INTRODUCTION

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HORMONAL EFFECTS on fetal lung development have provided important tools for understanding the cellular and molecular mechanisms that orchestrate and time this complex epithelial-mesenchymal cascade (22). However, the paradoxical observation that the lung can develop structurally and functionally in explant culture in the absence of added hormones (6, 8, 16) has left open the question as to what is the nature of the central regulatory mechanism(s) mediating alveolar differentiation. One potential clue is the observation that fluid distension and the resultant stretching of lung tissue are necessary for normal lung development (1, 5, 17), leaving open the question as to whether the stretch-mediated and hormone-regulated pathways share common regulatory elements. The existence of a common mechanism was suggested by the study of Skinner et al. (20), in which cortisol was shown to potentiate the effect of prostaglandin (PG) E₂, a stretch-activated factor (25), on fetal rat lung fibroblast adenosine 3',5'-cyclic monophosphate production. In this context, Hume et al. (9-11) and Ballard et al. (2) have identified PGE₂ as an endogenous mediator of the automaturational mechanism of lung explant development. We now provide experimental evidence that both dexamethasone (Dex) and stretch stimulate PGE₂ synthesis by type II cells and surfactant substrate (triglyceride) release by fibroblasts, integrating key epithelial-mesenchymal interactions involved in surfactant phospholipid metabolism in a timely manner.

	METHODS AND MATERIALS

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Reagents. PGE₂, Dex, bumetanide, indomethacin, phloretin, triolein, and phospholipid standards were all purchased from Sigma Chemical (St. Louis, MO). [9,10-³H(N)]triolein (30 Ci/mmol) and [5,6,8,9,10,11,12,14,15-³H(N)]arachidonic acid (100 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Trypsin (1:250), Dulbecco's modified Eagle's medium (DMEM), and antibiotics for tissue culture were obtained from GIBCO (Grand Island, NY). Collagen type I was purchased from Collaborative Biomedical Products (Bedford, MA). A PGE₂ enzyme-linked immunosorbent assay was bought from Cayman Chemical (Ann Arbor, MI).

Explant culture of fetal rat lung. Time-mated (vaginal smear positive = day 0) Sprague-Dawley rats were obtained from Charles River Breeders (Kingston, NY). The pups were weighed to confirm that they were within the appropriate weight-for-age limits established in this laboratory. The dams were killed by decapitation, and the pups were removed from the uterus by laparotomy and kept on ice. The lungs were removed en bloc in a laminar flow hood with sterile technique, dissected free of adjoining structures, and put into ice-cold sterile Hanks' balanced salt solution without calcium or magnesium (HBSS). Lungs (2/well) were planted in 24-well plates (1-cm diameter) in a minimal volume of culture medium for 60 min at 37°C in 5% CO₂-95% air to promote adherence to the plastic surface. At the end of the incubation period, the medium used to transfer the explants to the wells was aspirated, and culture medium was added to cover the explants (1 ml). The explants were cultured in Waymouth MB-752/1 medium containing penicillin (100 µg/ml), streptomycin (100 µg/ml), and amphotericin B (5 µg/ml) (7).

Technique for harvesting lung fibroblasts by differential adherence. Five to ten time-mated dams were used per preparation depending on the number of experimental variables to be tested. The fetal lungs were removed into HBSS as described in Explant culture of fetal rat lung. The HBSS was decanted, and five volumes of 0.05% trypsin were added to the lung preparation. The lungs were dissociated in a 37°C water bath with a Teflon stirring bar to disrupt the tissue mechanically. Once the tissue was dispersed into a unicellular suspension (~20 min), the cells were pelleted at 500 g for 10 min at room temperature in a 50-ml polystyrene centrifuge tube. The supernatant was decanted, and the pellet was resuspended in DMEM containing 20% fetal bovine serum (FBS) to yield a mixed cell suspension of ~3 × 10⁸ cells (determined by a Coulter particle counter, Hialeah, FL). The cell suspension was then added to culture flasks (25 or 80 cm²) for 30-60 min to allow for differential adherence of lung fibroblasts (21). These cells are >95% pure fibroblasts on the basis of vimentin staining.

