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Journal of Physiology (2002), 542.1, pp. 245-253
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.014274
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ABSTRACT |
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The factors that control the differentiation of alveolar epithelial cells (AECs) into type-I and type-II cells in vivo are largely unknown. As sustained increases in fetal lung expansion induce type-II AECs to differentiate into type-I cells, our aim was to determine whether reduced fetal lung expansion can induce type-I AECs to trans-differentiate into type-II AECs. Chronically catheterised fetal sheep were divided into two age-matched control groups and three experimental groups (n = 5 for each). The experimental groups were exposed to either: (1) 10 days of increased lung expansion induced by tracheal obstruction (TO), (2) 10 days of TO followed by 5 days of reduced lung expansion induced by lung liquid drainage (LLD), or (3) 10 days of TO followed by 10 days of LLD. Following 10 days of TO, 5 days of LLD reduced the proportion of type-I AECs from 89.4 ± 0.9 % to 68.4 ± 2.8 %, which was similar to control values (64.8 ± 0.5 %), and increased the proportion of type-II AECs from 1.9 ± 0.3 % to 21.9 ± 2.8 %, which remained below control values (33.4 ± 1.7 %). The same treatment increased surfactant protein (SP)-A, SP-B and SP-C mRNA levels (expressed as a percentage of control values) from 26.7 ± 6.0 %, 40.0 ± 7.3 % and 10.3 ± 1.8 % to 78.1 ± 10.3 %, 105.8 ± 12.7 % and 121.0 ± 14.1 %, respectively. Similar results were obtained after 10 days of LLD, which followed 10 days of TO. These results indicate that the phenotypes of type-I and type-II AECs are strongly influenced by the basal degree of lung expansion in fetal sheep. Furthermore, the coincident increase in type-II AEC proportions and SP mRNA levels in response to LLD suggests that type-I AECs can trans-differentiate into functional type-II cells, and hence are not terminally differentiated.
(Received 29 November 2001; accepted after revision 30 April 2002)
Corresponding author S. B. Hooper: Department of Physiology, Monash University, Victoria, 3800, Australia. Email: stuart.hooper{at}med.monash.edu.au
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INTRODUCTION |
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After birth, the efficient exchange of respiratory gases is dependent upon the alveolar epithelium containing appropriate proportions of type-I and type-II alveolar epithelial cells (AECs). Type-I AECs are large, flattened cells that have long cytoplasmic extensions that extend over much of the surface area of the lung, providing the vast majority of the epithelial component of the air-blood gas barrier (Schneeberger, 1997). Type-II AECs are rounded in shape and contain cytoplasmic organelles (lamellar bodies), which are the intracellular storage sites for surfactant. In addition to producing and releasing surfactant, type-II AECs are thought to be the progenitor cell type that gives rise to both phenotypes (Mason & Shannon, 1997). Although both AEC types are critical for respiratory function after birth, little is known about the factors that control differentiation into either phenotype in vivo. Previous research has focussed predominantly on the endocrine regulation of AECs, although more recently, attention has focussed on the impact of mechanical forces on AEC phenotypes (Gutierrez et al. 1998, 1999; Flecknoe et al. 2000; Wirtz & Dobbs, 2000).
Development of the fetal lung is critically dependent upon the volume of lung liquid retained within the future airways. This liquid is secreted across the pulmonary epithelium into the future airspaces and, although it leaves the lungs via the trachea, its efflux is restricted by the upper airway (Hooper & Harding, 1995; Harding & Hooper, 1996). This promotes liquid retention within the future airways, which maintains the lungs in a constantly distended state. Obstruction of the trachea in fetal sheep prevents the efflux of lung liquid and causes the lungs to hyperexpand with accumulated liquid (Nardo et al. 1998). This is a potent stimulus for fetal lung growth and structural development and has profound effects on AEC phenotypes. Previous studies (Alcorn et al. 1977; Piedboeuf et al. 1997; De Paepe et al. 1998; Flecknoe et al. 2000) have shown that sustained alterations in lung expansion in fetal sheep greatly alter the proportions of type-I and type-II AECs in vivo, producing coincident changes in surfactant protein gene expression (Lines et al. 1999; Flecknoe et al. 2000). As a result, there is now good in vitro (Shannon et al. 1992; Gutierrez et al. 1998) and in vivo (Flecknoe et al. 2000) evidence to indicate that prolonged increases in cell stretch stimulate differentiation of type-II into type-I AECs, via an intermediate cell type (Flecknoe et al. 2000). We have recently shown that the proportion of type-II AECs decreases from ~30 % to 
2 % during a 10 day period of tracheal obstruction (TO) in fetal sheep, whereas the proportion of type-II AECs increases from ~60 % to ~90 % (Flecknoe et al. 2000).
