Alveolar Homeostasis Research Articles

Evidence of a functional CFTR Cl(-) channel in adult alveolar epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the fetal lung, but during lung development it gradually disappears in cells of future alveolar spaces. Recent studies have implicated the CFTR in fluid transport by the adult alveolar epithelium, but its presence has not been demonstrated directly. This study re-evaluated CFTR expression and activity in the adult pulmonary epithelium by using freshly isolated rat alveolar type II (ATII) cells. CFTR mRNA was detected by semiquantitative polymerase chain reaction on the day of cell isolation but was rapidly reduced by 60% after 24 h of cell culture. This was paralleled by a similar decrease of surfactant protein A expression and alkaline phosphatase staining, markers of the ATII cell phenotype. CFTR expression increased significantly on day 4 in cells grown on filters at the air-liquid interface compared with cells submerged or grown on plastic. Significantly higher CFTR expression was detected in distal lung tissue compared with the trachea. The CFTR was also found at the protein level in Western blot experiments employing lysates of freshly isolated alveolar cells. Whole cell patch-clamp experiments revealed cAMP-stimulated, 5-nitro-2-(3-phenylpropylamino)-benzoate-sensitive Cl(-) conductance with a linear current-voltage relationship. In cell-attached membrane patches with 100 microM amiloride in pipette solution, forskolin stimulated channels of approximately 4 pS conductance. Our results indicate that 50-250 of functional CFTR Cl(-) channels occur in adult alveolar cells and could contribute to alveolar liquid homeostasis.

Effect of cyclosporin A on alveolar bone homeostasis in a rat periodontitis model.

The administration of cyclosporin A has been associated with significant bone loss and increased bone remodeling. The present investigation was designed to evaluate the effects of cyclosporin A on alveolar bone of rats subjected to experimental periodontitis, using serum, stereometric and radiographic analysis. Twenty-four rats were divided into one of the following groups with six animals each: group I, control rats; group II, in which the animals received a cotton ligature around the lower first molars; group III, in which the rats received a cotton ligature around the lower first molars and were treated with 10 mg/(kg body weight day) of cyclosporin A; group IV, in which the rats were treated with 10 mg/(kg body weight day) of cyclosporin A. At the end of experimental period, at 30 days, animals were killed and the serum calcium and alkaline phosphatase levels were measured in all groups. The distance from the alveolar bone crest to the cemento-enamel junction was measured radiographically for each mesial surface of the lower first molars of each rat. After histological processing, the stereological parameters: volume densities of multinucleated osteoclasts (V(o)), alveolar bone (V(b)), marrow (V(m)), and relation of eroded surface/bone surface (Es/Bs) were assessed at the mesial region of the alveolar bone. Significant decreases in serum calcium were observed in those groups that received cyclosporin A therapy. No significant changes in serum alkaline phosphatase were observed. The therapy with cyclosporin A combined with the ligature placement decreased the V(b) and increased the V(o), V(m) and Es/Bs at the mesial surface of lower first molars. On the other hand, the radiographic data showed that cyclosporin A therapy diminished the alveolar bone loss at the mesial surface of the lower first molars. Therefore, within the limits of this study, we suggest that cyclosporin A at immunosuppressive levels can bring about an imbalance in the alveolar bone homeostasis in rats. However, in the presence of inflammatory stimulation, the inhibition of the immune system by cyclosporin A may decrease the initial periodontal breakdown.

Regulation of postnatal lung development and homeostasis by estrogen receptor beta.

Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor alpha (ERalpha) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ERbeta is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERbeta gene (ERbeta(-/-)) revealed decreased numbers of alveoli in adult female ERbeta(-/-) mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ERbeta(-/-) mice, and direct transcriptional regulation of these genes by ERbeta was demonstrated. This suggests that estrogens act via ERbeta in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ERbeta as a previously unknown regulator of postnatal lung development and homeostasis.

Identification of three genes of known function expressed by alveolar epithelial type I cells.

To identify genes of known function expressed by type I (AT1) cells, changes in gene expression during transdifferentiation of alveolar epithelial cells (AEC) in primary culture from type II (AT2) to type I-like cell phenotype were evaluated. Total RNA from AEC on Day 0 or Day 8 was hybridized to a rat microarray for screening. Eight upregulated genes on Day 8 were selected for further investigation. Northern analysis confirmed upregulation of three of these genes, PAI-1, P2X4, and P15INK4B. The corresponding proteins were evaluated in cultured AEC and results correlated with expression in AT1 cells. In AEC monolayers, all three proteins increased between Day 1 and Day 8. In mixed populations of freshly isolated rat lung cells, concurrent labeling with the AT1 cell-specific antibody, VIIIB2, localized these proteins to AT1 cells. In whole lung, all three proteins were detected in alveolar epithelium in a location consistent with expression in AT1 cells. Identification of novel AT1 cell genes of known function suggests an active role for AT1 cells in alveolar homeostasis. Furthermore, expression of these gene products in AT1-like cells, in freshly isolated AT1 cells, and AT1 cells in whole lung indicates that AT1-like cells reflect many of the properties of AT1 cells in situ.

Protein transport across the lung epithelial barrier.

