Maintenance of the mouse type II cell phenotype in vitro
Ward R.
Rice,
Juliana J.
Conkright,
Cheng-Lun
Na,
Machiko
Ikegami,
John M.
Shannon, and
Timothy E.
Weaver
Division of Pulmonary Biology, Children's Hospital Medical
Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
The purpose
of this study was to identify culture conditions for maintenance of
isolated mouse type II cells with intact surfactant protein (SP) and
phospholipid production. Type II cells were isolated from 6-wk-old mice
and cultured on Matrigel matrix-rat tail collagen (70:30 vol/vol) in
bronchial epithelial cell growth medium minus hydrocortisone plus 5%
charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under
these conditions, type II cells actively produced surfactant
phospholipids and proteins for at least 7 days. Synthesis and secretion
of surfactant phospholipids and SP-A, -B, -C, and -D declined on
day 1 of culture but recovered by day 3, reaching
levels comparable to or exceeding freshly isolated cells by day
5. Abundant lamellar bodies were readily apparent in cells
examined on days 5 and 7, and a surfactant pellet
was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered
surface tension to 5.2 ± 2.3 mN/m. This is the first description
of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.
surfactant; secretion; lung
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INTRODUCTION |
ISOLATED ALVEOLAR
type II cells in primary culture have provided insight into the
function of this important cell type in the lung. However, the rapid
loss of the type II cell phenotype has limited the usefulness of this
system. Manipulation of culture substratum and media has identified
conditions that support the synthesis of surfactant proteins (SPs) and
phospholipids in primary cultures of rat type II cells. Key substratum
components include elements of extracellular matrix, such as the
basement membrane extracted from Engelbreth-Holm-Swarm (EHS) tumor
(commercially available as Matrigel), which contains laminin, type IV
collagen, and heparin sulfate proteoglycan (24, 26).
Interaction of type II cells with extracellular matrix is thought to
promote a native cuboidal cell shape, which is important for type II
cell function in vitro (25, 27). Keratinocyte growth
factor (KGF; fibroblast growth factor-7) has been identified as a
critical component of the culture medium, which likely reflects the
importance of epithelial-mesenchymal interactions in vivo (28,
36). In addition to effects of media and substratum, the culture
of type II cells at an air-liquid interface (by limiting the amount of apical medium and rocking the culture dish) has also been shown to
enhance maintenance of the rat type II cell phenotype in vitro (7, 35).
Although considerable advances have been made in optimizing culture
conditions for rat type II cells, comparable progress for mouse type II
cell culture is lacking. The development of such a culture system is
important, since it would allow the study of type II cells from a large
number of transgenic mouse lines in which the SP-C promoter has been
used to target transgene expression to the distal epithelium. An
important step toward achieving this goal was the development of a
reliable method for isolation of mouse type II cells in high yield and
purity (4). These cells have been maintained in mixed
culture for 7-14 days, but surfactant phospholipid and protein
synthesis and secretion were not examined (32). In the
present study, we describe culture conditions that preserve key aspects
of the type II cell phenotype for up to 7 days in culture, including
the synthesis and secretion of a functional surfactant complex.
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MATERIALS AND METHODS |
Materials.
Dispase was purchased from Fisher (Cincinnati, OH). CD45 and CD32 were
purchased from BD PharMingen (San Diego, CA). Matrigel was
obtained from BD Biosciences (Franklin Lakes, NJ). Human KGF was
purchased from Peprotech (Rocky Hill, NJ). Bronchial epithelial cell
basal medium (BEBM) and bronchial epithelial cell growth medium (BEGM)
were obtained from Clonetics (Walkersville, MD). BEGM is BEBM that also
contains bovine pituitary extract, triiodothyronine, retinoic acid,
insulin, hydrocortisone, transferrin, epidermal growth factor,
epinephrine, gentamicin, and amphotericin. FCS was purchased from
HyClone (Logan, UT). All other chemicals were obtained from
Sigma-Aldrich (St. Louis, MO). Rat tail collagen was prepared as
previously described (19).
Isolation and culture of murine alveolar type II cells.
