serum and dexamethasone on SP-A and SP-D
secretion.
We determined in earlier studies (26) that rat serum was
mitogenic for rat type II cells and might provide a more compact cuboidal epithelium. In addition, rat serum provides a source of
exogenous fatty acids and increases lipid synthesis for the formation
of lamellar bodies (6). Rat serum enhanced the
proliferative effects of KGF, which could be seen visually or
quantitated as the amount of DNA per well. As shown in Fig.
2A, rat
serum increased the amount of DNA per well compared with KGF alone;
dexamethasone inhibited this effect. The values for DNA per well with
1% CS-FBS alone were about half that for 1% CS-FBS plus KGF (data not
shown). Addition of dexamethasone to medium containing KGF and rat
serum increased the amount of SP-A in the apical medium more than
twofold when normalized to DNA (Fig. 2B). Rat serum plus KGF
stimulated accumulation of SP-D in the apical medium as much as rat
serum plus KGF and dexamethasone (Fig. 2C). In all of these
experiments, the amounts of SP-A and SP-D protein were normalized to
DNA values, because the amount of DNA per well varied with the culture
conditions. Hence, in the presence of KGF, rat serum, and
dexamethasone, no further enhancement of SP-A or SP-D secretion was
seen by coculture with fibroblasts.
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Secretion of phospholipids, SP-A, and SP-D.
To determine whether SP-A and SP-D were secreted along with the
phospholipids of surfactant in these cultures, the type II cells were
incubated with [3H]choline for 16 h on day
6 of air-liquid culture to label phosphatidylcholine. Secretion
was evaluated over a 3-h period on day 7. In preliminary studies, we determined that the addition of dexamethasone to KGF and
rat serum increased the percentage of agonist-induced phospholipid secretion and that a short duration of exposure to dexamethasone was
preferable to a long exposure. Dexamethasone (108 M) was
therefore added for the 2 days before the addition of [3H]choline. As can be seen in Fig.
6, there was an increase in phospholipid
secretion with terbutaline, ATP, and tetradecanoyl phorbol acetate
without any concomitant increase in SP-A or SP-D. Hence, in these
cultures, there was agonist-induced regulated secretion of
phospholipids and apparent constitutive secretion of SP-A and SP-D. To
decrease the possibility of any nonspecific adsorption of SP-A to the
culture vessel, two of the experiments had 3 mg/ml of bovine serum
albumin added to the media, which did not affect the results.
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DISCUSSION |
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The purpose of this study was to develop a culture system with which we could study secretion of SP-A and SP-D. This required a polarized monolayer that afforded access to both the basolateral and apical fluids. To accomplish these goals, we used a combination of KGF, rat serum, and in most studies, dexamethasone or coculture with fibroblasts to produce a monolayer of differentiated type II cells. These cells maintained high levels of surfactant protein mRNAs and secreted SP-A and SP-D into the apical medium. In short-term incubations, there was regulated secretion of phospholipids. Importantly, only constitutive secretion of SP-A and SP-D could be demonstrated in these cultures.
The pathway of secretion of SP-A has been controversial. Via immunogold techniques, SP-A has been identified in lamellar bodies and hence is thought to be secreted with lamellar bodies. There is some in vitro data to support this concept (11, 52); however, there are differences in the content of SP-A reported for isolated lamellar bodies (31, 35, 49). The consensus is that the content of SP-A in lamellar bodies is low and much lower than that found in alveolar surfactant. Several investigators have found little evidence for SP-A secretion with phospholipids in vitro and have suggested that secretion of newly synthesized SP-A is independent of phospholipid secretion and that the SP-A found in lamellar bodies occurs only after endocytosis of previously secreted SP-A (32, 39, 47). Our data support this concept that SP-A is secreted independent of surfactant phospholipids. In our experiments, we demonstrated agonist-induced phospholipid secretion without a concomitant increase in SP-A secretion. SP-D has always been thought to be secreted independently of lamellar bodies (7, 50). There are no agonists known to induce SP-D secretion in culture. However, there remain some limitations in the interpretation of these observations. In these experiments, SP-A and SP-D were measured via ELISA. The accumulation in the medium reflects the balance of secretion and reuptake or adsorption to the cells or culture-vessel surfaces. The failure of finding an increase in SP-A concentration with secretagogues strongly supports the concept that SP-A is secreted independently of phospholipid. However, we cannot absolutely exclude regulated secretion that was masked by the binding of secreted SP-A to the culture vessel, cells, or gel. We performed the secretion studies with and without 3 mg/ml of bovine serum albumin to minimize any adsorption and found no difference in the accumulation in the medium. We previously demonstrated that this concentration of albumin blocks the adsorption of SP-A to plastic surfaces. In addition, it is possible that cell culture per se influenced the secretory pathway for SP-A in terms of processing, transport, or subcellular compartmentalization. The type II cells in culture may process SP-A by routes different from type II cells in vivo. This concern cannot be addressed in our studies.
