Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep

S. J. Flecknoe, M. J. Wallace, M. L. Cock, R. Harding, and S. B. Hooper

Department of Physiology, Monash University, Victoria 3800, Australia

Submitted 11 September 2002 ; accepted in final form 27 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because basal lung expansion increases toward term and is reduced after birth, we hypothesized that these changes would be associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and postnatal sheep. Type I AECs increased from 4.8 ± 1.3% at 91 days to 63.0 ± 3.6% at 111 days of gestation, remained at this level until term, and decreased to 44.8 ± 1.8% after birth. Type II AECs increased from 4.3 ± 1.5% at 111 days to 29.6 ± 4.1% at 128 days of gestation, remained at this level until term, and then increased to 52.9 ± 1.5% after birth. Surfactant protein (SP)-A, -B and -C mRNA levels increased with increasing gestational age before birth, but the changes in SP expression after birth were inconsistent. Thus before birth type I AECs predominate, whereas after birth type II AECs predominate, possibly due to the reduction in basal lung expansion associated with the entry of air into the lungs.

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


THE ALVEOLAR EPITHELIUM comprises type I and type II alveolar epithelial cells (AECs), both of which play critical roles in the respiratory function of the lung. Type I AECs are large flattened cells with elongated cytoplasmic extensions that, collectively, form a large surface area for gas exchange (24). Type II AECs are rounded in shape, synthesize and release surfactant, and are considered to be the main progenitor cell type; they give rise to new type II cells by division and to type I cells by transdifferentiation (19, 26). Although it was previously considered that type I cells are terminally differentiated, recent in vitro (7) and in vivo (9, 10) studies suggest that type I cells are capable of transdifferentiation into type II cells. Thus it is possible that both AEC types have the potential to transdifferentiate, but the factors that determine the transdifferentiation are not clear. As both cell types are critical for the respiratory function of the lung after birth, it is important to understand how the lung attains the correct proportions of type I and type II AECs during fetal development and after birth.

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.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Lung tissue was collected from fetal sheep at the gestational ages of 91 (n = 4), 105 (n = 4), 111 (n = 4), 120 (n = 4), 128 (n = 5), 132 (n = 5), 138 (n = 5), and 142 days (n = 4), from lambs at 2 (n = 4) and 8 wk (n = 4) after birth and from adult sheep at 2 yr of age (n = 5); full term is 145-147 days of gestation in this breed of sheep, and sexual maturity is reached within their first year. The ewes, fetuses, and lambs were all painlessly killed by an intravenous injection of pentobarbital sodium and weighed, and their lungs were removed and weighed before the left main bronchus was ligated and the left lung was removed; portions of the left lung were frozen in liquid nitrogen for biochemical analysis. The right lung was fixed at 20 cmH2O via the lung lumen with 4% paraformaldehyde and 4% glutaraldehyde; tissues collected from fetuses at 105 days of gestation were not fixed in glutaraldehyde and could not, therefore, be analyzed by electron microscopy (EM) for AEC proportions. All ewes, fetuses, and lambs used in this study were either sham-operated or unoperated controls that were not subjected to experimental manipulation. All procedures performed on animals were approved by the Monash University Animal Welfare Committee according to guidelines established by the Australian NH & MRC.

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).



View larger version (108K):
[in this window]
[in a new window]
 
Fig. 1. Electron micrographs of alveolar epithelial cells (AECs) at different stages of gestation. Cells that are at an early stage of differentiation from a stem cell into either a type I (A, C) or type II (B) AEC are depicted in A-C. D: a fully differentiated type I cell. E: a differentiated type II cell with numerous lamellar bodies. Arrows indicate the position of tight junctions between adjacent cells, arrowheads indicate cytoplasmic extensions, and the bar represents 2 µm; n, nucleus; lb, lamellar bodies; g, glycogen; c, capillary.

 

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 [{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
AEC Phenotypes

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).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Changes in the proportions of undifferentiated alveolar epithelial stem cells ({bullet}) and intermediate AECs ({circ}) before (from 91 to 142 days of gestation) and after birth (from 2 wk to 2 yr of age). For each cell type, values that do not share a common letter are significantly different from one another.

