Department of Physiology, Monash University, Victoria 3800, Australia
Submitted 31 October 2003 ; accepted in final form 28 July 2004
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ABSTRACT |
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lung liquid; surfactant proteins; fetus
Recent studies indicate that the degree of fetal lung expansion, which influences the degree of strain imposed on individual AECs, plays an important role in regulating AEC differentiation in vivo (15, 1719, 40). Sustained increases in fetal lung expansion promote differentiation of AECs into the type I cell phenotype, whereas sustained lung deflation promotes differentiation into the type II cell phenotype, most probably due to transdifferentiation of type I AECs into type II AECs (19). These findings are the corollary of earlier in vitro studies (14, 43) and have led to the concept that type I AECs are not terminally differentiated but can transdifferentiate into type II AECs, depending upon the mechanical strain they experience (14, 19, 43).
Although corticosteroids are considered to be important regulators of type II AEC differentiation, the reported in vivo effects of corticosteroids are contradictory. It is well established that corticosteroids induce structural maturation of the fetal lung, causing thinner alveolar walls, increased air space volumes, and reduced tissue volumes, leading to a marked increase in lung tissue compliance (reviewed in Ref. 10). The idea that corticosteroids also induce type II AEC differentiation is mainly predicated on their ability to stimulate surfactant and surfactant protein (SP) synthesis (6, 34, 37). However, this idea is not consistent with in vivo studies that either have blocked intracellular signaling of cortisol or have reduced endogenous cortisol levels (11, 13). Fetal hypophysectomy, which reduces circulating cortisol levels, causes an approximate fivefold increase in type II AEC numbers, and this increase is prevented by the infusion of ACTH or corticosteroids into hypophysectomized fetuses (13). Similarly, in glucocorticoid receptor (GR) knockout mice, the abolition of signaling via the GR markedly increases the proportion of type II AECs at the expense of type I AECs (11). Furthermore, it is now evident that, at least in fetal sheep, type II AECs differentiate before the preparturient increase in endogenous cortisol levels (18). Combined, these findings clearly indicate that type II AEC differentiation is not dependent on corticosteroid activation of the GR.
In the fetus, reductions in circulating cortisol levels (13) and abolition of cortisol signaling via the GR (11) markedly impair structural development of the lung, leading to a reduction in lung expansion [from 40 to <20 ml/kg in fetal sheep (47)]. On the other hand, cortisol infusions increase fetal lung expansion, most likely via a cortisol-mediated increase in lung tissue compliance (9, 46). As type II AECs are increased in GR null fetal mice and hypophysectomized fetal sheep and in view of the relationship between lung expansion and AEC differentiation, we hypothesized that the effects of endogenous corticosteroids on AEC differentiation are secondary to an increase in fetal lung expansion. Specifically, we hypothesized that elevated cortisol levels would accelerate structural development of the fetal lung, thereby increasing lung compliance and lung expansion (46), resulting in increased differentiation of AECs into the type I cell phenotype. We also hypothesized that sustained lung deflation would prevent the cortisol-mediated effect on type I AECs, resulting in increased differentiation into the type II phenotype. To characterize the role of physiological levels of corticosteroids in the differentiation of AECs, we chose to infuse increasing doses of cortisol into the fetus over a 9-day period; this mimics the preparturient increase in circulating cortisol levels late in gestation. We also chose to perform this study at a stage of gestation when the majority of AECs have fully differentiated. In contrast, previous studies have examined the effects of high doses of synthetic corticosteroids at a developmental stage when most AECs were undifferentiated. Hence, our studies have avoided the potential complicating effect of cortisol on the differentiation of undifferentiated AECs and have determined the effect of physiological doses of cortisol on AECs.