Isolation of fetal type II cells by Nycodenz gradient. The minced lungs from four to six litters were pooled, washed with 50 ml of a mixture of 140 mM NaCl, 5 mM KCl, 2.5 mM Na₂HPO₄, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 6 mM glucose, and 0.2 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, pH 7.4 (solution I), and filtered through 100-µm nylon mesh. The minced lungs were transferred to a trypsinizing flask containing 40 ml of a mixture of 140 mM NaCl, 5 mM KCl, 2.5 mM Na₂HPO₂, 10 mM HEPES, 2.0 mM CaCl₂, and 1.3 mM MgSO₄, pH 7.4 (solution II) with elastase (30 orcein-elastase units/ml) and deoxyribonuclease (250 µg/ml) and stirred for 20 min at 37°C. The digestion was terminated by adding 5 ml of carbon-stripped (hormone-depleted) FBS. The cell suspension was filtered sequentially through 2- and 4-ply gauze and then 37- and 15-µm nylon mesh and was washed with additional solution II to a final volume of 40 ml. The cells were centrifuged at 130 g for 10 min at 20°C and resuspended in DMEM with 2% FBS for the addition to Nycodenz gradients (26). Identity of fetal type II cells was documented by staining for the presence of glycogen and by the reaction with specific type II cell markers, Maclura pomifera lectin, and antibody to cytokeratins 8 and 18 and was found to be ~90% pure by these criteria.

Organotypic culture. Organotypic cultures were prepared as previously described (23).

Cell stretch apparatus. This technique is an adaptation of the method of Wirtz and Dobbs (27). Freshly isolated rat fetal lung type II cells (day 20 postconception) were seeded onto Silastic membranes (Dow Corning, Midland, MI) coated with type I collagen. The Silastic membranes were mounted in a 25-mm filter holder (Gelman Sciences, Ann Arbor, MI), and DMEM (0.7 ml) was added to the upper chamber to maintain the cell layer. To stretch the cell monolayer, a 0.5-ml bolus of air was introduced into the lower chamber through a plastic tube that was then clamped to maintain positive pressure. The preparations were then placed in a CO₂ incubator at 37°C in an atmosphere of 5% CO₂-95% air for 3 h. At the end of the incubation, the conditioned media were collected and frozen at 70°C until analyzed for PGE₂. The cell layers were harvested from the Silastic membranes with 0.1% trypsin; an aliquot of the cell suspension was taken to determine cell number.

Uptake and release of [³H]triolein by fibroblasts. Culture medium was aspirated from confluent fibroblast cultures and replaced with DMEM containing 20% triglyceride (vol/vol) mixed with [³H]triolein (5 µCi/ml) that was prepared by first drying the [³H]triolein under a stream of nitrogen, resuspending it in 50 µl of ethanol, and then adding the DMEM plus serum and vortexing the mixture thoroughly. The cells were incubated at 37°C in 5% CO₂-95% air for 4-12 h and subsequently analyzed for their triglyceride content. For the triglyceride release assay, the fibroblasts were incubated with the [³H]triolein mixture for 24 h, washed three times with 3 ml of DMEM to remove excess radiolabeled solution, and then incubated with PGE₂ (200 ng/ml) in DMEM at 37°C in 5% CO₂-95% air for 2 h. At the end of the incubation, the media were aspirated and analyzed for triglyceride content (18).

Rate of [³H]arachidonic acid incorporation into [³H]PGE₂. This assay was based on the method of Polgar and Taylor (19). Briefly, type II cells in monolayer culture were incubated with [³H]arachidonic acid (5 µCi/ml) in DMEM in a CO₂ incubator for 24 h. At the end of the incubation, the culture medium was extracted with ethyl acetate, the extract was evaporated under a stream of nitrogen at 55°C, and the extract was chromatographically separated by thin-layer chromatography in ethyl acetate-isooctane-acetic acid-water (11:5:2:10). The area on the chromatogram corresponding to authentic PGE₂ was scraped from the plate, and its radioactive content was determined by liquid scintillation spectrometry. PGE₂ represented at least 80% of the radioactivity on the chromatogram.

PGE₂ assay. PGE₂ in culture medium was assayed with a commercial enzyme-linked immunosorbent assay.

