Department of Physiology, Monash University, Victoria 3800, Australia
Submitted 11 September 2002 ; accepted in final form 27 May 2003
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ABSTRACT |
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type I alveolar epithelial cell; type II alveolar epithelial cell; alveolar stem cell; lung volume; lung development; surfactant protein A; surfactant protein B; surfactant protein C
Recent studies showing that type I cells have the potential to transdifferentiate into type II cells indicate that the degree of strain experienced by AECs is a critical determinant of their phenotype (7, 9, 10). For instance, in sheep, a species in which AECs differentiate before birth, sustained increases in fetal lung expansion induce type II cells to transdifferentiate into type I cells via an intermediate cell type (9). On the other hand, sustained reductions in lung expansion promote an increase in type II AEC proportions, most probably via transdifferentiation of type I into type II AECs (10). These studies indicate that the basal degree of lung expansion is an important determinant of the relative proportions of each AEC phenotype within the alveolar epithelium.
During fetal development, the future airways of the lung are filled with
liquid, which is secreted across the pulmonary epithelium into the lung lumen
(15). This liquid leaves the
lungs via the trachea, although its efflux is retarded by adduction of the
fetal glottis during apnea
(12) and by contraction of the
diaphragm during fetal breathing movements
(15). These fetal activities
promote the retention of liquid within the future airways, which increases in
volume from 30 ml/kg at 115 days of gestation to 35-45 ml/kg near term in
fetal sheep (13). Thus in late
gestation the fetal lungs are maintained in a distended state, which is
essential for their growth and development
(13,
15). However, at birth, the
removal of liquid from the airways and the entry of air into the lungs reduce
resting lung volumes, because the distending influence of lung liquid is lost
and the creation of surface tension at the air-liquid interface increases the
lung's recoil properties (13,
15). As a result,
end-expiratory lung volumes decrease from 35-45 ml/kg
(3,
13,
17,
21) in fetal sheep late in
gestation to 25-30 ml/kg in air-breathing newborn lambs
(8,
16).
In view of the relationship between basal lung expansion and AEC phenotype, we hypothesized that the proportion of type I and type II AECs would change in relation to the changes in lung expansion during late gestation and after birth. Specifically, we hypothesized that the proportion of type I AECs would predominate in late gestation and increase toward term with increasing lung volume, whereas type II cells would predominate after birth due to a decrease in the basal degree of lung expansion. We also hypothesized that the expression of the surfactant proteins (SP)-A, -B, and -C would change in parallel with the change in type II AEC proportions. Consequently, in separate groups of sheep, we have measured the proportion of each AEC phenotype and the expression of SP-A, -B, and -C between 91 days of gestation and term and then after birth at 2 wk, 8 wk, and 2 yr of age.
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METHODS |
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Analytical Methods
Histological analysis. We chose to identify AECs by morphological criteria using EM as previously described (9, 10), rather than by light microscopy in conjunction with stains for specific cell markers. We did this because it is currently unclear which markers should be used to definitively identify the stem, intermediate, and type I AECs in sheep (see below). Previous studies have shown that, in sheep (1) and humans (4), the nuclear diameters of type I and type II AECs are similar, and, therefore, the chances of counting a nuclear profile of each type are equal by EM. We have made similar observations in our studies, with average nuclear diameters of 4-6 µm for both type I and type II AECs (unpublished observations).
After fixation, the right lung was processed for EM (9). The right lung was separated into the upper, middle, and lower lobes, and then each lobe was accurately cut into 5-mm slices. Every second slice from each lobe was further subdivided into three sections. We then chose six sections at random from each lobe and cut the tissue into cubes (2 x 2 x 2 mm), taking care to avoid major airways and blood vessels. One tissue cube from each section was then selected for further processing (i.e., six cubes per lobe for each of the three lobes; 18 tissue cubes per animal). The tissue cubes were washed in 0.1 M cacodylate buffer, incubated in 2% OsO4 (in 0.1 M cacodylate buffer), and embedded in epoxy resin. At least three epoxy resin/tissue blocks were randomly chosen from each animal. Ultrathin sections (70-90 nm) were cut with a diamond knife, mounted on 200-mesh copper grids, and stained with aqueous uranyl acetate and Reynolds lead citrate. All sections were coded, and the observer was blinded to the age of the animal.