Cells were prepared from 6-wk-old female C57B/6 mice by a modification
of the method of Corti et al. (4). Mice were anesthetized with 0.2 ml Nembutal by intraperitoneal injection. The abdominal cavity
was opened, and mice were exsanguinated by severing the inferior vena
cava and the left renal artery. The trachea was isolated and cannulated
with a 20-gauge luer stub adapter. The diaphragm was cut, and the chest
plate and thymus were removed. With the use of a 21-gauge needle fitted
on a 10-ml syringe, lungs were perfused with 10-20 ml 0.9% saline
via the pulmonary artery. Dispase (3 ml) was rapidly instilled through
the cannula in the trachea followed by 0.5 ml agarose (45°C). Lungs
were immediately covered with ice for 2 min to gel the agarose. After
this incubation, lungs were removed from the animals and incubated in 1 ml dispase for 45 min (25°C). Lungs were subsequently transferred to
a 60-mm culture dish containing 7 ml of HEPES-buffered DMEM and 100 U/ml DNAse I, and lung tissue was gently teased from the bronchi. The cell suspension was filtered through progressively smaller cell strainers (100 and 40 µm) and nylon gauze (20 µm). Cells were collected by centrifugation at 130 g for 8 min (4°C) and
placed on prewashed 100-mm tissue culture plates that had been coated for 24-48 h at 4°C with 42 µg CD45 and 16 µg CD 32 in
PBS. After incubation for 1-2 h at 37°C, type II cells were
gently panned from the plate and collected by centrifugation.
Type II cells were resuspended in culture media and cultured under
conditions detailed in RESULTS. The media were changed
after the 1st day of culture and every 2 days thereafter.
For experiments requiring cell harvest, matrixes were solubilized by
incubating cultures with dispase containing 1 mg/ml collagenase at
37°C for 60 min.
SP synthesis and secretion.
Type II cells were labeled with [35S]cysteine/methionine
in MEM (cysteine/methionine deficient) containing 2% dialyzed FBS for 4 h and immunoprecipitated exactly as previously described
(16). Total labeled protein was determined by
trichloroacetic acid precipitation, and equal counts per minute of
protein were precleared with normal rabbit serum. Lysates were
sequentially immunoprecipitated by adding 30 µl protein G-Sepharose
(Zymed, San Francisco, CA) and 5 µl of anti-rat SP-A antibody
(8), pro-SP-C antibody (31), mature SP-B
antibody (12, 34), or SP-D antibody (37).
SDS-PAGE and autoradiography were performed as previously described
(16). For Western blotting, gels were electrophoretically
transferred to nitrocellulose and probed with the same antibodies used
for immunoprecipitation or with antibody directed against recombinant, mature SP-C (21).
For secretion experiments, cells were used after 7 days of culture. The
cells were washed three times with BEGM to remove extracellular
surfactant, and secretagogues or inhibitors were added at time
0. Media were removed after 3 h, and cells were rinsed with
0.5 ml of fresh media. The media samples were combined, and cells were
removed by centrifugation (130 g for 8 min). Surfactant pellets were then isolated by centrifugation of media (14,000 g for 30 min) and examined by Western blotting as noted
above. Protein secretion was quantitated by scanning densitometry and expressed relative to control values as 100%.
Surfactant phospholipid synthesis and secretion.
For analysis of phospholipid synthesis, cells were incubated for 24 or
48 h with 1 µCi [14C]acetate/ml. After the cells
were washed to remove free radiolabel, lipids were extracted with
methanol, lipid, and aqueous phases generated with chloroform and 0.2 M
KCl, and the lipid phase evaporated to dryness. After resuspension in
chloroform, samples were spotted on preactivated silica gel plates for
phospholipid determinations. Plates were run in the first dimension
with chloroform-methanol-glacial acetic acid-water (195:75:24:12
vol/vol/vol/vol) and in the second dimension with
tetrahydrofuran-methylal-methanol-2 M ammonium hydroxide
(166:114: 31:17 vol/vol/vol/vol; see Ref. 2). Plates were dried, and phospholipids were visualized with iodine and compared
with phospholipid standards. Phospholipids were harvested from the
plates, and radioactivity was determined. Results for each phospholipid
were expressed relative to total radioactivity.
[3H]phosphatidylcholine secretion was assessed as
previously described for rat type II cells (20). Murine
cells were labeled with 1 µCi/ml [3H]choline for
48 h before assay after 7 days in culture. Cells were washed to
remove free label, and secretagogues or inhibitors were added at
time 0. Media were removed after 3 h, and cells were
rinsed with 0.5 ml of fresh media. The media samples were combined, and
cells were removed by centrifugation (130 g for 8 min).