 

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).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Changes in the proportions of type I ({bullet}) and type II ({circ}) alveolar epithelial cells before (from 91 to 142 days of gestation) and after birth (from 2 wk to 2 yr of age). For each cell type, values that do not share a common letter are significantly different from one another.

 

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).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Changes in surfactant protein (SP)-A (A), SP-B (B), and SP-C (C) mRNA levels in lung tissue before (from 91 to 142 days of gestation) and after birth (from 2 wk to 2 yr of age). Values are expressed as a percentage of 128-day (d) mRNA expression. In each panel, values that do not share a common letter are significantly different from one another.

 

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.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In view of the relationship between fetal lung expansion and AEC differentiation (9, 10), we hypothesized that the proportion of type I and type II AECs would change in parallel with the changes in basal lung expansion that occur during late gestation and at birth. We found that during the early canalicular stage of fetal lung development [91 days of gestation (1)], most (~94%) AECs were undifferentiated AE stem cells with very few type I (~4%) or type II (~1%) cells. By 111 days of gestation, a substantial proportion of the AECs (~63.0%) had differentiated into the type I cell phenotype, although very few type II AECs were present at this stage (~4%). However, by 120 days of gestation, which coincides with the beginning of the alveolar stage of lung development (1), the proportion of type II AECs had increased to ~25%. Despite the increase in lung liquid volume from 120 days of gestation until near term (15), the proportions of type I and type II cells did not change during this period. However, within 2 wk of birth, the proportion of type I AECs decreased from ~63% to ~44%, whereas the proportion of type II cells almost doubled from ~30% to ~53%; these proportions persisted into adult life. Our findings demonstrate that type I and type II cell differentiation occurs rapidly (10-20 days) at defined, yet different, stages of gestation in fetal sheep. Type I AECs differentiate at an earlier stage of development (91-111 days of gestation) than type II AECs, which rapidly appear over a 9-day period (111-120 days). The changes in the proportions of type I and type II AECs that occur soon after birth are consistent with the changes in end-expiratory lung expansion that occur at birth (13, 15), and these proportions persist into adult life.

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.


    DISCLOSURES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was funded by the National Health and Medical Research Council of Australia.


    ACKNOWLEDGMENTS
 
We are indebted to Alison Thiel for expert technical assistance, particularly in the extraction of RNA samples and the generation of Northern blots, as well as Samantha Louey for assistance in obtaining lung tissue from 8-wk- and 2-yr-old sheep.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. B. Hooper, Dept. of Physiology, Monash Univ. P. O. Box 13F, Victoria 3800, Australia (E-mail: stuart.hooper{at}med.monash.edu.au).