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METHODS |
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All experimental procedures on animals were approved by the Monash University Animal Ethics Committee. Aseptic surgery was performed on 22 pregnant ewes and their fetuses (Merino x Border-Leicester) at 109.4 ± 0.9 days of gestation (term is 147 days) to implant fetal and maternal vascular catheters and bidirectional fetal tracheal catheters. After the recovery period, the descending tracheal catheter was used to drain lung liquid to chronically reduce the degree of fetal lung expansion. A minimum of 5 days was allowed for the ewe and fetus to recover from surgery before experiments began. To assess fetal well-being, measurements of fetal arterial blood pH, partial pressure of CO2, partial pressure of O2, and percent saturation of O2 were made on every second day, using an ABL510 blood gas and acid-base analyzer (Radiometer, Copenhagen, Denmark).
Ewes and their fetuses were divided into four groups; 1) saline-only group in which saline was infused intravenously into the fetus for 9 days [122131 days gestational age (GA), n = 5], 2) cortisol-only group in which cortisol was infused intravenously into the fetus at increasing doses (see below) for 9 days (122131 days GA, n = 5), 3) LLD (lung liquid drain) + saline group in which fetal lung liquid was drained by gravity into a sterile bag for 20 days (111131 days GA) and fetuses received a saline infusion as in group 1 (n = 5), and 4) LLD + cortisol group in which lung liquid was drained by gravity into a sterile bag for 20 days (111131 days GA) and fetuses received a cortisol infusion as in group 2 (n = 5). In all cortisol-infused fetuses, increasing doses of cortisol (hydrocortisone sodium succinate, Solu Cortef; Upjohn Pty) were infused into a fetal jugular vein over the 9-day period: 1.5 mg/day on 122123 days GA, 2.5 mg/day on 124125 days GA, 3.0 mg/day on 126127 days GA, 3.5 mg/day on 128129 days GA, and 4.0 mg/day on 130131 days GA. The cortisol dose was prepared daily in a constant volume of heparinized saline and was delivered at 1.2 ml/h; saline-infused fetuses were administered equal volumes of heparinized saline, delivered at the same rate. Fetal blood samples (2 ml) were collected from all animals every 23 days for the measurement of fetal plasma cortisol concentrations. All infusions continued until the time of the autopsy (131 days of gestation).
Just before autopsy, the fetal lungs were drained of liquid via the descending tracheal catheter. The ewe and fetus were painlessly killed by an overdose of pentobarbitone sodium administered to the ewe (130 mg/kg iv). The fetal lungs were removed and weighed before the left bronchus was ligated and the left lung removed distal to the ligature. Portions of the left lung were frozen in liquid nitrogen and stored at 70°C for subsequent analysis. The right lung was fixed at 20 cmH2O via the trachea with 4% paraformaldehyde.
Analytical Methods
Analysis of AEC phenotypes.
We chose to identify AECs according to morphological criteria identified by transmission electron microscopy (TEM) as previously described (1719), rather than by light microscopy in conjunction with staining for specific cell markers. We chose this method of analysis because it is currently unclear which markers should be used to categorically identify AECs in sheep. Although type II AECs are often identified using one of the SPs as a cell marker, a recent study demonstrated that SPs are expressed in the fetal sheep lung before morphologically distinct type II AECs appear (18). Furthermore, T1, which has been used as a type I AEC marker in rats and mice, has not been identified in sheep, whereas aquaporin 5 expression, another marker for type I AECs, is reduced when type I AEC proportions are increased in response to an increase in fetal lung expansion (17, 29). Previous studies have also shown that, in sheep (5) and humans (12), the nuclear diameters of type I and type II AECs are similar, and, therefore, the chances of counting a nuclear profile of each cell type are equal using TEM; our observations (unpublished) confirm the findings of these earlier studies. Of particular importance are the findings that the nuclear diameters of type I and type II AECs are not affected by changes in plasma cortisol concentrations (25) or by changes in the basal level of lung expansion (S. J. Flecknoe and S. B. Hooper, unpublished observations).
After fixation, the right lung was processed for TEM as previously described (1719). Briefly, the right lung was cut into 5-mm slices. Each slice was further subdivided into three sections; we chose six sections at random and cut them into cubes (2 x 2 x 2 mm), taking care to avoid major airways and blood vessels. 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. At least three epoxy resin/tissue blocks were randomly chosen from each animal. Ultrathin sections (7090 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 experimental group.