	RESULTS

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Day 20 postconception fetal rat lung explants were maintained in culture for up to 3 days in the presence of either bumetanide (1 × 10⁵ M), an inhibitor of chloride ion secretion, indomethacin (5 × 10⁸ M), and/or PGE₂ (200 ng/ml) as indicated (Fig. 1). At the time of assay, the cultures were incubated with [³H]triolein to monitor for saturated phosphatidylcholine (SPC). As can be seen in Fig. 1, during the course of the 3-day culture period, there was a threefold increase in triolein incorporation into SPC by the explants. Treatment of the explants with PGE₂ further enhanced the rate of [³H]triolein incorporation into SPC (30%). In contrast to the effects of time in culture and PGE₂ treatment, both bumetanide and indomethacin blocked the spontaneous increase in triglyceride incorporation into SPC; the inhibitory effects of both bumetanide and indomethacin were completely reversed by exogenous PGE₂.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   [3H]triolein incorporation into saturated phosphatidylcholine (SPC) by fetal rat lung explants. Day 17 postconception fetal rat lung explants were cultured for 3 days (d3) in serum-free medium plus bumetanide (Bu; 5 × 105 M), indomethacin (IND; 5 × 108 M), prostaglandin E2 (PGE2; 200 ng/ml), Bu/PGE2, or Bu/IND and subsequently incubated with [3H]triolein for 15 h. dpm, Disintegrations/min. Values are means ± SD of 5 experiments; n = 20 lungs. Significantly different from control culture (day 0): * P < 0.05; ** P < 0.01 (by 2-way analysis of variance).

PGE₂ synthesis by type II cells. When primary cultures of fetal rat type II cells were incubated with [³H]arachidonic acid, the predominant metabolite (>80%) was PGE₂, which was largely found in the extracellular medium (>95%). As can be seen in Fig. 2A, PGE₂ synthesis was observed at all gestational ages (16-21 days postconception), although there were obvious developmental changes: there was basal synthesis of [³H]PGE₂ between days 16 and 18 postconception; beginning on day 20 postconception, there was a 2.5-fold increase in de novo PGE₂ synthesis (P < 0.05) that increased to 4-fold by day 21 postconception (P < 0.01). A similar developmental pattern was observed when conditioned medium from confluent type II cell monolayers was assayed for PGE₂ content (Fig. 2B): basal production of PGE₂ (100-140 ng · 10⁶ cells¹ · 24 h¹) was observed between days 16 and 19 postconception, followed by a saltatory increase on days 20 and 21 postconception (324 ± 102 and 609 ± 170 ng · 10⁶ cells¹ · 24 h¹, respectively).

View larger version (14K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig. 2.   Ontogeny of PGE2 synthesis by fetal rat lung type II cells. A: type II cells were incubated with [3H]arachidonic acid (5 µCi/ml for 2 h), and media were analyzed for [3H]PGE2. Values are means ± SD of 5 experiments; n = 20 lungs. Significantly different from previous day: * P < 0.05; ** P < 0.01 (by 1-way analysis of variance). B: type II cells were incubated in serum-free medium for 24 h, and conditioned medium was harvested and assayed for PGE2 content by enzyme-linked immunosorbent assay. Values are means ± SD of 3-5 experiments; n = 15-20 lungs. Significantly different from previous day: * P < 0.05; ** P < 0.01 (by 1-way analysis of variance).

Effect of PGE₂ on triglyceride release by fetal rat lung fibroblasts. Using a concentration of PGE₂ comparable to the amount of PGE₂ produced by day 20 postconception type II cells/24 h (i.e., 200 ng/ml), we observed a saltatory increase in triglyceride release by fetal rat lung fibroblasts (Fig. 3) beginning on day 19 postconception, which then increased progressively on days 20 (6-fold) and 21 postconception (10- to 14-fold). Basal triglyceride release remained unchanged over this entire period in the absence of added PGE₂ (<5% of intracellular triglyceride). Release of triglyceride by day 21 postconception fibroblasts in response to PGE₂ increased linearly over a 15-h time course (Fig. 4); coincubation with phloretin (1 × 10⁵ M), a PGE₂-receptor antagonist, completely blocked the effect of PGE₂ on triglyceride release by these cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Ontogeny of PGE2-stimulated triglyceride release by fetal rat lung fibroblasts. Fibroblasts were incubated with [3H]triolein (5 µCi/ml + 200 µg of nonradiolabeled triglyceride) for 24 h and subsequently challenged with (+) or without () PGE2 (200 ng/ml) for 15 h. [3H]triglyceride released into medium was quantitated. Each data point represents mean ± SD of 3-5 experiments; n = 15-20 lungs. Significantly different from previous day: * P < 0.05; ** P < 0.01 (by 1-way analysis of variance).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Phloretin inhibition of PGE2-stimulated triglyceride release by fibroblasts. Day 21 fibroblasts were preincubated with [3H]triolein (5 µCi/ml + 200 µg of nonradiolabeled triglyceride) for 24 h. Cells were subsequently challenged with PGE2 (200 ng/ml) with and without phloretin (1 × 105 M) for up to 15 h. Culture medium was analyzed for [3H]triglyceride. Each data point represents mean ± SD of 2 experiments; n = 10 lungs. Significantly different from previous data point: * P < 0.05; ** P < 0.01; *** P < 0.001 (by 1-way analysis of variance).