Although it was widely considered that type-I AECs are terminally differentiated (Schneeberger, 1997), it has been suggested that type-I AECs retain the ability to trans-differentiate into type-II cells in vitro (Shannon et al. 1992; Danto et al. 1995; Dobbs et al. 1997; Uhal, 1997). However, it is not known whether type-I cells retain this ability to trans-differentiate in vivo. In view of our previous study (Flecknoe et al. 2000), we hypothesised that if type-I AECs retain the ability to trans-differentiate in vivo, a prolonged reduction in fetal lung expansion should induce type-I AECs to differentiate into type-II AECs. Thus, our aim was to determine the effects of reduced lung expansion, following a period of lung overexpansion, on the proportion of AEC phenotypes and on surfactant protein gene expression in fetal sheep. Before deflating the fetal lungs, fetuses were subjected to 10 days of increased lung expansion, induced by TO, so that at the onset of lung liquid drainage (LLD) most (~90 %) AECs would be of the type-I cell phenotype (Flecknoe et al. 2000). Following the increase in lung expansion, we examined the effects of both 5 and 10 days of LLD to determine whether lung deflation induced a gradual increase in type-II AEC number and surfactant protein (SP) gene expression. We hypothesised that if lung deflation stimulated type-I to type-II AEC trans-differentiation via an intermediate cell type, the proportion of this intermediate cell type may increase transiently before type-II AEC numbers are restored to control levels.
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METHODS |
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Experimental procedures
All experimental procedures on animals were approved by the Monash University Animal Ethics Committee.
Aseptic surgery was performed on 25 pregnant Merino 
Border-Leicester ewes at ~113 days of gestation (GA; term is ~147 days) under general anaesthesia (1.5 % halothane in O2). Two large-bore saline-filled catheters were inserted into the mid-cervical trachea of each fetus and were connected externally to form an exteriorised tracheal loop (Hooper et al. 1988). Catheters were also implanted into a fetal jugular vein, carotid artery and amniotic sac. Procaine penicillin was administered to each fetus (400 mg I.M.) before it was returned to the uterus and all catheters were externalised via a small incision in the ewe's right upper flank. Animals were allowed to recover for at least 5 days before experimentation began. Fetal well-being was assessed by examining fetal arterial blood pH, partial pressure of CO2, partial pressure of O2 and percent saturation of O2; these parameters were measured on alternate days, using an ABL510 blood gas and acid-base analyser (Radiometer, Copenhagen, Denmark).
Animals were randomly assigned to one of five groups (n = 5 for each). Overexpansion of the fetal lung was induced by TO, which was achieved by occluding the exteriorised tracheal loop. Underexpansion of the lung was induced by continuously draining the lung of luminal liquid (LLD) into an external bag via gravity. The five groups were: (1) 10 days of TO (10d TO, 118-128 days GA); (2) no treatment for 10 days, (control 128d GA, 118-128 days GA); (3) 10 days of TO followed by 5 days of LLD (10d TO/5d LLD, 118-132 days GA); (4) 10 days of TO followed by 10 days of LLD (10d TO/10d LLD, 118-138 days GA); (5) no treatment for 20 days (control, 118-138 days GA). In the 10d TO/5d LLD and 10d TO/10d LLD fetuses, the volume of liquid drained was measured daily during the period of LLD
Eight hours before killing, [3H]thymidine (1 mCi kg-1 I.V.; Amersham, UK) was administered to each fetus to assess lung cell division rates using autoradiography on histological sections. Immediately before the ewes and fetuses were humanely killed with an overdose of pentobarbital sodium (I.V.), the fetal lungs were drained of luminal liquid. The fetus was removed and weighed and the fetal lungs, heart and liver were collected and weighed. Following ligation of the left main bronchus, the left lung was removed and portions were frozen in liquid N2 for biochemical analysis. The right lung was fixed at 20 cmH2O, via the airways, using 4 % paraformaldehyde in 0.1 M phosphate buffer. After fixation, the trachea was ligated and the lung placed into a container of fresh fixative for 24 h at 4 °C.
Analytical methods
Histological analysis. Rather than using light microscopy in conjunction with stains for specific cell markers, we chose to identify AECs directly using electron microscopy. This is because it is not possible to identify the intermediate AECs (see below) using specific markers as it is not known which markers are present on these cells. Furthermore, it is not known how long these markers persist after the cells cease expressing these proteins as a result of differentiating from one type into another. Indeed, the results of our previous study suggest that the surfactant proteins persist for a number of days after the time when surfactant protein expression levels are markedly reduced (Lines et al. 1999).