AECs were identified under a TEM (Joel 100s). For each animal, a minimum of 100 AECs were classified, and we determined the proportions of each phenotype by counting the number of nuclear profiles of each type (17, 19). At least three different sections were viewed per animal and only one section per tissue block was analyzed. Identification of AECs depended on clear visualization of the basement membrane with all AECs localized on 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 (see Fig. 1). Very few stem cells, easily identifiable by their abundant cytoplasmic glycogen, were present at this stage of gestation (18). Type I AECs had flattened cytoplasmic extensions, flattened nuclei, little perinuclear cytoplasm, and few cytoplasmic organelles. 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. The intermediate cells were a heterogenous group that displayed characteristics of both type I and type II AECs. Their classification depended on the presence of marked cytoplasmic extensions, but these cells also contained lamellar bodies and usually had apical surface microvilli (17).
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Statistical analysis. All data are expressed as means ± SE, and the level of significance used was P < 0.05, unless otherwise stated. Differences in the proportions of type I, type II, and intermediate AEC types between groups were determined by a one-way ANOVA followed by a Fischer least-square-difference post hoc test; differences between each cell type were not compared. The total integrated density of each SP-A, SP-B (the density of the two SP-B transcripts were summed), and SP-C transcript on the Northern blot 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, with each experimental group expressed as a percentage of the mean value obtained from the saline-only group of animals. Differences in SP gene expression between experimental groups were determined by a one-way ANOVA followed by a Fischer least-square-difference post hoc test.
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RESULTS |
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Fetal body weights were not different between any of the groups at the time of autopsy (saline-only, 3.4 ± 0.3 kg; cortisol-only, 3.6 ± 0.1 kg; LLD + saline, 3.0 ± 0.4 kg; LLD + cortisol 3.1 ± 0.2 kg). However, the wet lung weights (g/kg body wt) of fetuses exposed to a period of lung liquid drainage (LLD + saline, 16.3 ± 1.0 g/kg; LLD + cortisol, 16.5 ± 0.4 g/kg) were significantly reduced compared with lung weights for saline-only (35.9 ± 0.7 g/kg) and cortisol-only (35.6 ± 2.0 g/kg) fetuses (8). Cortisol infusions had no effect on lung weights.
AEC Phenotypes
Alveolar epithelial stem cells. The proportion of alveolar epithelial stem cells, expressed as a percentage of the total number of AECs counted, was too low in each of the experimental groups to allow statistical comparison. Less than 1% of the AECs counted in each of the groups were of an undifferentiated alveolar epithelial stem cell phenotype (saline-only, 0.18 ± 0.1%; cortisol-only, 0.28 ± 0.2%; LLD + saline, 0.12 ± 0.1%; LLD + cortisol, 0.0 ± 0.0%).
Type I AECs. In fetuses not exposed to LLD, the proportion of type I AECs, expressed as a percentage of the total number of AECs counted, was significantly lower in cortisol-infused fetuses (cortisol-only, 59.0 ± 2.2%) compared with saline-infused fetuses (saline-only, 70.4 ± 2.4%) (Fig. 2A). In saline-infused fetuses, the proportion of type I AECs was significantly lower in LLD fetuses (LLD + saline, 34.2 ± 3.7%) compared with fetuses not exposed to LLD (saline-only, 70.4 ± 2.4%). The infusion of cortisol into LLD fetuses (LLD + cortisol) tended to reduce the proportion of type I AECs below that (to 26.6 ± 2.3%) seen in LLD + saline fetuses (34.2 ± 3.7%), although this difference failed to reach significance (P = 0.070).