Lipids were extracted with methanol from the cell and media samples,
lipid and aqueous phases were generated with chloroform and 0.2 M KCl,
and the lipid phases evaporated to dryness. Lipid disintegrations per
minute (dpm) present in the media and cell samples were determined, and
percent phospholipid secretion was calculated as dpm media/(dpm
media + dpm cells) × 100%. Lactate dehydrogenase activity
was determined in each sample as a measure of cytotoxicity. None of the
agents tested had a significant effect on lactate dehydrogenase release
by the cells.
Quantity and surface activity of secreted surfactant.
Media was collected between days 1 and 3,
3 and 5, and 5 and 7 of
culture. The collected media were centrifuged at 150 g for 15 min to remove cells. The supernatant was then centrifuged at 48,400 g for 15 min. The surfactant pellet was resuspended in 0.9%
saline, and the centrifugation was repeated to remove residual medium
from the surfactant pellet. To assess phospholipid content, the
surfactant pellet was suspended in 0.9% saline, and an aliquot was
extracted with chloroform-methanol (2:1) for the phosphorus assay
(9). The surface activity of secreted surfactant was measured with a captive bubble surfactometer at 37°C. The
concentration of each sample was adjusted to 9 nmol phospholipid/µl
and 3 µl of surfactant were applied to the air interface by
microsyringe. Surface tension was measured every 10 s for 300 s to establish equilibrium surface tension before initiation of bubble
pulsation. The minimum surface tension after 35% bubble volume
reduction was measured at the fifth pulsation. The average bubble
volume was 8.7 ± 0.5 µl.
Statistics.
Differences among groups were determined by ANOVA and Newman-Keuls test
using GB-Stat. All values are expressed as means ± SE, and
significance was taken as P < 0.05.
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RESULTS |
Primary culture of murine type II epithelial cells.
Yields of type II cells from 6-wk-old C57B/6 mice varied from 4 to
6 × 106 cells/mouse. The purity of type II cell
preparations was typically >90%, as assessed by modified PAP stain
(5), electron microscopy, and immunostaining for SP-C.
Viability was >95%, as assessed by Trypan blue exclusion. Isolated
mouse type II cells were initially grown under conditions similar to
those optimized for the culture of rat type II cells (28).
Cells were seeded on Matrigel and cultured in DMEM containing 10 ng/ml
KGF. Under these conditions, SP-A, SP-B, SP-C, and SP-D were detected
by Western blot analysis of media samples or type II cell lysates after
7 days of culture, and DNA content of the cultures increased from
3.3 ± 0.1 µg/well on day 1 (n = 4)
to 9.1 ± 0.5 µg/well on day 7 (n = 4). The addition of hydrocortisone inhibited SP-C production by the
cells and significantly decreased the DNA content on day 7 (7.2 ± 0.7 µg/well, n = 4). Production of SP-A,
SP-B, SP-C, and SP-D was also maintained for 7 days when BEGM
containing KGF and without hydrocortisone (a normal component of BEGM)
was substituted for DMEM (Fig. 1). DNA content of the cultures under these conditions was 10.6 ± 1.0 µg/well on day 7 (n = 4), which was not
significantly different from the value obtained for cultures in DMEM
containing KGF. However, cells cultured for 7 days in BEGM contained
abundant lamellar bodies with typical concentric lamellae appearing
similar to freshly isolated cells and consistent with ongoing synthesis
of surfactant phospholipids and proteins (Fig.
2). Cells cultured in DMEM for 7 days
contained fewer lamellar bodies with disorganized and condensed lamellae. These cells also lost their cuboidal shape by 7 days. Therefore, BEGM was used for the remainder of the experiments. To
facilitate the formation of monolayers for secretion experiments, various concentrations of rat tail collagen were added to the Matrigel.
Consistent monolayers that maintained production of SP-C were observed
when type II cells were cultured on Matrigel-rat tail collagen (70:30
vol/vol) in BEGM (minus hydrocortisone) plus 5% charcoal-stripped FBS
and 10 ng/ml KGF (Fig. 3). Under these conditions, the cells would form monolayers suitable for secretion experiments and continued to produce SP-C (Fig.