Effects of Dex on PGE₂ synthesis and action. Day 20 postconception fetal rat type II cells in a monolayer culture were exposed to Dex (1 × 10⁷ M/15 h) in serum-free medium for 15 h and subsequently incubated with [³H]arachidonic acid for 2 h (Fig. 5). Analysis of the culture medium revealed a threefold increase in [³H]PGE₂ synthesized as a result of Dex treatment. Assay of the same media for PGE₂ content revealed a 70% increase in production in response to Dex (290 ± 80 vs. 415 ± 38 ng · 10⁶ cells¹ · 24 h¹; P < 0.02 for control vs. Dex cultures by Student's t-test). Treatment of day 20 postconception fetal rat lung fibroblasts with Dex (1 × 10⁷ M/15 h) resulted in a 60% increase in triglyceride release in response to PGE₂ (Fig. 6). Note that there was only a 5-10% triglyceride release in the control and Dex-treated cultures.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of dexamethasone (Dex) on PGE2 synthesis by fetal rat lung type II cells. Type II cells were incubated with Dex (1 × 107 M) in serum-free MEM for 15 h and subsequently incubated with [3H]arachidonic acid (5 µCi/ml for 2 h). Cells and medium were analyzed for [3H]PGE2. Values are means ± SD of 3 experiments; n = 15 lungs. * Significantly different from control cells, P < 0.01 (by Student's t-test).

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Effect of Dex on PGE2-mediated triglyceride release by fetal rat lung fibroblasts. Fibroblasts were initially incubated with [3H]triolein (5 µCi/ml + 200 µg of nonradiolabeled triolein) for 24 h, followed by a 15-h exposure to Dex (1 × 107 M). Cells were subsequently challenged with PGE2 (200 ng/ml) for 15 h, and culture medium was analyzed for [3H]triglyceride released. Values are means ± SD of 3 experiments; n = 15 lungs. * Significantly different from control cells, P < 0.01. ** Significantly different from Dex-treated cells, P < 0.05 (both by Student's t-test).

Effect of stretch on type II cell PGE₂ synthesis. Both static and stretched day 20 type II cells exhibited progressive increases in [³H]arachidonic acid incorporation into [³H]PGE₂ over a 3-h period (Fig. 7), although the stretched cells synthesized approximately twice as much PGE₂ as nonstretched cells per unit time. Because a similar increase has been observed when type II cells are cultured on plastic (data not shown), we assume this is a reflection of baseline PG synthesis unrelated to culture on Silastic. The stretched cells were evaluated for damage and found to be >98% viable by trypan blue dye exclusion; assay for lactic dehydrogenase content of conditioned medium revealed no differences between static and stretched cultures.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effect of stretch on type II cell PGE2 synthesis. Type II cells were cultured on collagen-coated Silastic membranes and stretched. Cells were incubated with [3H]arachidonic acid (5 µCi/ml) for the times indicated, and cells and media were analyzed for [3H]PGE2 synthesized. Values are means ± SD of 5 experiments; n = 15 lungs. Significantly different from control cells: * P < 0.05; ** P < 0.01 (by Student's t-test).

8

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Effect of IND on fibroblast triglyceride incorporation into type II cell SPC. Day 20 postconception fetal rat lung fibroblasts (fib) were prelabeled with [3H]triglyceride (5 µCi/ml + 200 µg of serum triglyceride/24 h) and recombined with day 20 postconception fetal rat type II cells (TII) in organotypic culture. Cultures were treated with IND (5 × 108 M) and PGE2 (200 ng/ml) as indicated. Type II cells were harvested at the times indicated and analyzed for [3H]SPC. Each data point represents mean ± SD of 3 experiments; n = 15 lungs. * Significantly different from TII cells, P < 0.0001 (by Student's t-test).