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Intermediate AECs. In saline-infused fetuses, the proportion of intermediate AECs was significantly increased in LLD fetuses (LLD + saline, 12.8 ± 2.0%) compared with fetuses not exposed to LLD (saline-only, 6.2 ± 1.6%). In the absence of LLD, the infusion of cortisol significantly reduced the proportion of intermediate AECs (to 2.6 ± 0.4%) compared with saline-only fetuses (Fig. 2C). In contrast, in fetuses exposed to LLD, the infusion of cortisol had no further effect on the proportion of intermediate AECs; they were similar in LLD + saline fetuses (12.8 ± 2.0%) and LLD + cortisol fetuses (12.6% ± 1.7%).
SP Gene Expression in Fetal Lung Tissue
SP-A mRNA levels. In saline-infused fetuses, LLD significantly increased the mRNA levels for SP-A (LLD + saline, 268.5 ± 56.3%) compared with fetuses not exposed to LLD (saline-only, 100.0 ± 8.2%). In the absence of LLD, the infusion of cortisol tended to increase SP-A mRNA levels (to 162.0 ± 6.5%) compared with saline-only fetuses, although this difference was not quite significant (Fig. 3A). Similarly, in fetuses exposed to LLD, the infusion of cortisol significantly increased SP-A mRNA levels (LLD + cortisol, 514.6 ± 104.6%) compared with saline-infused fetuses (LLD + saline, 268.5 ± 56.3%).
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SP-C mRNA levels. In saline-infused fetuses, LLD significantly increased the mRNA levels for SP-C (LLD + saline, 279.2 ± 24.7%) compared with fetuses not exposed to LLD (saline-only, 100.0 ± 5.7%). In the absence of LLD, cortisol infusions increased SP-B mRNA levels (to 162.7 ± 13.9%) compared with saline-only fetuses (Fig. 3C). Similarly, in fetuses exposed to LLD, cortisol infusions significantly increased SP-C mRNA levels (LLD + cortisol, 419.0 ± 49.9%) compared with saline-infused fetuses (LLD + saline, 279.2 ± 24.7%).
Representative Northern blots for SP-A, -B, and -C are shown in Fig. 4.
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DISCUSSION |
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Although previous studies examining the effect of cortisol on differentiated AECs are contradictory, the prevailing view in the literature is that cortisol stimulates differentiation of AECs into the type II cell phenotype. This view has mainly arisen from studies demonstrating that corticosteroids stimulate surfactant secretion and SP synthesis (6, 33, 39), as well as studies that report that increased corticosteroid concentrations increase type II AEC numbers (1, 24, 44, 48). On the other hand, hypophysectomy in fetal sheep and abolition of the GR in fetal mice result in an increase in the proportion of type II AECs (11, 13). The results of these latter studies (11, 13) indicate that cortisol is not necessary for type II cell differentiation, which is consistent with the recent finding that type II AEC differentiation during late gestation is independent of the preparturient increase in circulating cortisol levels (18).
In view of the discrepancies in the literature on the reported effects of corticosteroids on AEC differentiation in vivo, we considered that the role of cortisol in AEC differentiation was unresolved and the reported data confusing. Differences in the timing of the treatments, the dose and type of corticosteroid administered, as well as the type of analysis used to identify and count differentiated epithelial cells may have contributed to this confusion. In particular, many of the studies examining the effects of corticosteroids have administered synthetic corticosteroids, which have a much higher bioactivity (30-fold), at a stage of lung development when the majority of AECs were of an undifferentiated phenotype (21, 23, 34, 48). Wang et al. (48) showed that the administration of prednisolone to fetal rabbits increased the proportion of type II AECs from 18.3 to 40.2% but also increased the proportion of type I AECs from 24.7 to 48.5%. As the proportion of undifferentiated AECs was markedly reduced from 57 to 11.3% by corticosteroid treatment, the predominant corticosteroid effect was a nonspecific stimulation of AEC differentiation from undifferentiated epithelial cells. This is consistent with the recent finding of Cole et al. (11), that the proportion of undifferentiated AECs is higher in GR knockout fetal mice, indicating that in the absence of cortisol signaling via the GR, AEC differentiation is delayed (11).