4). Although cell aggregates comprised up
to 10% of the cultures (Fig. 3), aggregation did not preclude
secretion experiments, since the apical surface of the remainder of the
cells was exposed to the media. The following experiments were
performed with cells cultured under these conditions.

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Fig. 1.
Secretion of surfactant proteins after 7 days of culture.
Type II cells were cultured for 7 days. Media was harvested, and cells
were lysed. Equal amounts of cell protein were subjected to
SDS-PAGE/Western analysis. Representative Western blots from 5 independent experiments are presented. MW, molecular weight. Lane
A, surfactant protein (SP)-A; lane B, SP-B; lane
C, SP-C; lane D, SP-D.
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Fig. 2.
Type II cell ultrastructure after primary cell culture. Freshly
isolated type II cells and cells cultured for 5 or 7 days in bronchial
epithelial cell growth medium (BEGM) were fixed and prepared for
transmission electron microscopy. Cells shown are representative of 3 separate experiments. A: magnification bar = 5 µm for
cells cultured for 7 days. B: magnification bar = 2 µm.
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Fig. 3.
Effect of matrix composition on cell growth. Cells were cultured
for 7 days in BEGM on 100% Matrigel (A) or 90:10 (vol/vol;
B), 80:20 (C), or 70:30 (D)
Matrigel-collagen. Results are representative of 5 independent
experiments. Magnification bar = 22 µm.
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Fig. 4.
Expression of SP-C by type II cells cultured for 7 days.
Cells were cultured as noted in BEGM on matrix for 7 days. Fresh cells
(left) or cells cultured for 7 days (right) were
then fixed and stained for SP-C expression and examined by confocal
microscopy. Results are representative of 5 independent
experiments.
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SP synthesis.
Freshly isolated type II cells were cultured for 0, 1, 3, 5, or 7 days,
and SPs in cell lysates and media were analyzed by Western blotting.
After 1 day of culture, levels of mature SP-B [relative molecular mass
(Mr) = 16 kDa] and SP-C
(Mr = 4 kDa) peptides in cell lysates
declined relative to that in freshly isolated type II cells (Fig.
5). SP-B and SP-C production recovered by
day 3, reaching levels equal to or exceeding levels observed in freshly isolated cells by day 5 of culture. Consistent
with secretion of surfactant by type II cells, a white surfactant
pellet was easily detected after centrifugation of media isolated on alternate days of culture. Western analyses of the surfactant pellet
isolated from media detected both SP-B and SP-C mature peptides (Fig.
5). Similar results were obtained for SP-A and SP-D (Fig. 1).

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Fig. 5.
Storage and secretion of SP-B and SP-C during type II
cell culture. Top: type II cells were cultured for 0, 1, 3, 5, and 7 days. Cells were lysed, and equal amounts of protein from each
time point were subjected to SDS-PAGE/Western analysis under reducing
(SP-C) or nonreducing (SP-B) electrophoretic conditions. Mature SP-B
[relative molecular mass (Mr) ~16 kDa] and
SP-C (Mr ~4 k) peptides were detected by
Western blotting. A representative Western blot from 5 independent
experiments is shown. Bottom: to determine if SP-B and SP-C
were secreted between 5 and 7 days of culture, the media was changed on
day 5, and a surfactant pellet was isolated by
centrifugation of media collected on day 7. The pellet was
resuspended in sample buffer and analyzed by SDS-PAGE/Western analysis;
4, 10, and 20% of the surfactant pellet was electrophoresed in
lanes 1, 2, and 3, respectively.
Secreted mature SP-B and SP-C peptides were detected in media by
Western blotting.
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Sustained intracellular levels of SP-B and SP-C after 3-7 days of
culture suggested ongoing synthesis of SPs. To confirm that SPs were
actively synthesized after 7 days in culture, type II cells were
metabolically labeled with [35S]methionine/cysteine for
the last 4 h of culture, and cell lysates were immunoprecipitated
for SP-A, SP-B, SP-C, and SP-D. All four SPs were detected (Fig.
6). Both nonglycosylated and glycosylated forms of SP-A and SP-D were immunoprecipitated; in addition, both proprotein and processed forms of SP-B and SP-C were detected. To
confirm the specificity of SP-B proprotein processing, type II cells
were cultured for 7 days, pulse-labeled for 1 h, and incubated in
chase medium for up to 4 h (Fig. 7).
