Inhibitors of choline transport in alveolar type II epithelial cells.

Isolated alveolar type II epithelial cells (granular pneumocytes) from rat lung accumulate free choline against a concentration gradient by an energy-dependent saturable transport process with apparent Km approximately 18 microM. In order to evaluate the structural requirements for choline transport by these cells, the inhibition of the initial rate of cellular uptake of [3H]choline (5 microM) by its analogue was measured. There was no significant inhibition of substrate uptake by analogues lacking an amino group while the presence of a quaternary nitrogen was most effective. N,N'-dimethylethanolamine (apparent Ki, 7 microM) and n-decylcholine (apparent Ki, 0.5 microM) were potent competitive inhibitors of choline transport. Substitution of the hydroxyl group in choline greatly diminished the inhibitory effect; fluorocholine, thiocholine, betaine, and betaine aldehyde showed little or no inhibition. This requirement for a hydroxyl group raises the possibility of hydrogen bonding of choline with the transport protein. The choline transport system in granular pneumocytes appears to differ from that in synaptosomes by the lower affinity of the carrier for substrate and for hemicholinium-3 and from that in erythrocytes by the role of the hydroxyl in the substrate molecule. The availability of inhibitory analogues for choline transport will facilitate isolation and study of the granular pneumocyte choline transport protein.     

Human parainfluenza virus type 1 but not Sendai virus replicates in human respiratory cells despite IFN treatment

Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-beta into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-beta, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-beta pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV.     

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.     

Isolation of highly pure alveolar epithelial type I and type II cells from rat lungs

There are no ideal cell lines available for alveolar epithelial type I and II cells (AEC I and II) at the present time. The current methods for isolating AEC I and II give limited purities. Here, we reported improved and reproducible methods for the isolation of highly pure AEC I and II from rat lungs. AEC I and II were released from lung tissues using different concentrations of elastase digestion. Macrophages and leukocytes were removed by rat IgG 'panning' and anti-rat leukocyte common antigen antibodies. For AEC II isolation, polyclonal rabbit anti-T1alpha (an AEC I apical membrane protein) antibodies were used to remove AEC I contamination. For AEC I isolation, positive immunomagnetic selection by polyclonal anti-T1alpha antibodies was used. The purities of AEC I and II were 91 +/- 4 and 97 +/- 1%, respectively. The yield per rat was approximately 2 x 10(6) for AEC I and approximately 33 x 10(6) for AEC II. The viabilities of these cell preparations were more than 96%. The protocol for AEC II isolation is also suitable to obtain pure AEC II (93-95%) from hyperoxia-injured and recovering lungs. The purified AEC I and II can be used for gene expression profiling and functional studies. It also offers an important tool to the field of lung biology.     

Alterations in pulmonary ultrastructure and morphometric parameters induced by parainfluenza (Sendai) virus in rats during postnatal growth.

The ultrastructural and morphometric effects of viral respiratory disease during postnatal lung growth were examined in weanling (22-day-old) and suckling (5-day-old) rats infected with parainfluenza Type 1 (Sendai) virus. Viral nucleocapsids and budding virions were identified by transmission electron microscopy in ciliated cells, mucous cells, and nonciliated bronchiolar epithelial cells of weanling rats at 5 days after inoculation and were associated with epithelial necrosis and erosion as well as hyperplasia of nonciliated bronchiolar epithelial cells. Interstitial pneumonia characterized in early stages by swelling and sloughing of Type 1 and Type 2 alveolar epithelial cells was also present at 5 and 7 days after inoculation. Lesions persisting at 30, 60, and 90 days after inoculation included multifocal connective tissue polyps in terminal bronchioles that partially obstructed bronchiolar lumens. Specific lung volume was greater (P less than 0.01) in weanling rats at 30 and 60 days following viral infection than in control rats, and specific alveolar surface area was 42% greater (P less than 0.01) in infected rats at 60 days after inoculation. Suckling rats infected during a phase of rapid postnatal lung growth at 5 days of age had 33% greater (P less than 0.02) specific alveolar surface area and 48% greater (P less than 0.001) mean terminal bronchiolar cross-sectional area when compared with control rats at 22 days of age. The results indicate that viral pulmonary infection during early life can induce acute and persistent alterations in pulmonary structure that could adversely affect lung function.     

Effects of parainfluenza type 3 virus on guinea pig pulmonary alveolar macrophage functions in vitro

The influence of parainfluenza type 3 (PI-3) virus on the release of inflammatory mediators by guinea pig pulmonary alveolar macrophages (PAMs) was investigated in vitro. Direct application of PI-3 virus dose-dependently stimulated the generation of chemiluminescence by PAMs and induced aggregation of PAMs. No significant effects of PI-3 virus on the release of linoleic acid metabolites by PAMs were detected. However, an increased release of the arachidonic acid metabolite thromboxane B₂ (TxB₂) was observed when PAMs were stimulated with PI-3 virus. PAMs were also cultured for 2 h or 18 h in the presence of PI-3 virus or control medium. The production of reactive oxygen species and the release of fatty acid metabolites by these PAMs were determined upon stimulation with opsonized zymosan particles or phorbol myristate acetate. The amounts of hydrogen peroxide and superoxide produced did not differ between virus and control medium-incubated PAMs. However, the PI-3 virus-treated PAMs generated twice as much chemiluminescence when compared to PAMs incubated with control medium. The 2-h incubation period with PI-3 virus also resulted in a decreased release of TxB₂ from the PAMs upon zymosan stimulation. The changes in the production of reactive oxygen species and the release of TxB₂ by PAMs could account for damage to the airways and bronchial hyperresponsiveness often seen after viral infection.     

