Spencer I. Danto

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

SummaryIsolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm2 by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 µA/cm2. With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.

Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF

We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na+-K+-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current ( I sc) compared with controls by day 5, with further increases occurring through day 8. Relative Na+-K+-ATPase α1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas α2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the β1-subunit of Na+-K+-ATPase did not increase, whereas cellular α1- and β1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for α-, β-, and γ-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase α1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.

Modulation of T1α expression with alveolar epithelial cell phenotype in vitro

T1α is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1α expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1α expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1α expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1(DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1α expression was quantified by Northern and Western blotting, respectively. Expression of T1α progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1α expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1α on days 4 and 8. T1α expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1α expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both alpha1 and beta1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha1- and beta1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha1- and beta1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+ transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.

Studies on the mechanisms of active ion fluxes across alveolar epithelial cell monolayers

To investigate the cell physiologic and biological properties of the alveolar epithelium, we studied rat alveolar epithelial cell monolayers grown on permeable supports in primary culture. Type II alveolar epithelial cells were disaggregated using elastase, and partially purified on a discontinuous metrizamide gradient. These isolated cells were plated onto tissue culture-treated Nuclepore membrane filters at 1.5×106 cells/cm2 and maintained in a humidified incubator (5% CO2 in air, 37° C). After 2 days in culture, the bathing media on both sides of the cell monolayers were changed to fresh culture medium, thus removing nonadherent cells (mostly leukocytes). These monolayers exhibit a high transmonolayer resistance (>2000 Ω-cm2) and actively transport ions. Radionuclide flux studies indicate that Na+ is the predominant ionic species absorbed actively under baseline conditions, accounting for about 80% of the total active ion transport. Cl− seems to be passively transported across the epithelium. However, when the epithelium is exposed to a beta-agonist (terbutaline), active absorption of Na+ is increased and active absorption of Cl− occurs. Although it is clear that both active Na+ and Cl− transport are dependent on Na+/K+-ATPase activity, and that Na+ enters cells predominantly through channels, the specific mechanisms by which Cl− enters and exits the alveolar epithelial cells remain unclear. The stimulated reabsorption of Na+ and Cl− may be important in helping to remove excess fluid from alveolar air spaces in the lung.

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 μM) produced ∼20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that α-, β- and γ-subunits of rat epithelial Na+ channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations ([Formula: see text]/[Formula: see text]≈ 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

Late appearance of a type I alveolr epithelialar cell marker during fetal rat lung development

Recent studies in fetal lung using immunological and molecular probes have revealed type I and type II cell phenotypic markers in primordial lung epithelial cells prior to the morphogenesis of these cell types. We have recently developed monoclonal antibodies specific for adult type I cells. To evaluate further the temporal appearance of the type I cell phenotype during alveolar epithelial cell ontogeny, we analyzed fetal lung development using one of our monoclonal antibodies (mAb VIII B2). The epitope recognized by mAb VIII B2 first appears in the canalicular stage of fetal lung development, at approx. embryonic day 19 (E19), in occasional, faintly stained tubules. Staining with this type I cell probe becomes more intense and more widespread with increasing gestational age, during which time the pattern of staining changes. Initially, all cells of the distal epithelial tubules are uniformly labelled along their apical and basolateral surfaces. As morphological differentiation of the alveolar epithelium proceeds, type I cell immunoreactivity appears to become restricted to the apical surface of the primitive type I cells in a pattern approaching that seen in the mature lung. We concurrently analyzed developing fetal lung with an antiserum to surfactant apoprotein-A (α-SP-A). Consistent with the findings of others, labeling of SP-A was first detectable in scattered cuboidal cells at E18. Careful examination of the doublelabeled specimens suggested that some cells were reactive with both the VIII B2 and SP-A antibodies, particularly at E20. Confocal microscopic analysis of such sections from E20 lung confirmed this impression. Three populations of cells were detected: cells labeled only with α-SP-A, cells labeled only with mAb VIII B2, and a smaller subset of cells labeled by both. We conclude that: (1) binding of mAb VIII B2 may be a marker of late (possibly terminal) type I cell differentiation; (2) it is likely to recognize a different epitope from another published type I cell mAb (SF-1), since mAb VIII B2 epitope appears at a much later developmental age; and (3) cells may co-express both type II (SP-A) and type I (mAb VIII B2 epitope) cell differentation antigens.