A clear precursor-product relationship was established between the
Mr = 40-42 kDa SP-B proprotein and the
Mr = 16 kDa mature SP-B dimer. Taken
together, these results indicate that SPs are synthesized,
posttranslationally processed, and secreted by isolated murine type II
cells after 7 days in culture.

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Fig. 6.
Synthesis of surfactant proteins after 7 days of
culture. Type II cells were cultured for 7 days and labeled with
[S35]cysteine/methionine for the last 4 h of
culture. The cell lysate was sequentially immunoprecipitated with
nonimmune serum [control (C)] followed by antibodies directed against
SP-B, SP-C, SP-A, and SP-D. Immunoprecipitates were analyzed by
SDS-PAGE/autoradiography under nonreducing (SP-B) or reducing (SP-C,
SP-A, and SP-D) electrophoretic conditions. Precursor forms of the
surfactant proteins (filled arrows) were overexposed to detect
processed forms of SP-B and SP-C (open arrows). Glycosylated forms of
SP-A and SP-D (filled arrowheads) and the acylated form of the SP-C
proprotein (open arrowhead) were also detected. A representative
autoradiogram from 3 independent experiments is shown.
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Fig. 7.
Synthesis of mature SP-B peptide after 7 days of culture.
Type II cells were labeled with [35S]cysteine/methionine
for 1 h on day 7 of culture. The labeling media were
removed, the cells were washed, and chase media containing excess
unlabeled cysteine and methionine were added for 0-4 h. Type II
cell lysates were immunoprecipitated and analyzed by
SDS-PAGE/autoradiography under nonreducing electrophoretic conditions.
The SP-B proprotein (Mr = 40-42 kDa;
filled arrow) and processing intermediate
(Mr ~ 25 kDa; open arrow) were
overexposed to detect the mature peptide
(Mr = 16 kDa; arrowhead). A representative
autoradiogram from 3 independent experiments is shown.
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Surfactant phospholipid synthesis and lamellar body formation.
Incorporation of [14C]acetate into newly synthesized
phospholipids was used to determine surfactant phospholipid composition (Table 1). Consistent with the results of
Western analyses of SPs (Fig. 5), incorporation of labeled acetate into
phospholipid was lowest at early culture time points and increased on
days 5 and 7 of culture (Table 1). At each time
point examined, phosphatidylcholine was the most abundant phospholipid
species, constituting 74.5% (day 3) to 82.3% (day
7) of total surfactant phospholipid. The proportion of minor
phospholipid species (phosphatidylserine, phosphatidylethanolamine,
phosphosphatidylinositol, and phosphatidylglycerol) was remarkably
consistent, ranging from 12.4% (days 5 and 7) to 14.6% (day 3) of total surfactant phospholipid.
Sphingomyelin levels were lowest on day 7, resulting in a
lecithin-to-sphingomyelin ratio that was higher on day 7 than at any other time point. Unexpectedly, the levels of
phosphatidylglycerol declined significantly after day 1 and
remained relatively low for the remainder of the culture period.
Surfactant phospholipid and protein secretion.
Because phosphatidylcholine synthesis was maximal on day 7 of culture, regulation of surfactant phospholipid and protein secretion was determined on this day. PMA stimulated secretion of surfactant phospholipid, SP-B, and SP-C, whereas compound 48/80 inhibited the
stimulated secretion (Table 2). The
-agonist terbutaline inhibited both basal secretion and
PMA-stimulated secretion of surfactant phospholipid, SP-B, and SP-C
when cells were cultured in BEGM (Table 2 and Fig.
8). Because terbutaline stimulates phospholipid secretion from rat type II cells cultured in DMEM, we
hypothesized the inhibitory effect of terbutaline, which we observed
was a result of epinephrine exposure for 7 days, since epinephrine is a
normal component of BEGM. We tested this hypothesis by culturing cells
in BEGM without epinephrine for 7 days. When cells were cultured in
BEGM without epinephrine for 7 days, terbutaline stimulated secretion
of SP-B, relative to control secretion, although addition of
terbutaline and PMA together resulted in greater secretion of SP-B than
either agonist added alone (Fig. 8).

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Fig. 8.