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Alcorn DG, Adamson TM, Maloney JE, and Robinson PM. A morphologic and morphometric analysis of fetal lung development in the sheep. Anat Rec 201: 655-667, 1981.[ISI][Medline]
  2. Bassett JM and Thorburn GD. Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 44: 285-286, 1969.[ISI][Medline]
  3. Cassin S and Perks AM. Estimation of lung liquid production in fetal sheep with blue dye dextran and radioiodinated serum albumin. J Appl Physiol 92: 1531-1538, 2002.[Abstract/Free Full Text]
  4. Crapo JD, Barry BE, Gehr P, Bachofen M, and Weibel ER. Cell number and cell characteristics of the normal human lung. Am Rev Respir Dis 126: 332-337, 1982.[ISI][Medline]
  5. Crapo JD, Young SL, Fram EK, Pinkerton KE, Barry BE, and Crapo RO. Morphometric characteristics of cells in the alveolar region of mammalian lungs. Am Rev Respir Dis 128: S42-S46, 1983.[ISI][Medline]
  6. Crone RK, Davies P, Liggins GC, and Reid L. The effects of hypophysectomy, thyroidectomy, and postoperative infusion of cortisol or adrenocorticotrophin on the structure of the ovine fetal lung. J Dev Physiol 5: 281-288, 1983.[ISI][Medline]
  7. Danto SI, Shannon JM, Borok Z, Zabski SM, and Crandall ED. Reversible transdifferentiation of alveolar epithelial cells. Am J Respir Cell Mol Biol 12: 497-502, 1995.[Abstract]
  8. Davey MG, Johns DP, and Harding R. Postnatal development of respiratory function in lambs studied serially between birth and 8 weeks. Respir Physiol 113: 83-93, 1998.[ISI][Medline]
  9. Flecknoe S, Harding R, Maritz G, and Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol 278: L1180-L1185, 2000.[Abstract/Free Full Text]
  10. Flecknoe SJ, Wallace MJ, Harding R, and Hooper SB. Determination of alveolar epithelial cell phenotypes in fetal sheep: evidence for the involvement of basal lung expansion. J Physiol 542: 245-253, 2002.[Abstract/Free Full Text]
  11. Gutierrez JA, Gonzalez RF, and Dobbs LG. Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro. Am J Physiol Lung Cell Mol Physiol 274: L196-L202, 1998.[Abstract/Free Full Text]
  12. Harding R, Bocking AD, and Sigger JN. Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol 61: 68-74, 1986.[Abstract/Free Full Text]
  13. Harding R and Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol 81: 209-224, 1996.[Abstract/Free Full Text]
  14. Harding R and Liggins GC. Changes in thoracic dimensions induced by breathing movements in fetal sheep. Reprod Fertil Dev 8: 117-124, 1996.[ISI][Medline]
  15. Hooper SB and Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22: 235-247, 1995.[ISI][Medline]
  16. Jakubowski AE, Billings K, Johns DP, Hooper SB, and Harding R. Respiratory function in lambs following prolonged oligohydramnios during late gestation. Pediatr Res 34: 611-617, 1993.[Abstract]
  17. Lines A, Hooper SB, and Harding R. Lung liquid production rates and volumes do not decrease before labor in healthy fetal sheep. J Appl Physiol 82: 927-932, 1997.[Abstract/Free Full Text]
  18. Lines A, Nardo L, Phillips ID, Possmayer F, and Hooper SB. Alterations in lung expansion affect surfactant protein A, B and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol 276: L239-L245, 1999.[Abstract/Free Full Text]
  19. Mason RJ and Shannon JM. Alveolar type II cells. In: The Lung: Scientific Foundations, edited by Crystal RG, West JB, Weibel ER, and Barnes PJ. Philadelphia, New York: Lippincott-Raven, 1997, p. 543-555.
  20. Nakamura T, Liu M, Mourgeon E, Slutsky A, and Post M. Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression. Am J Physiol Lung Cell Mol Physiol 278: L974-L980, 2000.[Abstract/Free Full Text]
  21. Pfister RE, Ramsden CA, Neil HL, Kyriakides MA, and Berger PJ. Volume and secretion rate of lung liquid in the final days of gestation and labour in the fetal sheep. J Physiol 535: 889-899, 2001.[Abstract/Free Full Text]
  22. Sanchez-Esteban J, Cicchiello LA, Wang Y, Tsai SW, Williams LK, Torday JS, and Rubin LP. Mechanical stretch promotes alveolar epithelial type II cell differentiation. J Appl Physiol 91: 589-595, 2001.[Abstract/Free Full Text]
  23. Sanchez-Esteban J, Tsai SW, Sang J, Qin J, Torday JS, and Rubin LP. Effects of mechanical forces on lung-specific gene expression. Am J Med Sci 316: 200-204, 1998.[ISI][Medline]
  24. Schneeberger EE. Alveolar type I cells. In: The Lung: Scientific Foundations, edited by Crystal RG, West JB, Weibel ER, and Barnes PJ. Philadelphia, New York: Lippincott-Raven, 1997, p. 535-542.
  25. Shannon JM, Jennings SD, and Nielsen LD. Modulation of alveolar type II cell differentiated function in vitro. Am J Physiol Lung Cell Mol Physiol 262: L427-L436, 1992.[Abstract/Free Full Text]
  26. Uhal BD. Cell cycle kinetics in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol 272: L1031-L1045, 1997.[Abstract/Free Full Text]