Alveolar epithelial cells were identified under a transmission EM (Joel 100s). For each animal, a minimum of 100 AECs were classified, and the number of nuclear profiles of each type was counted (9, 10). In a previous study (9), we performed multiple AEC counts on the same group of fetuses and found that increasing the number of AECs counted (to >200) did not alter the proportion of each AEC phenotype obtained following the counting of 100 AECs. We viewed at least three different sections per animal, ensuring that only one section per tissue block was analyzed. Identification of AECs depended on clear visualization of the basement membrane, with all AECs localized to the luminal surface of this membrane. AECs were categorized as one of four phenotypes: stem cells, type I AECs, type II AECs, and intermediate AECs. Alveolar epithelial stem cells (AE stem cells) were rounded in shape and contained abundant cytoplasmic glycogen; they did not contain lamellar bodies or have any evidence of a cytoplasmic extension (see Fig. 1 and below). Previous studies may have referred to this cell type as either "progenitor cells" or "immature alveolar type II cells." Type I AECs had elongated cytoplasmic extensions, flattened nuclei, little perinuclear cytoplasm, and few cytoplasmic organelles. Cytoplasmic extensions are defined as peripheral regions of cytoplasm that extend along the luminal surface of the epithelial cell basement membrane, with both apical and basolateral membranes running in parallel to each other and separated by a thin layer of cytoplasm, <0.5 µm in thickness (Fig. 1). Type II AECs were rounded in shape with a rounded nucleus and had microvilli on their apical surface and abundant cytoplasmic organelles, including lamellar bodies (Fig. 1). The intermediate cells were a heterogeneous group that displayed characteristics of both type I and type II AECs. Their classification depended on the presence of a flattened nucleus and marked cytoplasmic extensions, but they also contained lamellar bodies and usually had microvilli on their apical surface (9).
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Surfactant Protein Gene Expression
SP-A, SP-B and SP-C mRNA levels in lung tissues from fetal sheep, lambs,
and ewes were quantified by Northern blot analysis as previously described
(18). Total RNA was extracted
from lung tissue, and 20 µg were denatured and electrophoresed in a 1%
agarose gel containing 2.2 M formaldehyde. The RNA was then transferred to a
nylon membrane (Duralon; Stratagene, La Jolla, CA) by capillary action and
cross-linked to it by ultraviolet light (Hoeffer UVC 500, Amrad). The membrane
was incubated in hybridization buffer for 3-4 h at 42°C. This was followed
by hybridization with the 32P-labeled SP-A, SP-B, or SP-C cDNA
probe (2 x 106 counts · min-1
· ml-1) for 24-48 h at 42°C in the same
hybridization buffer. These ovine-specific cDNA probes have been described
previously (18) and were
labeled with [-32P]dCTP by the random-priming technique
(Oligolabeling kit, Pharmacia).
After hybridization with the labeled probe, the membranes were washed, sealed in airtight bags, and exposed to a storage phosphor screen for 24-48 h at room temperature. To standardize the amount of total RNA loaded onto each lane, the blot was stripped by washing in 0.01x standard saline-sodium citrate containing 0.5% SDS at 80°C for 30 min and was reprobed with a 32P-labeled cDNA probe for 18S rRNA. We quantified the relative levels of SP-A, SP-B, and SP-C mRNA and 18S rRNA by measuring the total integrated density of each band using a phosphorimager and Image-Quant software (Molecular Dynamics, Sunnyvale, CA).
Data Analysis
Data are expressed as the means ± SE, and the level of significance used was P < 0.05. The changes in the proportions of each AEC type were determined separately by oneway ANOVA. In all Northern blots, the total integrated density of each SP-A, SP-B (the density of the two SP-B transcripts were summed), and SP-C transcript was divided by the total integrated density of the 18S rRNA band for that sample (lane) to account for minor differences in total RNA loading between lanes. As a result, the band densities are presented as a ratio of the 18S rRNA band density and, therefore, have no units. To compare values from different Northern blots, we expressed values from each age group as a percentage of the mean value obtained from the same 128-day fetuses that were run on all blots. For each surfactant protein, differences between age groups were determined by one-way ANOVA following log10 transformation to normalize the data.
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RESULTS |
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Undifferentiated AE stem cells. At 91 days of gestation, most epithelial cells (93.8 ± 2.0%) were undifferentiated AE stem cells, but the proportion of this cell type was reduced to 30.3 ± 3.2% by 111 days of gestation (Fig. 2). The proportion of undifferentiated AE stems cells continued to decrease with increasing fetal age, reaching 2.5 ± 0.4% at 120 days, 1.0 ± 0.4% at 128 days, 0.2 ± 0.2% at 132 days, and 0.4 ± 0.2% at 138 days; too few stem cells could be counted at these ages for statistical comparison. No stem cells were observed at 142 days' gestation, in 2- and 8-wk-old lambs, or in 2-yr old sheep (Fig. 2).