Following fixation, the right lung was processed for light and electron microscopy as described previously (Flecknoe et al. 2000; Nardo et al. 2000). Briefly, the right lung was cut into 5 mm slices and approximately nine slices were chosen at random. These slices were further subdivided into three sections, of which two were selected randomly and fixed for a further 24 h in 4 % formaldehyde, 0.1 M phosphate buffer, 15 % picric acid (Zamboni's solution) at 4 °C and then embedded in paraffin wax. The remaining section from each slice was cut into cubes (2 mm 2 mm 2 mm), taking care to avoid major airways and blood vessels, and immersion-fixed in 4 % glutaraldehyde overnight. The tissue cubes were then washed in 0.1 M cacodylate buffer, incubated in 2 % OsO4 (in 0.1 M cacodylate buffer) and embedded in epoxy resin (Procure 812). At least three epoxy resin/tissue blocks were chosen randomly from each animal. Ultrathin sections (70-90 nm) were cut using a diamond knife, mounted on 200 mesh copper grids, and stained with uranyl acetate and lead citrate. All sections were coded and the observer was blind to the experimental group.
Alveolar epithelial cells were identified using a transmission electron microscope (Jeol 100s) and the proportions determined as described previously (Flecknoe et al. 2000). A minimum of 100 AECs were classified and counted per animal, with only AECs displaying a nuclear profile counted. This method of analysis is similar to that used in a previous study in which it was shown that the size of the nucleus of type-I and type-II cells is similar in humans (Crapo et al. 1982). As we have observed similar results in sheep (unpublished observations), the chance of counting each cell type in a section of ovine lung is similar. At least three different sections were viewed, ensuring that only one section per tissue block was analysed. AECs were categorised into one of four groups (stem cells, type-I AECs, type-II AECs and intermediate AECs; Fig. 2), based on their morphological appearance (Flecknoe et al. 2000). Identification of AECs depended upon clear visualisation of the basement membrane, and all AECs had to be located on the luminal surface of this membrane. Alveolar epithelial stem cells were cuboidal in shape and contained abundant cytoplasmic glycogen. Type-I AECs had flattened cytoplasmic extensions, flattened and elongated nuclei, little perinuclear cytoplasm and few cytoplasmic organelles. Type-II cells were rounded with a rounded nucleus and had apical surface microvilli and abundant cytoplasmic organelles, including lamellar bodies. The intermediate cells comprised a heterogeneous group that displayed characteristics of both type-I and type-II AECs (Flecknoe et al. 2000). Their classification depended upon the presence of a flattened elongated nucleus, marked cytoplasmic extensions and lamellar bodies, although the presence of apical surface microvilli varied (Fig. 2).
Autoradiography. Autoradiography at the light microscope level was used to assess lung cell division, particularly of type-II AECs in response to prolonged LLD (Nardo et al. 2000). Paraffin sections (4 µm) were mounted onto plain glass slides, with each slide containing lung tissue from a control fetus and either a 10d TO/5d LLD or 10d TO/10d LLD fetus. Photographic emulsion (K2, Ilford, UK) was diluted 1:1 with distilled water (dH2O) at 40 °C in a darkroom. Slides were dipped into the emulsion, placed on a cooled metal plate for 3 h and stored in light-proof boxes for ~4 weeks at 4 °C. The slides were developed (Kodak D-19; diluted 1:1 with dH2O) for 5 min and then placed in a stop bath (1 % acetic acid) for 30 s. The emulsion was fixed (Hypam, Ilford) for 2 min and the slides washed for 10 min in running water. Tissue sections were counterstained using haemotoxylin and eosin.
Viewed with the aid of a light microscope, silver grains labelled the nuclei of cells that had incorporated [3H]thymidine into their DNA during DNA synthesis. The total labelling index was calculated by counting the number of labelled cells and expressing them as a proportion of the total number of cells counted (approximately 1500 cells per animal) (Nardo et al. 2000).
Northern Blot Analysis. Fetal lung SP-A, SP-B and SP-C mRNA levels were quantified by Northern Blot analysis (Lines et al. 1999); we have shown previously that these proteins can be differentially regulated (Lines et al. 2001) by alterations in fetal lung expansion. Briefly, total RNA was extracted from fetal lung tissue and 20 µg was 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, USA) by capillary action and cross-linked to it using ultraviolet light (Hoeffer UVC 500, Amrad). The membrane was then incubated in hybridisation buffer (50 % (v:v) deionised formamide, 7 % (w:v) SDS, 5 saline-sodium phosphate-EDTA, and 0.1 mg ml-1of denatured and fragmented salmon sperm DNA)for 3-4 h at 42 °C. This was followed by hybridisation with the 32P-labelled SP-A, SP-B or SP-C cDNA probe (2 106 counts min-1 ml-1) for 24-48 h at 42 °C in the same hybridisation buffer. These ovine-specific cDNA probes have been described previously (Lines et al. 1999) and were labelled with [
-32P]dCTP by the random-priming technique (Oligolabeling Kit, Pharmacia).