This culture system represents a significant improvement over previous culture systems for maintaining rat type II cells. The system has been very reproducible, and the type II cells are highly differentiated. Another significant improvement is the rate of phospholipid secretion with and without agonists. In current experiments, the mean secretion in 3 h was 9.2% of total phosphatidylcholine with a combination of agonists and 3.2% under basal conditions (see Fig. 6). In our previous report (52), basal secretion was 1.1% and rose to 5.0% with ATP and terbutaline. By comparison, rat type II cells cultured for a week on a collagen substratum under air-liquid conditions showed a basal rate of phospholipid secretion of 0.2% over 3 h that rose to 0.4% with ATP (12). Another difference from other culture systems is that the type II cells proliferate to form the monolayer. These cells are therefore highly viable but may have properties that are closer to the phenotype of the hyperplastic type II cells seen in interstitial lung diseases or after lung injury.
The factors that are important for this culture system are the substratum, KGF, rat serum, and dexamethasone. The substratum is important to keep the cells from spreading. We found that the proportion of EHS to rat-tail collagen determines whether type II cells form a monolayer or spherules. KGF was also critical for these studies. We compared KGF to HGF, FGF-10, and HB-EGF. Only KGF produced an obvious increase in the number of cuboidal cells that could be seen by phase microscopy, and only KGF increased the amounts of SP-A and SP-D in the apical fluid. The observation that KGF was different from HGF and HB-EGF is not surprising, because these factors work through different receptors. Previous studies with type II cells cultured on EHS matrix demonstrated that KGF increased the expression of surfactant proteins (46, 48). However, the difference between KGF and FGF-10 was surprising. Both are thought to recognize the same receptor (20, 29). Presumably, there are costimulatory molecules or other subtle differences in the receptor signaling that are presently not known that account for this difference. For example, FGF-10 binds heparin more avidly than KGF, and the expression of heparin sulfate proteoglycans, which are important for binding of FGFs to their receptors, may be altered in these cultured type II cells. FGF-10 will stimulate thymidine incorporation in cultured rat type II cells and will also stimulate obvious type II cell proliferation when administered intratracheally in vivo (data not shown). The precise differences in how KGF and FGF-10 affect type II cells will require additional studies. However, it is known that there are significant differences in the biological properties of FGF-10 and KGF in developing lungs (3, 34). In fetal lungs, FGF-10 has more chemotactic properties to direct the migration of the forming epithelium and is a weak mitogen. In contradistinction, KGF is a more potent mitogen but is not a chemotactic factor for the epithelium.
Rat serum was clearly mitogenic, and it enhanced the proliferative effect of KGF and also increased the plating efficiency of rat type II cells on these matrices. In our earlier studies on lipid metabolism, rat serum increased the incorporation of acetate into saturated phosphatidylcholine (6). However, the specific factors in rat serum that account for these findings are not known and are probably multiple because of the complexity of the proteins and lipids in serum. Earlier studies demonstrated that rat serum contains a significant amount of HGF (22). Although it is possible that HGF contributed to the proliferative effects of rat serum in our studies, it could not account for all the findings. Rat serum alone did not increase the mRNA levels of surfactant proteins. However, rat serum increases the proliferative effects of KGF. In an earlier study (33), it was noted that FBS was required for the effect of KGF on thymidine incorporation. Clearly, additional studies on the interaction between rat serum and KGF are warranted; however, they are beyond the scope of this report.
The effects of corticosteroids on type II cells are complex. It has
been known for some time that there are significant dose- and
time-dependent effects of corticosteroids on the expression of SP-A in
developing lungs (28). Dexamethasone also inhibits epithelial proliferation when administered toward the end of gestation. Our in vitro observations are consistent with these results. A high
concentration of dexamethasone (107 M) markedly inhibited
proliferation. Dexamethasone also stimulates some but not all markers
of type II cell differentiation (46). For example,
dexamethasone increases expression of SP-B and fatty acid synthase
(2, 38, 46). In our studies, there was increased expression of SP-B when dexamethasone was added from day 1 through day 7 in culture, whereas there was little effect on
the other surfactant protein mRNA levels. Dexamethasone improved the
ultrastructural appearance of lamellar inclusions such that they
appeared more organized, less condensed, and similar to type II cells
in vivo.
Some questions remain unanswered. We did not evaluate whether the
effects of KGF and rat serum are direct or indirect by inducing the
production of unidentified autocrine growth factors for type II
cells. For example, it is known in other systems that KGF
increases secretion of HB-EGF (1, 8). In unpublished
studies we have demonstrated that alveolar type II cells express HB-EGF
and that HB-EGF mRNA is increased with exogenous KGF, although not as
markedly as with HB-EGF itself or transforming growth factor-.
In summary, we have developed a reproducible method for producing and maintaining alveolar type II cells in vitro in which the cells form a polarized monolayer and the collection of both apical and basolateral secretions is possible. These cells secrete surfactant phospholipids in a regulated manner, but secretion of SP-A and SP-D appears to be independent and constitutive. We believe that this culture system will prove to be very useful in defining the physiological properties of alveolar type II cells.
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ACKNOWLEDGEMENTS |
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The authors thank Lynn Cunningham for the immunocytochemistry; Gail Smith for the electron microscopy; and Dennis Voelker and Mandy Evans for providing the SP-A and SP-D and the corresponding antibodies for the SP-A and SP-D ELISAs. Additionally, the authors appreciate the help from Zung Tran in performing the statistical analyses.
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FOOTNOTES |
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These studies were funded by National Institutes of Health Grants HL-29891 and HL-56556.
Present address for J. M. Shannon: Division of Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Address for reprint requests and other correspondence: R. J. Mason, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: masonb{at}njc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00027.2001
Received 31 January 2001; accepted in final form 2 October 2001.
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