In view of the potentially conflicting effects of cortisol on the differentiation of undifferentiated AECs, we chose to administer cortisol to fetal sheep at a GA that coincides with a time when most AECs have differentiated into type I or type II AECs; at the start of our infusions (122 days) only 2% of AECs are of an undifferentiated phenotype (18). Furthermore, we chose to administer gradually increasing doses of cortisol to mimic the preparturient increase in fetal plasma cortisol concentrations. In saline-infused fetuses, cortisol concentrations remained at 12 ng/ml, whereas in cortisol-infused fetuses, circulating cortisol concentrations gradually increased to
34 ng/ml. As the induction of normal labor in fetal sheep requires that endogenous fetal plasma cortisol concentrations are increased >40 ng/ml for at least 3 days (16, 31), the dose of cortisol we administered achieved plasma cortisol levels that were marginally less than that expected before normal labor in fetal sheep. Thus, in the present study, we investigated the effect of a preparturient-like increase in fetal plasma cortisol levels on the phenotype of mature type I and type II AECs.
The results of the present study confirm the potent effect that reductions in lung expansion have on type I and type II AEC proportions (4, 19). Prolonged LLD reduced the proportions of type I cells by >50%, from 70 to
34%, and doubled the proportion of type II AECs from
23 to
54%. However, in LLD fetuses, the infusion of cortisol tended to further decrease the proportion of type I AECs from
34 to
27% (P = 0.070) and to increase the proportion of type II AECs from
54 to
60% (P = 0.066). These data indicate that cortisol may have a small stimulatory effect on type II AEC differentiation, which is independent of any associated changes in lung expansion. This is consistent with the finding that the administration of cortisol to fetuses not exposed to LLD significantly increased the proportion of type II cells (from
24 to
38%) and reduced the proportion of type I cells.
In view of the reduction in the proportion of intermediate AECs in fetuses exposed to cortisol but not LLD, it is possible that the cortisol-induced increase in type II AEC proportions, compared with saline-infused controls, resulted from a reduction in the transdifferentiation of type II into type I AECs. Intermediate AECs are thought to be cells that are at an intermediate stage of differentiation between type I and type II AECs and have been observed in a number of previous studies (24, 19, 35, 48). Indeed, this cell type is transiently elevated during a period of rapid transdifferentiation of type II AECs into type I AECs in response to an increase in fetal lung expansion (17). As 6% of the AECs are of an intermediate phenotype at this stage of gestation in control fetuses (Fig. 1C), transdifferentiation of AECs from one phenotype into another is likely to be a normal process that occurs during lung development. Consequently, the significant reduction in the proportion of intermediate AECs in cortisol-only fetuses is consistent with a reduction in transdifferentiation of type II into type I AECs by the cortisol infusion rather than an increase in transdifferentiation of type I into type II AECs. On the other hand, the increased proportion of intermediate cells in both groups of LLD fetuses most probably reflects an increased rate of AEC transdifferentiation, from type I into type II AECs. It is unlikely that the increase in type II AEC proportions induced by cortisol resulted from increased type II AEC proliferation as corticosteroids are generally considered to decrease cellular proliferation (24), although not at the dose we used (46). Furthermore, LLD also causes a marked inhibition of lung cell proliferation, particularly of type II AECs (19).
The findings that a preparturient-like cortisol infusion during the early alveolar stage of lung development increased the proportion of type II AECs and decreased the proportion of type I AECs are not consistent with our previous study (18). We have previously shown that the proportions of type I and type II AECs do not change between 120 and 142 days of gestation, despite the naturally occurring preparturient increase in fetal plasma cortisol concentrations after 135 days (18). Although the reasons for this inconsistency are unclear, it is possible that the timing of corticosteroid exposure is a critical factor. Indeed, the lack of change in the proportions of AECs in the last 10 days of normal gestation may be related to the greater degree of structural maturity of the lung at the onset of the preparturient increase in cortisol. Thus, in late gestation, any stimulatory effect on type II differentiation may have been offset by an associated increase in lung expansion that was greater than the increase induced by the cortisol infusion at the earlier GA.