After hybridisation with the labelled probe, the membranes were washed, sealed in airtight bags and exposed to a storage phosphor screen for 24-48 h at room temperature. To standardise the amount of total RNA loaded onto each lane, the blot was stripped by washing in 0.01 standard saline-sodium citrate, containing 0.5 % SDS, at 80 °C for 30 min, and was reprobed with a 32P-labelled ovine cDNA probe for 18S rRNA. The relative levels of SP-A, SP-B and SP-C mRNA were quantified by measuring the total integrated density of each band using ImageQuant (Molecular Dynamics, Sunnyvale, CA, USA).
DNA assay. The total DNA content of fetal lung tissue was measured using an established fluorometric assay (Hooper et al. 1993).
Statistical analysis. All data are expressed as the mean ± S.E.M. and, unless stated otherwise, the level of significance used was P 0.05. Differences in the proportions of type-I, type-II and intermediate AEC types between groups were determined using a one-way ANOVA; differences between each cell type were not compared. Similarly, differences in total lung DNA contents and [3H]thymidine labelling index were also determined using a one-way ANOVA. In all Northern blots, each lane contained a sample of RNA from a different fetus. The total integrated density of each SP-A, SP-B (the densities of the two SP-B transcripts were summed) or SP-C transcript was divided by the total integrated density of the 18S rRNA band for each lane. This gave a corrected density for each band that accounted for minor RNA loading differences between lanes. SP-A, SP-B and SP-C mRNA levels are presented as a ratio with the optical density of the 18S rRNA and as such have no units. Statistical comparisons between control and experimental groups, run on the same blot, were performed using Student's unpaired t tests. To make comparisons between northern blots, each experimental group was expressed as a percentage of the mean control value obtained from control samples run on the same blot. Differences between groups were then determined using one-way ANOVA.
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RESULTS |
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LLD rates
Following 10 days of TO in the 10d TO/10d LLD group of fetuses, the rate of LLD increased from 5.5 ± 0.4 ml h-1 at 128 days to 17.7 ± 2.0 ml h-1 at 138 days GA (Fig. 1). Expressed in relation to estimated fetal body weight (Lumbers et al. 1985), the rate of LLD increased from 2.1 ± 0.2 ml h-1 kg-1 at 128 days to 4.7 ± 0.7 ml h-1 kg-1 at 138 days GA; normal lung liquid production rates are 3.3-4.4 ml h-1 kg-1 at this stage of gestation in fetal sheep, as reported previously (Harding & Hooper, 1996), but measurements were not made in this study. In 10d TO/5d LLD fetuses, LLD rates were similar over the first 5 days of drainage to those measured in 10d TO/ 10d LLD fetuses (Fig. 1).
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Figure 1. Change in lung liquid drainage (LLD) rate following experimental perturbation of fetal lung liquid volumes in fetal sheep The volume of lung liquid drained, expressed in ml h-1 (circles) and ml h-1 kg-1 of fetal body weight (triangles) averaged over a 24 h period, from fetuses exposed to either 10 days of tracheal obstruction (TO) followed by 5 days of LLD (10d TO/5d LLD; open symbols) or 10 days of TO followed by 10 days of LLD (10d TO/10d LLD; filled symbols). The volumes recorded on the 1st day of LLD do not include the liquid drained from the lungs immediately following the TO period. Each parameter values that do not share a common letter are significantly different from one another. | ||
Lung weights and DNA contents
Lung weights, in relation to fetal body weight, in both 10d TO/5d LLD and 10d TO/10d LLD fetuses were significantly less than lung weights from 128d and 138d control fetuses (Table 1). In contrast, lung weights in the 10d TO fetuses were significantly greater than in both control groups and LLD groups of fetuses (Table 1).
Total lung DNA content, expressed in relation to fetal body weight, was significantly increased in 10d TO fetuses compared with age-matched (128d) control fetuses (Table 1). In contrast, total lung DNA contents in 10d TO/5d LLD and 10d TO/10d LLD fetuses were not different from either the 128d or the 138d control fetuses. In control fetuses, there was a trend for the total lung DNA content to be greater at 128 than at 138 days of gestation (Table 1).