Regulation of SP-B secretion. Cells were cultured for 7 days in BEGM with or without epinephrine, as noted, on matrix. SP-B
secretion was followed for 3 h in the absence or presence of
phorbol 12-myristate 13-acetate (PMA; 100 nM), terbutaline (terb; 10 µM), or both agents. Secretion was analyzed by Western analysis as
noted in MATERIALS AND METHODS. Results are representative
of 3 independent experiments. t, Time.
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Surface properties of secreted surfactant.
Media were collected between days 1 and 3,
3 and 5, or 5 and 7 of
culture, and surfactant was isolated by centrifugation. The quantity of
phospholipid in media was 36.2 ± 0.9, 22.1 ± 3.4, and
18.6 ± 0.9 nmol/ml for media collected on days 3,
5, and 7, respectively. To assess the surface
properties of the secreted surfactant, the media were changed on
day 3 and collected on day 5. A surfactant pellet
was isolated by centrifugation and washed three times to remove serum
proteins. The minimum surface tension detected with the captive bubble
surfactometer was 5.2 ± 2.3 mN/m and ranged from 1.7 to 9.5 mN/m
(n = 5).
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DISCUSSION |
Primary cultures of rat type II epithelial cells maintained on
plastic dishes and in media containing FBS rapidly lose markers associated with the type II cell phenotype in vivo. Synthesis of both
SPs (14, 24, 33) and phospholipids (17, 29) decreases with time in culture, with downregulation of SP expression occurring within hours of cell isolation. In addition to the loss of SP
expression, the ability to sort transfected SP to the lamellar body is
lost (15), suggesting that the regulated secretory pathway is also rapidly downregulated with time in culture. Consistent with
this hypothesis, the number of lamellar bodies decreases during
culture, and the cells become refractory to some secretagogues (29). These striking biochemical changes are accompanied
by altered cell morphology, most notably a transition from the cuboidal epithelium seen in vivo to a flattened cell shape in vitro.
A variety of culture conditions have been shown to retard the loss of
the type II cell phenotype in vitro. Culture of freshly isolated rat or
guinea pig type II cells on components of extracellular matrix or
amniotic membrane and under conditions that promote cuboidal cell shape
preserve many characteristics of type II cells, including SP expression
and the synthesis of surfactant phospholipids (22,
24-26). Accordingly, in the present study, we found
Matrigel to be essential for sustained expression of SPs in cultured
mouse type II cells. However, culture of type II cells on Matrigel
resulted in the formation of multicellular aggregates unsuitable for
secretion experiments in which cell apices were inwardly directed.
Therefore, rat tail collagen was added to the Matrigel to produce a
30:70 mixture (collagen-Matrigel, vol/vol) that promoted formation of monolayers necessary for secretion experiments. As previously reported
for rat type II cells, addition of human KGF to the culture medium
enhanced cell proliferation and SP expression (28, 36). Both DMEM and BEGM supported cell proliferation in the presence of KGF
to the same extent. However, fewer lamellar bodies were noted, and many
cells lost their cuboidal shape when the cells were cultured in DMEM
for 7 days and examined by electron microscopy. Therefore, BEGM was
used for routine culture of mouse type II cells to maintain phenotype.
These optimized culture conditions resulted in ongoing synthesis and
secretion of SP-A, SP-B, SP-C, and SP-D for up to 7 days. SP levels
consistently declined after 1 day in culture but began to recover by
day 3 of culture, reaching levels comparable to or exceeding
those in freshly isolated cells by days 5 and 7. We hypothesize the decreases are the result of type II cell injury after exposure to protease during the isolation procedure. Synthesis of
surfactant phospholipids was maintained during culture, with results
comparable to those previously reported for rat type II cells cultured
on EHS matrix (11). The level of newly synthesized phosphatidylcholine (75-82%; Table 1) detected over a 7-day
period compared very favorably to the phosphatidylcholine content in mouse bronchoalveolar lavage fluid, which ranged from 75 to 85% of
total surfactant phospholipid (1, 3, 10, 13). Consistent with the ongoing synthesis of surfactant phospholipids and proteins, typical lamellar bodies were readily detected in cultured type II
cells; moreover, surfactant secretion was stimulated by secretagogues, reflecting an intact regulated secretory pathway. Importantly, surfactant secreted by cultured type II cells exhibited excellent surface tension-reducing properties in vitro. Taken together, these
results indicate that key aspects of the type II cell phenotype, including surfactant synthesis, secretion, and function, are maintained for at least 7 days of primary culture. This is the first description of the synthesis and secretion of a functional surfactant complex by
mouse type II cells in primary culture.