	DISCUSSION

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In the present series of experiments, we have observed that fluid distension of the developing lung in explant culture (3, 14, 15) is associated with increased triglyceride incorporation into surfactant phospholipids, a process that was blocked by the addition of either bumetanide or indomethacin, implicating fluid distension and PG synthesis in this complex cellular mechanism. The addition of exogenous PGE₂ to these cultures in the presence or absence of bumetanide stimulated triglyceride incorporation into SPC, indicating that PGE₂ stimulates this mechanism through a stretch-related process. We subsequently found that type II cells synthesize and produce PGE₂ beginning on days 19-20 postconception and that PGE₂ [at a concentration similar to that produced by static day 20 postconception type II cells (200 ng/ml)] stimulates triglyceride release from cultured fetal rat lung fibroblasts. This sequence provides a cellular mechanism for type II cell PGE₂-mediated mobilization of triglycerides from lung fibroblasts. Phloretin inhibition of PGE₂-stimulated triglyceride release by fibroblasts indicates that the PG effect is receptor mediated (4a). Furthermore, both the synthesis of PGE₂ by type II cells and its stimulation of triglyceride release by PGE₂ are enhanced by exposure to Dex. Thus the timing of PGE₂ elaboration and augmentation by steroids and stretch is coincident with the spontaneous surge of surfactant production in utero (7, 23). Additionally, when type II cells are stretched in culture, the de novo synthesis of PGE₂ is stimulated. These data suggest that fluid distension stimulates PGE₂ elaboration by type II cells, triggering the incorporation of triglyceride into surfactant phospholipids.

Lung tissue has the capacity to differentiate spontaneously in vitro, forming all of the cellular elements of the mature lung (2-5, 9-11), particularly the ontogeny of the alveolar type II cells (2, 4) that flatten and develop surfactant synthetic capacity. This process of cellular development occurs in conjunction with fluid dilatation of the terminal air sacs that has been found to play a central role in cellular growth and differentiation. Fluid distension of developing alveoli has been shown to be necessary for normal lung growth and differentiation both in vivo (6-8) and in vitro (9, 10, 12). The distension of the alveoli and the attendant stretching of the cells lining them has been implicated in cell growth (12, 13) and differentiation (24), particularly as they relate to PG synthesis (2, 8-11).

In the present series of studies, the explant experiments indicate that automaturation, as reflected by the spontaneous increase in incorporation of triglyceride into surfactant phospholipid with time in culture, can be inhibited by either bumetanide, which blocks chloride ion secretion and alveolar fluid formation (14, 15), or indomethacin, an inhibitor of PG synthesis. Furthermore, the inhibitory effects of both bumetanide and indomethacin can be overcome by the addition of exogenous PGE₂ to the explant cultures, suggesting that the effect of alveolar dilatation is mediated, at least in part, by PGs. Similar effects of PGE₂ on the structural maturation of human fetal lung were shown by both Ballard et al. (2) and Hume et al. (9-11) with regard to formation of alveolar lumina and cellular maturation. We have now addressed one of the underlying mechanisms that mediate the stretch-dependent cell-cell interactions leading to augmented surfactant synthesis.

In previous studies, Nunez and Torday (18) found that the accumulation of triglycerides by fetal lung fibroblasts is a glucocorticoid-regulated mechanism that may be important for surfactant phospholipid synthesis because these triglycerides can act as a substrate for type II cell surfactant phospholipid synthesis in cell culture as found by Torday et al. (23). The transit of triglyceride from fibroblasts to type II cells in an organotypic culture is glucocorticoid stimulated (18), inferring that either the synthesis of PGE₂, its action on fibroblast triglyceride release, or both are stimulated by glucocorticoids. In the present series of experiments, we have shown that Dex stimulates both PGE₂ synthesis by type II cells and fibroblast triglyceride release in response to PGE₂, which would explain the observed effect of Dex on triglyceride mobilization in the organotypic cultures. These effects of Dex on PGE₂ synthesis and action in the fetal lung are somewhat surprising at first because steroids are generically anti-inflammatory (and prostanoids are proinflammatory). However, this is true in mature, homeostatically regulated systems, whereas the present studies are of a developmental mechanism. In support of this seemingly paradoxical effect of steroid on the developing rat lung, Tsai et al. (24) have observed Dex stimulation of PG synthesis by fetal rat lung in vivo, and Skinner et al. (20) have observed that cortisol enhances the fetal rat lung fibroblast adenosine 3',5'-cyclic monophosphate response to PGE₂.

In the present series of experiments, we have demonstrated that 1) triglyceride metabolism by fetal lung explants depends on alveolar fluid distension and PG synthesis, 2) type II cells develop the ability to synthesize PGE₂, 3) fibroblasts develop the capacity to respond to PGE₂, 4) fibroblasts develop the capacity to respond to PGE₂ with regard to triglyceride secretion, 5) Dex stimulates both 2 and 3, and 6) stretch stimulates de novo PGE₂ synthesis by type II cells. Therefore, we conclude that fluid distension plays a central role in the organized development of the primordial lung, a process that can be modulated by steroid exposure.