AEC phenotypes
The total numbers of AECs counted per animal were 110.2 ± 8.1 in 128d control fetuses, 105.8 ± 2.3 in 138d control fetuses, 100.0 ± 1.6 in 10d TO fetuses, 119.0 ± 13.4 in 10d TO/5d LLD fetuses and 129.0 ± 21.2 in 10d TO/10d LLD fetuses. In control fetuses, the proportions of AEC types were similar at 128 and 138 days GA. At both ages, the majority (128d: 64.8 ± 0.5 % and 138d: 63.2 ± 2.3 %) of AECs were of the type-I cell phenotype (see Fig. 2B). Similarly, the proportions of type-II AECs (see Fig. 2A) were not different at 128 (28.5 ± 2.2 %) and 138 days (33.4 ± 1.7 %) GA. Very few stem AECs (~1 %) were observed at the GAs examined, whereas the remainder of the AECs were identified as intermediate cells (see Fig. 2C).
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Figure 2. Ultrastructural characteristics of type-I, type-II and intermediate alveolar epithelial cells (AECs) Electron micrographs of the nuclear region of a type-II AEC (A), a type-I AEC (B) and an intermediate AEC (C). Type-II AECs (A) are rounded in shape with rounded nuclei, and contain lamellar bodies and apical-surface microvilli. Type-I cells (B) have flattened nuclei, little perinuclear cytoplasm and long cytoplasmic extensions (indicated by arrows) that can extend into adjacent alveoli. The cells identified as intermediate AECs (C) display characteristics of both type-I and type-II cells. They have flattened nuclei and elongated cytoplasmic extensions (indicated by arrows) as well as lamellar bodies and apical-surface microvilli. The bar in each micrograph represents 1 µm. | ||
Type-I AECs. Expressed as a percentage of the total number of AECs counted, the proportion of type-I AECs was significantly increased from 63.1 ± 2.3 % in 128d control fetuses to 89.5 ± 0.8 % following 10 days of TO (10d TO; Fig. 3A). However, following the 10 day TO period, 5 days of LLD reduced the proportion of type-I AECs to 63.4 ± 2.8 % (10d TO/5d LLD), which was similar to values in both the 128d (63.1 ± 2.3 %) and 138d (62.2 ± 2.4 %) control fetuses (Fig. 3A). After 10 days of LLD (in 10d TO/10d LLD fetuses), the proportion of type-I cells was reduced compared with that measured after 5 days of LLD (10d TO/5d LLD: 63.4 ± 2.8 % vs. 10d TO/10d LLD: 57.9 ± 3.5 %), but was not different from control values (Fig. 3A).
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Figure 3. Changes in the relative proportions of type-I, type-II and intermediate AECs following experimental perturbation of fetal lung liquid volumes in fetal sheep The proportions of type-I (filled bars) and type-II (open bars) AECs (A) and intermediate AECs (B) measured in control fetuses at 128 days (128d control) and 138 days (138d control) of gestation and in fetuses exposed to 10 days of tracheal obstruction (TO; 10d TO), 10 days of TO followed by 5 days of LLD (10d TO/5d LLD) and 10 days of TO followed by 10 days of LLD (10d TO/10d LLD). For each cell type, values that do not share a common letter are significantly different from one another. | ||
Type-II AECs. The proportion of type-II AECs was markedly reduced from 28.5 ± 2.2 % in 128d control fetuses to 1.8 ± 0.4 % by 10 days of TO (Fig. 3A). Following 10 days of TO, 5 days of LLD increased the proportion of type-II cells to 21.9 ± 2.8 % (10d TO/5d LLD), although these values remained below both the 128d (28.5 ± 2.2 %) and 138d (33.4 ± 1.7) control values (Fig. 3A). However, after 10 days of LLD (in 10d TO/10d LLD fetuses), the proportion of type-II AECs had increased to 35.6 ± 3.1 %, which was similar to that in 138d age-matched control fetuses (Fig. 3A).
Intermediate AECs. At 10 days of TO, the proportion of intermediate cells (7.7 ± 0.9 %) was similar to that observed in age-matched control fetuses (128d controls: 5.7 ± 1.3 %). Following 10 days of TO, 5 days of LLD increased the proportion of intermediate AECs to 9.1 ± 0.9 % (10d TO/5d LLD; Fig. 3B). By 10 days of LLD (10d TO/10d LLD), the proportion of intermediate cells (6.5 ± 0.8 %) was not different from the value in 128d age-matched controls (3.0 ± 1.4 %) but was higher than the value in 138d age-matched controls.