Surfactant recovered from the media of cultured murine type II cells
was able to reduce surface tension to a relatively low value of 5.2 mN/m, quite similar to the value of 5 mN/m obtained for surfactant
secreted from isolated rat type II cells cultured on plastic for
22 h (6). However, these values are somewhat higher
than reported values for native and replacement surfactants (<1 mN/m;
see Ref. 23). This result may be due in part to incomplete removal of serum proteins (an important component of the culture medium) from the isolated surfactant pellet. Serum proteins are known
to markedly inhibit the surface tension-reducing properties of
surfactant (30). It is also possible that changes in
phosphatidylglycerol or other phospholipid components of surfactant
contributed to the higher minimum surface tension upon bubble
compression. In this regard, further optimization of the culture
medium, such as addition of linoleic acid/albumin complex to increase
phosphatidylglycerol levels (11), may improve the surface
tension lowering properties of the surfactant secreted by cultured type
II cells.
Regulation of surfactant phospholipid secretion has been extensively
studied in isolated rat type II cells, but these experiments were
performed in type II cells that had lost or were actively losing their
differentiated phenotype. Examination of the regulation of SP secretion
has not been possible with previously described culture systems. The
present system has allowed for the first time a direct comparison of
the regulation of surfactant phospholipid secretion with SP secretion
in cultured type II cells that are well differentiated. When cells were
cultured in BEGM, PMA stimulated secretion of surfactant phospholipid,
SP-B, and SP-C, whereas compound 48/80 inhibited the PMA-stimulated
secretion of phospholipid and proteins. The
-agonist terbutaline
inhibited secretion of both surfactant phospholipid and SP-B and -C
when cells were cultured in BEGM. When cells were cultured in BEGM
without epinephrine (a normal component of BEGM), terbutaline
stimulated SP secretion. These latter results are consistent with
studies in rat type II cells and strongly suggest epinephrine in the
BEGM altered the effect of terbutaline on surfactant secretion. Why
chronic exposure to epinephrine alters the type II cell response to
-agonists is presently unclear. Although aggregation of the cells
was noted using the present culture system, the majority of cells
formed a monolayer. Overall, the results of secretion experiments in these cultured murine type II cells were similar to those previously reported for isolated rat type II cells and support the hypothesis that
SP-B and SP-C secretion are coregulated with phospholipid secretion.
This is in contrast to the constitutive secretion of SP-A and SP-D from
isolated rat type II cells (18). The amount of
phospholipid, SP-B, and SP-C secreted after stimulation is likely the
sum of component accumulation in lamellar bodies via both the
biosynthetic and endocytic (recycling) pathways. A careful analysis of
the rates of synthesis and recycling for individual surfactant
components will be required to assess the relative contributions of
these two pathways to lamellar body content.
In summary, culture of murine type II cells on Matrigel-rat tail
collagen substratum and in media supplemented with KGF and carbon-stripped FCS resulted in maintenance of the hallmark features of
the type II cell phenotype for at least 7 days. Surfactant phospholipids were synthesized and packaged into typical lamellar bodies. SP-B and SP-C were synthesized, appropriately processed, and
secreted with phospholipids. Most importantly, secreted surfactant exhibited excellent surface tension-reducing properties. These culture
conditions should facilitate analyses of type II cells isolated from
transgenic and knockout mice.
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ACKNOWLEDGEMENTS |
The expert technical assistance of Mary Falconieri, Emily Martin,
and LeDong Ray is gratefully acknowledged.
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FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Specialized Center of Research Grants HL-56387 (W. R. Rice),
HL-57144 (J. M. Shannon), HL-61646 (M. Ikegami), and HL-56285 (T. E. Weaver).
Address for reprint requests and other correspondence:
W. R. Rice, Div. of Pulmonary Biology, Children's Hospital
Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: ricew0{at}chmcc.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.
March 22, 2002;10.1152/ajplung.00302.2001
Received 2 August 2001; accepted in final form 15 March 2002.
 |
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