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Type I AECs. The proportion of type I AECs increased from 4.8 ± 1.3% at 91 days of gestation to 63.0 ± 3.6% at 111 days of gestation. Similar proportions of type I AECs were maintained throughout the rest of gestation (120 days, 66.9 ± 3.2%; 128 days, 64.8 ± 0.5%; 132 days, 71.6 ± 2.6%; 138 days, 63.2 ± 2.3%; 142 days, 68.9 ± 3.6%). In contrast, at 2 wk after birth, the proportion of type I AECs had decreased to 44.8 ± 1.8% and remained at this level at 8 wk and 2 yr of age (45.5 ± 2.9%) (Fig. 3).
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Type II AECs. At 91 days of gestation, only 1.2 ± 0.9% of all AECs was of the type II cell phenotype. A similar proportion of type II AECs was present at 111 days (4.3 ± 1.5%), but by 120 days, the proportion of type II AECs had increased to 25.1 ± 3.9% (Fig. 3). The proportion of type II AECs remained at similar levels throughout the rest of gestation (128 days, 28.5 ± 2.2%; 132 days, 22.4 ± 3.1%; 138 days, 33.4 ± 1.7; 142 days, 30.0 ± 3.7%). In contrast, the proportion of type II AECs increased from 30.0 ± 3.7% at 142 days of gestation to 52.9 ± 1.5% at 2 wk following birth and remained at this level at 8 wk and at 2 yr of age (53.4 ± 2.7%) (Fig. 3).
Intermediate AECs. At 91 days of gestation, only 0.1 ± 0.1%
of AECs was of an intermediate phenotype. Although this level did not change
significantly for the remainder of gestation (111 days, 2.4 ± 1.1%; 120
days, 5.5 ± 0.7%; 128 days, 5.7 ± 1.3%; 132 days, 5.8 ±
2.1%; 138 days, 3.0 ± 1.4%; 142 days, 1.1 ± 0.4%), it tended to
increase between 120 and 132 days of gestation before declining again by 138
days (Fig. 2). After birth, the
proportions of intermediate AECs remained at low levels (2 wk, 2.2 ±
0.9%; 8 wk, 1.2 ± 0.5%), but these cells were still observed at 2 yr of
age (1%; Fig. 2).
Surfactant Protein mRNA Levels
SP-A. Expressed as a percentage of the mean value for 128-day fetuses, SP-A mRNA levels were low at 91 (15.8 ± 1.6%), 105 (16.5 ± 1.1%), and 111 days (17.8 ± 1.3%) of gestation, before increasing to 100.0 ± 10.7% at 128 days of gestation. SP-A mRNA levels continued to increase after 128 days of gestation to reach 208.4 ± 67.5 and 400.2 ± 80.9% of the 128-day value at 138 and 142 days of gestation, respectively. By 8 wk after birth, SP-A mRNA levels had increased to 728.0 ± 56.2% of the 128-day value and remained at similar values (646.0 ± 65.5%) at 2 yr of age (Fig. 4A).
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SP-B. Relative to the mean 128-day value, SP-B mRNA levels were low at 91 (37.5 ± 4.1%), 105 (45.8 ± 6.6%), and 111 (40.6 ± 7.0%) days of gestation and increased to 100.0 ± 2.6% at 128 days. SP-B mRNA levels continued to increase after 128 days to reach 168.7 ± 22.7 and 264.2 ± 40.6% of the 128-day value at 138 and 142 days of gestation, respectively. At 2 wk after birth, SP-B mRNA levels had decreased to 170.0 ± 11.6% of the 128-day value and remained at similar values at 8 wk after birth (160.5 ± 8.4%) and at 2 yr of age (135.0 ± 5.6%), which was not significantly different from values at 128 days of gestation (Fig. 4B).
SP-C. Relative to the 128-day values, SP-C mRNA levels were low at 91 (22.8 ± 3.0%), 105 (26.1 ± 4.5%), and 111 days (34.0 ± 10.9%) of gestation and increased to 100.0 ± 4.3% at 128 days of gestation. SP-C mRNA levels continued to increase after 128 days of gestation to reach 217.1 ± 55.1 and 223.7 ± 40.0% of the 128-day value at 138 and 142 days of gestation, respectively. At 8 wk after birth, SP-C mRNA levels had increased to 332.0 ± 19.4% of the 128-day value (Fig. 4C) but then decreased to 223.0 ± 29.1% at 2 yr of age.