SP expression
SP-A mRNA levels. The mRNA level for SP-A in fetal lung tissue, expressed as a percentage of the mean control value, was reduced from 100.0 ± 23.6 % in 128d control fetuses to 26.7 ± 6.0 % by 10 days of TO (10d TO; Fig. 4A). Following 10 days of TO, 5 days of LLD (10d TO/5d LLD) significantly increased SP-A levels to 78.1 ± 10.3 %, which tended to be lower than, but was not significantly different from, control values (100.0 ± 22.9 %) run on the same blot. In fetuses exposed to 10 days of TO followed by 10 days of LLD (10d TO/10d LLD), SPA mRNA levels (104.2 ± 33.9 %) were similar to control values (100.0 ± 22.9 %).
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Figure 4. Surfactant protein (SP) mRNA levels following experimental perturbation of fetal lung liquid volumes in fetal sheep SP-A (A), SP-B (B) and SP-C (C) mRNA levels in lung tissue collected from control fetuses at 128 days (128d control) and 138 days (138d control) of gestation and from fetuses exposed to 10 days of tracheal obstruction (TO; 10d TO), 10 days of TO followed by 5 days of LLD (10d TO/5d LLD) and 10 days of TO followed by 10 days of LLD (10d TO/10d LLD). In each panel, values indicated by an asterisk are significantly different from control values. | ||
SP-B mRNA levels. Ten days of TO reduced SP-B mRNA levels in lung tissue from a mean control value of 100.0 ± 10.8 % (128d controls) to 40.0 ± 7.3 % (10d TO; Fig. 4B). Following 10 days of TO, 5 days of LLD increased SP-B levels to 105.8 ± 10.7 % (10d TO/5d LLD) which was similar to 138d control values (100.0 ± 10.5 %). In lung tissue from 10d TO/10d LLD fetuses, SP-B mRNA levels (122.3 ± 10.6 %) appeared to be greater than in 138d age-matched control fetuses (100.0 ± 10.5 %), although the difference was not quite significant (P = 0.11; Fig. 4B).
SP-C mRNA levels. Ten days of TO reduced the mRNA levels for SP-C in lung tissue from 100.0 ± 10.6 % in 128d control fetuses to 10.3 ± 1.8 % (Fig. 4C). Following 10 days of TO, 5 days of LLD increased SP-C mRNA levels to 121.0 ± 14.1 % (10d TO/5d LLD). In fetuses exposed to 10 days of TO, followed by 10 days of LLD (10d TO/10d LLD) SP-C mRNA levels (138.6 ± 20.2 %) appeared to be greater than in 138d control fetuses (100.0 ± 17.5 %), but this difference did not quite reach significance.
Autoradiographic analysis
Expressed as a proportion of the total number of cells counted, the proportion of pulmonary cells labelled by the incorporation of [3H]thymidine into DNA was 1.2 ± 0.1 % in control fetuses. In fetuses exposed to 10 days of TO, followed by either 5 days (10d TO/5d LLD; 1.3 ± 0.2 %) or 10 days (10d TO/10d LLD; 0.9 ± 0.2 %) of LLD, the labelling index of lung cells was similar to that observed in control fetuses. Based on our analysis by light microscopy, the labelling index of type-II AECs was similar in control fetuses (0.32 ± 0.05 %) and in 10d TO/5d LLD fetuses (0.27 ± 0.08 %), but was significantly reduced in 10d TO/10d LLD (0.17 ± 0.04 %) fetuses.
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DISCUSSION |
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In the study presented here, the basal degree of lung expansion was increased in experimental fetuses by obstructing the trachea for 10 days, so that the lungs would contain a high proportion of type-I AECs and few ( 2 %) type-II AECs (Flecknoe et al. 2000) before the onset of lung deflation. After 5 days of lung deflation, which reduces the basal degree of lung expansion, the proportion of type-I AECs was reduced from ~90 % to a level that was similar to that in control fetuses (~63 %). In contrast, the proportion of type-II AECs increased from ~2 % to 22 %, but remained below control values. Following a further 5 days of lung deflation, the proportions of type-I and type-II AECs had returned to control values. The changes in the proportions of AEC types observed in response to lung deflation are consistent with our hypothesis that a prolonged reduction in the basal degree of fetal lung expansion induces trans-differentiation of type-I into type-II AECs via an intermediate cell type. In support of this finding, the increase in type-II cell numbers induced by sustained lung deflation was accompanied by an increase in SP-A, SP-B and SP-C mRNA levels, with each returning to control levels within the first 5 days of lung deflation. Thus, our results indicate that a sustained period of lung deflation, following 10 days of lung overexpansion, promotes the morphological and functional reappearance of type-II AECs in fetal sheep, most probably via trans-differentiation of type-I AECs.