Corticosteroids are known to increase the rate of fetal lung liquid secretion (46) and to increase the ability of the lung to reabsorb liquid in response to epinephrine (7, 46, 47). These effects are thought to be mediated by increased expression of proteins regulating transepithelial water and ion flux; these include ion and water channels as well as Na+-K+-ATPase. However, it is unlikely that a cortisol-mediated increase in lung liquid secretion alone will significantly affect fetal lung expansion as this is principally regulated by the transthoracic pressure gradient and upper-airway resistance (22). Increases or decreases in lung liquid secretion rates simply cause corresponding changes in lung liquid efflux via the trachea, resulting in little change in lung liquid volume (22). Instead, increases in lung expansion following corticosteroid exposure are most likely secondary to a cortisol-induced alteration in lung tissue structure, resulting in an increase lung compliance and a reduction in lung tissue recoil (41). Thus, to retain a 12 mmHg transthoracic pressure gradient at rest (45), which is regulated by the fetal upper-airway resistance (22), lung expansion will increase.
Although we have demonstrated that cortisol may have a direct stimulatory effect on type II AEC differentiation, the effect of altered lung expansion on AEC differentiation was much greater and has the potential to cause profound changes in AEC proportions (17, 19). Recently, much attention has focused on the role of strain in the regulation of AEC phenotypes both in vivo (17, 19) and in vitro (14, 43) as increased strain stimulates type II to type I cell transdifferentiation. On the other hand, reduced strain is thought to stimulate type I to type II AEC transdifferentiation, which is supported by both in vitro (17, 19) and in vivo (19) studies, indicating that type I AECs may not be terminally differentiated. Although the mechanisms by which strain influences the phenotype of AECs are unknown, they may involve the direct transmission of force from the extracellular to the intracellular compartment, via extracellular matrix receptors, which are mechanically coupled to intracellular structural filaments (30).
The mRNA levels for all three of the SPs were increased by the infusion of cortisol, both in the presence and absence of LLD. In particular, in LLD fetuses the cortisol infusion caused a marked and significant increase in SP-A, SP-B, and SP-C expression that was greater than the percentage increase in type II cell proportions. It is likely, therefore, that cortisol had an additional stimulatory effect on SP expression that exceeded that which could be accounted for by an increase in type II cell proportions. This finding is consistent with previous in vitro studies that have shown that cortisol has a direct stimulatory effect on SP expression (20, 27, 49). However, it is unknown whether the observed changes in SP expression will result in changes in SP protein levels, although we have previously shown that a reduction in SP-A expression causes a reduction in SP-A protein levels after a delay of 4 days (28). It is also of interest that, in saline-infused LLD fetuses, the percentage increases in SP-A (169%) and SP-C, (179%), but not SP-B (40%), expression were greater than the percentage increase in type II AEC proportions (122%). Thus it is possible that LLD may increase the expression of these SP-A and SP-C per type II cell, although the mechanisms involved are unknown.
In conclusion, we have shown that an increase in fetal plasma cortisol concentrations, within physiological levels, can induce a small increase type II AEC proportions and a small decrease in type I AEC proportions via mechanisms that are independent of associated changes in fetal lung expansion. However, the effect of reduced lung expansion on AEC proportions was much greater (over 2-fold) than the effects of cortisol, indicating that the degree of basal lung expansion is one of the primary physiological determinants of AEC phenotypes before birth. The mechanisms by which cortisol affects type II AEC differentiation are currently unknown but could involve either increased type I to type II AEC transdifferentiation or reduced type II to type I AEC transdifferentiation. As the increase in type II AEC proportions observed in cortisol-only fetuses was associated with a decrease in the proportion of intermediate AECs, it is possible that the increase in type II AEC proportions primarily resulted from a decrease in type II to type I AEC transdifferentiation.
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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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