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DISCUSSION |
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Previous in vitro studies have shown that sustained cell stretch may be an
important determinant of AEC phenotype
(7,
11,
25). More recently we have
provided evidence to indicate that, in vivo, sustained increases in fetal lung
expansion induce type II AECs to transdifferentiate into type I AECs via an
intermediate cell type (9). In
contrast, sustained reductions in fetal lung expansion increase the proportion
of type II AECs, most probably via the transdifferentiation of type I into
type II cells (10). The
findings of these studies indicate that sustained increases in lung expansion
promote differentiation into the type I cell phenotype, whereas reduced lung
expansion promotes differentiation into the type II cell phenotype. As the
basal degree of lung expansion in the fetus is greater than in the newborn
(13,
15), it is not surprising that
type I AECs predominate before birth, accounting for 65% of all AECs;
this finding is consistent with previous studies in fetal sheep
(1,
6,
9). Furthermore, the large
increase in the proportion of type I AECs between 91 and 111 days of gestation
coincides with the exponential-like increase in potential air space volume of
the lung at this stage of development in sheep
(1). The stimulus for this
increase in type I AEC proportions is unknown, but as the increase occurs in
advance of the increase in type II cell numbers it is likely to result from
direct differentiation of undifferentiated stem cells into type I cells, as
previously suggested (1)
(Fig. 1). Indeed, a number of
the type I cells identified at this stage of gestation had significant amounts
of perinuclear cytoplasm and contained some glycogen but were identified as
type I cells due to the presence of cytoplasmic extensions (see
Fig. 1). It is possible that
this increase in type I AEC numbers is causally related to the increase in
potential air space volume that occurs at this stage of gestation
(1). This, in turn, may be
determined by the ability of the fetal glottis and fetal breathing movements
to restrict lung liquid efflux and, therefore, maintain an internal distending
pressure on the lung.
In contrast to type I AECs, few morphologically distinct type II AECs were
present at 111 days (4%), after which the proportion of type II cells
rapidly increased to
25% by 120 days of gestation. The marked increases
in mRNA levels for SP-A (fivefold), SP-B (2.5-fold), and SP-C (threefold) over
this gestational age period (111-128 days of gestation) are consistent with
the increase in the proportion of type II AECs at this time. However,
expression of the surfactant proteins, particularly SP-B and SP-C, was also
evident well before a significant number of morphologically distinct type II
AECs (containing lamellar bodies) could be identified. This raises the
interesting question as to whether expression of the surfactant proteins can
be reliably used to distinguish between AE stem cells and type II AECs early
in gestation. The mechanisms for the rapid increase in type II cell
proportions between 111 and 120 days of gestation are unknown, but it is
unlikely that corticosteroids are involved, as plasma cortisol concentrations
do not begin to increase until
135 days of gestation in fetal sheep
(2). Whatever the mechanisms,
our data indicate that the period between 111 and 120 days of gestation is
critical for type II cell differentiation in fetal sheep in vivo.
Our finding that the proportions of type I and type II AECs did not change
significantly between 120 days of gestation and near term (142 days) was
unexpected considering the large increase in lung luminal volume that occurs
over this time (13). In fetal
sheep, lung liquid volumes increase from 30 ml/kg (
70 mls) at 115
days of gestation to 35-45 ml/kg (
170 ml) near term
(13). If the mechanical load
experienced by AECs is the primary determinant of their phenotype, as
previously proposed (9,
10), these data indicate that
despite the increase in lung luminal volume, the mechanical load experienced
by individual AECs does not change between 120 days of gestation and term.
This could result from lung growth as well as from the considerable amount of
structural remodeling that occurs in the lung over this period of development,
resulting in marked changes in tissue mechanics.
Our study is the first to document the changes in the proportions of type I
and type II AECs before and after birth, and our data are consistent with
previous studies reporting the relative proportions of type I and type II AECs
in fetal sheep before birth (1,
6) as well as in adult humans
(4) and rats
(5). We have demonstrated that
the proportion of type II AECs increases whereas the proportion of type I AECs
decreases after birth, and we propose that these changes result from an
associated reduction in the basal degree of lung expansion. Although
discrepancies exist in the literature as to the precise timing for the
decrease in lung liquid volume around the time of birth, most recent studies
agree that fetal lung liquid volumes are maintained at 35-45 ml/kg up until
1-3 days before labor in sheep
(3,
13,
15,
17,
21). However, the oldest
gestational age examined in this study was 142 days, which is 5 days
before labor in the breed of sheep we used and 2-4 days before the period when
fetal lung liquid volumes arguably decrease. Our previous studies indicate
that, unless there is an external complicating factor (e.g., oligohydramnios),
fetal lung liquid volumes do not decrease before labor onset
(17). The presence or absence
of oligohydramnios, which can cause reductions in lung liquid volumes
(13), was not examined at the
time of lung liquid volume measurement in some recent studies focusing on this
question (3,
21).