We have shown previously that after 10 days of TO, ~90 % of all AECs are of the type-I phenotype, whereas less than 2 % are of the type-II phenotype (Flecknoe et al. 2000). These changes in AEC phenotype proportions are consistent with the profound changes in SP-A, SP-B and SP-C mRNA levels that occur in response to TO (Lines et al. 1999). Thus, in this study we chose to overexpand the fetal lungs for 10 days before deflating them, so that the lungs in these fetuses would contain predominantly type-I AECs. As a result, the observed increase in the proportion of type-II AECs in response to fetal lung deflation could only have arisen from (1) proliferation of the remaining small pool of type-II AECs in addition to selective apoptosis of type-I AECs, (2) trans-differentiation of type-I into type-II AECs or (3) differentiation from undifferentiated stem cells. However, as there are so few stem cells ( 1 %) at this stage of gestation in fetal sheep (Flecknoe et al. 2000; this study), we consider the latter to be the least likely explanation.
It is well established that sustained underexpansion of the fetal lung as a result of LLD reduces cellular proliferation in that organ (Alcorn et al. 1977; Moessinger et al. 1990; Hooper et al. 1993; Nardo et al. 1995). Thus, it is highly unlikely that the proliferation of a small population of type-II AECs during LLD could have given rise to the approximately 10-fold increase in type-II cell numbers observed after 5 days of LLD and the approximately 15-fold increase after 10 days of LLD. In support of this contention, we found that the labelling index of pulmonary cells tended to be reduced in fetuses exposed to LLD following a period of TO. More specifically, our analysis revealed that the labelling index of type-II AECs was reduced by ~47 % in fetuses exposed to 10 days of lung deflation. Although the resolution of light microscopy is insufficient to identify all type-II cells within a field of view, as the analysis was the same for each group, the relative changes in the proportion of labelled type-II cells are likely to reflect the true changes. In addition to the changes in labelling index, we found no evidence of increased apoptosis in type-I AECs, as assessed with the aid of an electron microscope. Thus, based on these findings, the most likely explanation for the simultaneous changes in the proportions of type-I and type-II AECs we observed is trans-differentiation of type-I into type-II AECs.
Previous studies have shown that the phenotype of AECs is strongly influenced by physical factors that either impose a mechanical strain on individual AECs or alter their shape (Alcorn et al. 1977; Shannon et al. 1992; Piedboeuf et al. 1997; Gutierrez et al. 1998; Flecknoe et al. 2000). Most of the in vivo studies have concentrated on the differentiation of type-II to type-I AECs, whereas few have examined the possibility of trans-differentiation of type-I into type-II AECs. This lack of investigation may have stemmed from the previously held belief that type-I cells are terminally differentiated (Schneeberger, 1997). However, in view of the findings of this study, and that of previous in vitro studies (Shannon et al. 1992; Danto et al. 1995; Uhal, 1997), the available evidence indicates that type-I AECs are not terminally differentiated, but are able to trans-differentiate into type-II cells via an intermediate cell type, depending upon the prevailing conditions.
The differentiation of cells into a specific phenotype is thought to result from a process that suppresses and/or activates a subset of genes that collectively determine the differentiated function of that cell (Brody & Williams, 1992). Whether or not the inactivation of genes is permanent was thought to determine whether the differentiation process was terminal. Our findings, and those of previous in vitro studies (Shannon et al. 1992; Danto et al. 1995; Uhal, 1997), support the concept that the type-I AEC is not terminally differentiated and can trans-differentiate both in vivo and in vitro. Thus, it is likely that both type-I and type-II AECs retain the ability to re-express the complement of genes that characterise each phenotype, and that the processes that determine each phenotype are not permanent, but are actively maintained. We suggest that one of the primary factors that regulate AEC phenotypes in vivo is the degree of mechanical strain experienced by individual cells. Thus, increased lung expansion presumably increases the proportion of AECs exposed to mechanical strain, which activates and suppresses the genes that collectively characterise the type-I cell phenotype. Conversely, deflation of the lung reduces the mechanical strain on AECs, which appears to activate and suppress the genes that collectively characterise the type-II cell phenotype. Other factors that are thought to regulate AEC phenotype in vitro include cell shape (Shannon et al. 1992), which relates to mechanical strain (Sims et al. 1992), exposure of the apical surface of AECs to air (Dobbs et al. 1997) and the presence of keratinocyte growth factor in the culture medium (Borok et al. 1998). The role of corticosteroids on AEC differentiation in vivo is unclear, but differences in endogenous cortisol concentrations could not explain the changes in AEC phenotypes observed in this study because alterations in lung expansion do not affect circulating cortisol concentrations (Boland et al. 1997; Boland & Hooper, unpublished observations).