Alterations in eicosanoid production by rat alveolar type II cells isolated after silica-induced lung injury.

Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-keto-PGF1 alpha or thromboxane B2 (TxB2). Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE2 and 6-keto-PGF1 alpha production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE2 between the normotrophic and the hypertrophic type II cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Adrenergic induced synthesis and secretion of phosphatidylcholine by isolated pulmonary alveolar type II cells

Rat pulmonary alveolar type II cells isolated by trypsinization and discontinuous density gradient ultracentrifugation were maintained in primary culture for 48 hours. The cultured type II cells responded to beta-adrenergic, but not cholinergic, agonists by an increase in the rate of synthesis and also secretion of 3H-phosphatidylcholine. The beta-adrenergic agonists, isoproterenol and terbutaline, 10 microM, caused a 1.7-fold increase in the rate of synthesis of 3H-phosphatidylcholine after a 4-hour incubation. At this time, there was also an increase in the cAMP content of the cultured cells. Terbutaline, 10 microM, caused a 4.9-fold increase in the rate of secretion of 3H-phosphatidylcholine after a 1-hour incubation. The beta-adrenergic effect on both synthesis and secretion by type II cells was blocked by propranolol. 8-Br-cAMP, 100 microM, but not 8-Br-cGMP, mimicked the beta-adrenergic effect on both synthesis and secretion of 3H-phosphatidylcholine. The increased rate of 3H-phosphatidylcholine induced by beta-adrenergic agonists was unaffected by colchicine. These data are consistent with the hypothesis that both synthesis and secretion of pulmonary surfactant are under adrenergic control operating through a beta-receptor and the adenylate cyclase system. These data also suggest that synthesis and secretion of pulmonary surfactant are independent processes. The possibility of other neural or hormonal mechanisms is not excluded.     

Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung

We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.     

Replication of parainfluenza type 3 virus in alveolar macrophages: evidence of in vivo infection and of in vitro temperature sensitivity in virus maturation.

Approximately 2% of cultured alveolar macrophages (AM), originally lavaged from the lungs of parainfluenza type 3 virus (PI-3V)-infected calves, were observed to contain viral antigen (by fluorescent antibody method) or viral nucleocapsids (by electron microscopy). Plaque assays, however, indicated that virus titers were generally low when cultures were incubated at 37 degrees C for 10 days. AM, obtained from "in vivo infected" and "noninfected" calves, were found to be equally susceptible to further in vitro PI-3V infection when cultures were incubated at 37 degrees C. AM that were obtained from the lungs of normal calves, cultured at 37 degrees C, and inoculated with PI-3V were observed to produce relatively high virus titers when the incubation temperature was shifted down to 32 degrees C. Results from hemagglutinin assays showed that considerable amounts of hemagglutinin were detected when AM cultures were incubated at 32 degrees C, but only limited amounts were detected at 37 degrees C. Results from electron microscopic examinations at both temperatures substantiated the results of plaque and hemagglutinin assays. The PI-3V, isolated from AM cultures incubated at 32 degrees C, grew well in Madin-Darby bovine kidney cells at 32 degrees C, but little virus was produced at 37 degrees C. In contrast, parent PI-3V grew equally well at both temperatures. The results are discussed in terms of host susceptibility, temperature-sensitivity and virus maturation, and surface viral antigens and persistent viral infection.     

The fate of protein subunits of parainfluenza (Sendai) virus after adsorption to NIL8 hamster embryo cells.

Adsorption of u.v.-inactivated Sendai virus on to NIL8 hamster cells causes fusion of the cells into polykaryocytes within 2 h. "Infected" cells were incubated at 37 degrees C for periods of 10 min to 8 h and their surface proteins iodinated with [125I] catalysed by peroxidase. Structural components of the viral envelope, such as haemagglutin-neuraminidase (HN) and probably also the fusion protein (F) were detected in the cell membrane for periods up to 4 h post infection.     

Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed.     

Expression of urokinase-type plasminogen activator by rat pulmonary alveolar epithelial cells

Intra-alveolar fibrin deposition accompanies many forms of inflammatory lung injury. Appropriate clearance of this fibrin matrix is important for normal healing and remodeling. The local generation of plasmin by the action of plasminogen activators (PAs) represents a pivotal step in the fibrinolytic process. To investigate whether the alveolar epithelium plays a role in the modulation of intra-alveolar fibrinolysis, we have studied PA regulation by rat pulmonary alveolar epithelial cells. We have found large quantities of PA activity both in conditioned media and cell lysates from epithelial monolayers in culture. Casein-plasminogen zymography reveals that this PA activity migrates as a tight doublet with an apparent mol wt of 45 kD, clearly distinct from rat tissue-type PA (tPA, greater than 68 kD). Analysis of freshly isolated type II alveolar epithelial cells demonstrates readily measurable PA activity in cell lysates, as well as expression of urokinase-type PA (uPA) mRNA on Northern blot analysis. Upregulation of PA activity occurs progressively with time in culture as the alveolar epithelial cells lose type II cell characteristics and become more flattened. Stimulation of alveolar epithelial cell monolayers with lipopolysaccharide or tumor necrosis factor increases levels of secreted PA activity. The relative abundance of uPA mRNA was shown to change in parallel with PA activity during in vitro differentiation or after exposure to inflammatory mediators. Thus, alveolar epithelial cells are likely an important source of uPA in the lung, the expression of which is influenced by the state of cellular differentiation as well as the presence of inflammatory mediators.(ABSTRACT TRUNCATED AT 250 WORDS)