The mechanism for the change in AEC proportions after birth is unknown but could have resulted only from type II cell proliferation, type I cell apoptosis, or from type I-to-type II AEC transdifferentiation. Although it was originally considered that type I AECs were terminally differentiated, recent in vitro and in vivo studies have provided evidence to suggest that type I cells are not terminally differentiated but can transdifferentiate into type II cells, particularly in response to reduced levels of lung expansion (10). Consequently, we suggest that a sustained reduction in basal lung expansion, associated with the onset of gaseous ventilation at birth, induces some type I AECs to transdifferentiate, possibly via an intermediate cell type, into type II AECs. A dependent relationship between reduced lung expansion and differentiation into the type II cell phenotype after birth may have functional advantages. In particular, it may increase the lung's potential to produce surfactant at a time when the recoil of the lung increases and the mechanism that opposes its collapse changes from the internal distension by liquid to external bracing by a semirigid structure (chest wall) that is relatively compliant at birth (8).
It is also possible that, with the onset of air breathing after birth, the
larger tidal volumes and the resultant increase in phasic stretch of the lungs
are responsible for the increase in type II cell proportions at this time. In
the fetus, individual breathing movements are essentially isovolumetric, with
a tidal volume of <1% of lung volume
(13). This is because the
viscosity of lung liquid is much greater than air and the fetal chest wall is
very compliant; as a result, parts of the chest wall are drawn in when the
diaphragm contracts (14).
However, after birth, tidal volumes increase to 20% of end-expiratory
lung volumes (functional residual capacity)
(8), indicating that the
breath-by-breath expansion of the lungs substantially increases at this time.
Previous studies have shown that phasic stretch of type II AECs in culture
increases the expression of the surfactant proteins
(20,
22,
23), although a direct effect
on type II cell differentiation was not examined.
Before 128 days of gestation, expression of SP-A, -B, and -C was low, which
is consistent with the low number of type II AECs at this stage of gestation,
but markedly increased with the appearance of morphologically distinct type II
AECs at 120 days of gestation. However, despite the fact that the
proportion of type II AECs did not increase further between 120 days and term,
SP-A, -B, and -C mRNA levels increased over this period; SP-A increased by
400%, SP-B by 250%, and SP-C by 220% between 128 and 142 days of gestation.
This increase in surfactant protein expression, without a corresponding
increase in type II AEC proportions, indicates that surfactant protein
expression per type II cell may increase over this period. The mechanisms
involved are unknown but could be related to the increase in endogenous fetal
cortisol levels at this time. However, the large increase in the proportion of
type II AECs after birth (from
30-53%) had an inconsistent effect on
surfactant protein expression. This increase in type II AEC proportions (77%
increase) at 2 wk of age was not accompanied by a similar increase in SP-A,
-B, or -C mRNA levels, although SP-A expression was increased at 8 wk and 2 yr
of age. After birth, we found that expression of SP-A, -B, and -C showed a
different pattern of expression. Compared with values measured in late
gestation (142 days), after birth SP-A mRNA levels increased, and SP-B mRNA
levels decreased, whereas SP-C mRNA levels were elevated at 8 wk but not at 2
wk or 2 yr of age. These data indicate that expression of the SP-A, -B, and -C
genes is differentially regulated after birth, although the potential
mechanisms are unclear.
Our study demonstrates, for the first time, that the relative proportions of type I and type II AECs reverse after birth. Before birth the type I cell phenotype predominates, whereas after birth the type II cell phenotype predominates, and these proportions persist into adult life. We suggest that the changes in the proportion of AEC phenotypes at birth result from an increase in lung recoil and a decrease in the degree of lung expansion at this time, possibly due to type I to type II cell transdifferentiation. In view of the effect of birth on AEC phenotypes, it will be important to determine the effect of preterm birth on the proportion of AECs, particularly in the very preterm infant. These infants can be born with few, if any, fully differentiated AECs, and, therefore, the majority of AECs must differentiate after birth in an environment completely different from that which they are exposed during in utero development.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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