Although it is not known whether the results of this study can be extrapolated to the air-filled lung after birth, there is some evidence to suggest that the phenomenon of AEC trans-differentiation is not restricted to the fetus. For example, most of the in vitro evidence supporting trans-differentiation of AECs was obtained from studies using adult AECs (Shannon et al. 1992; Borok et al. 1998; Gutierrez et al. 1998). Furthermore, it is evident that the proportion of type-II AECs is increased within the fibrotic lesions associated with pulmonary fibrosis (Kasper & Haroske, 1996). It is possible that fibrosis reduces the mechanical load experienced by individual AECs due to the excessive production of collagen.
Our rationale for including the 5 day LLD group of fetuses (10d TO/5d LLD) was to observe the progressive changes in the proportion of all AEC types over the 10 day lung deflation period, particularly the intermediate cell type. We have observed previously a transient increase in this cell type during a mechanical stimulus (TO) that induces type-II to type-I AEC trans-differentiation. Therefore, we hypothesised that if lung deflation induces type-I to type-II AEC differentiation via an intermediate cell type, we would observe a similar transient increase in the proportion of this cell type following LLD (Flecknoe et al. 2000). Although we observed a transient increase in the proportion of intermediate cells at 5 days of LLD, it is likely that the greatest increase occurred much earlier during LLD. This is because most (~66 %) of the AECs differentiating into type-II cells had already done so by 5 days of LLD. Indeed, the proportion of type-II AECs increased 11-fold (to ~22 %) over the first 5 days of LLD. Whether or not the complement of genes expressed by intermediate cells is similar depending upon whether the cell is at an intermediate stage of differentiation between type-I and type-II cells, or vice versa, is unknown. In our previous study, we showed that at 2 days of TO, the proportion of type-II AECs was halved, whereas the proportion of type-I cells was not altered. The decrease in type-II AEC numbers, without an increase in type-I cell numbers, was due to a large increase in the proportion of intermediate cells at this time (Flecknoe et al. 2000). As the mRNA levels for SP-A, SP-B and SP-C were greatly reduced at 2 days of TO (Lines et al. 1999), we have suggested that although these intermediate cells contained lamellar bodies, they were functionally distinct from type-II cells and were unlikely to express the SPs (Flecknoe et al. 2000). On the other hand, in the present study we found that SP expression was restored to control values in 10d TO/5d LLD fetuses, but the proportion of type-II cells remained ~25 % below control values. Thus, at 5 days of LLD, either type-II cells expressed SPs at higher levels than controls, or the intermediate cells also expressed the SPs. We suggest the latter, and consider that activation or suppression of the SPs occurs early in the differentiation of AECs from one phenotype to the other. Thus, we predict that the complement of genes expressed at any one time in intermediate cells may depend upon whether the cell is differentiating into a type-I or a type-II cell. However, confirmation of this prediction requires an in situ hybridisation analysis using electron microscopy.
Sustained increases in fetal lung expansion, as induced by TO, are a potent stimulus for fetal lung growth and it has therefore been suggested that TO can be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia (Hooper & Harding, 1995; Harding & Hooper, 1996). However, the finding that sustained over-expansion of the fetal lung reduces type-II AEC numbers (Flecknoe et al. 2000) and greatly reduces SP expression (Piedboeuf et al. 1997; Lines et al. 1999) has raised concerns regarding the therapeutic potential of this procedure. Our findings indicate that any changes in AEC proportions resulting from increased expansion of the fetal lung can be rapidly reversed by lung deflation without a detrimental effect on lung growth. Indeed, as the majority of conditions that cause fetal lung hypoplasia result from prolonged periods of lung deflation, it is likely that hypoplastic lungs will have increased proportions of type-II cells (Benachi et al. 1999). Thus, a closely regulated period of TO may induce lung growth and restore the normal proportions of AECs.
The results of our study provide compelling evidence to indicate that the degree of sustained lung expansion, which then imparts a mechanical load onto AECs, is an important determinant of the phenotype of both type-I and type-II AECs. Furthermore, the most likely explanation for the change in proportions of AECs we observed in response to sustained lung deflation is trans-differentiation of type-I cells into type-II AECs. Thus, our study provides novel in vivo evidence that supports the hypothesis that type-I AECs are not terminally differentiated.
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Acknowledgements
We are indebted to Alex Satragno for his assistance with the surgical preparation of animals and to Alison Thiel for her expert technical assistance, particularly with the RNA extractions. This work was funded by the National Health and Medical Research Council of Australia (NH & MRC).
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