Alveolar lining fluid normally contains proteins of important physiological, antioxidant, and mucosal defense functions [such as albumin, immunoglobulin G (IgG), secretory IgA, transferrin, and ceruloplasmin]. Because concentrations of plasma proteins in alveolar fluid can increase in injured lungs (such as with permeability edema and inflammation), understanding how alveolar epithelium handles protein transport is needed to develop therapeutic measures to restore alveolar homeostasis. This review provides an update on recent findings on protein transport across the alveolar epithelial barrier. The use of primary cultured rat alveolar epithelial cell monolayers (that exhibit phenotypic and morphological traits of in vivo alveolar epithelial type I cells) has shown that albumin and IgG are absorbed via saturable processes at rates greater than those predicted by passive diffusional mechanisms. In contrast, secretory component, the extracellular portion of the polymeric immunoglobulin receptor, is secreted into alveolar fluid. Transcytosis involving caveolae and clathrin-coated pits is likely the main route of alveolar epithelial protein transport, although relative contributions of these internalization steps to overall protein handling of alveolar epithelium remain to be determined. The specific pathways and regulatory mechanisms responsible for translocation of proteins across lung alveolar epithelium and regulation of the cognate receptors (e.g., 60-kDa albumin binding protein and IgG binding FcRn) expressed in alveolar epithelium need to be elucidated.

Absorption of intact albumin across rat alveolar epithelial cell monolayers.

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral (ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical (ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of approximately 1.6 microM and maximal transport rate of approximately 0.15 fmol.cm(-2).s(-1). At 15 and 4 degrees C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an approximately 60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung.

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix.

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.

Effect of mechanical loading on periodontal cells.

Mechanical loading is an important regulatory factor in alveolar bone homeostasis, and plays an essential role in maintaining the structure and mass of the alveolar processes throughout lifetime. A better understanding of the cellular and molecular responses of periodontal cells is a prerequisite for further improvements of therapeutic approaches in orthodontics, periodontal and alveolar bone repair and regeneration, implantology, and post-surgical wound healing. The purpose of this review is to provide an insight into some cell culture and animal models used for studying the effects of mechanical loading on periodontal cells, and into the recent developments and utilization of new in vivo animal models. There has been an increased awareness about the need for improvement and development of in vivo models to supplement the widely used cell culture models, and for biological validation of in vitro results, especially in the light of evidence that developmental models may not always reflect bone homeostasis in an adult organism. Due to the limitations of in vivo models, previous studies on mechanical regulation of alveolar bone osteoblasts and cementoblasts mostly focused on proliferative responses, rather than on the stimulation of cell differentiation. To address this problem, we have recently characterized and implemented a mouse osteoinductive tooth movement model for studying mechanically induced regulation of osteoblast- and cementoblast-associated genes. In this model, a defined and reproducible mechanical osteogenic loading is applied during a time course of up to two weeks. Regulation of gene expression in either wild-type or transgenic animals is assessed by a relative quantitative measurement of the level of target mRNAs directly within the subpopulations of periodontal cells. To date, results demonstrate a defined temporal pattern of cell-specific gene regulation in periodontal osteoblasts mechanically stimulated to differentiate and deposit bone matrix. The responses of osteoblast-associated genes to mechanical loading were 10- to 20-fold greater than the increase in the numbers of these cells, indicating that the induction of differentiation and an increase of cell function are the primary responses to osteogenic loading. The progression of the osteoblast phenotype in the intact mouse periodontium was several-fold faster compared with that in cultured cells, suggesting that the mechanical signal may be targeting osteoblast precursors in the state of readiness to respond to an environmental challenge, without the initial proliferative response. An early response of alkaline phosphatase and bone sialoprotein genes was detected after 24 hrs of treatment, followed by a concomitant stimulation of osteocalcin and collagen I between 24 and 48 hrs, and deposition of osteoid after 72 hrs. Although cementoblasts constitutively express biochemical markers similar to those of osteoblasts, distinct responses of osteocalcin, collagen I, and bone sialoprotein genes to mechanical loading were observed in the two cell phenotypes. This finding indicates that differential genetic responses to mechanical loading provide functional markers for distinction of the cementoblast and osteoblast phenotypes.

Modulation of pulmonary NA+ pump gene expression during cold storage and reperfusion.

Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein. Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured. There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA. Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.

Ethanol Ingestion Impairs Alveolar Epithelial Glutathione Homeostasis and Function, and Predisposes to Endotoxin-Mediated Acute Lung Injury

Ethanol Ingestion Impairs Alveolar Epithelial Glutathione Homeostasis and Function, and Predisposes to Endotoxin-Mediated Acute Lung Injury

Differential effects of isoflurane and i.v. anaesthetic agents on metabolism of alveolar type II cells

Alveolar type II (ATII) cells perform many important functions within the lung, including surfactant metabolism. We have investigated the effects of isoflurane and different i.v. anaesthetics on cell metabolism in primary cultures of rat ATII cells. Biosynthesis of phosphatidylcholine, the main phospholipid component of surfactant, was decreased in cells exposed to isoflurane in a dose- and time-related manner. This effect was fully reversible within 2 h after isoflurane removal. Lactate dehydrogenase (LDH) release, an index of cell damage, was increased with isoflurane exposure (1%) over a long incubation period (8-12 h). Enhanced lactate production, reflecting an increase in glycolytic metabolism, was also observed in isoflurane exposed cells. In contrast, metabolism of ATII cells was moderately affected by i.v. anaesthetics. Our data suggest differential metabolism of alveolar homeostasis depending on the anaesthetic agent used.

SP-D FORMS SURFACE-ACTIVE TUBULAR AGGREGATES OF DIPALMITOYLPHOSPHATIDYLCHOLINE-PHOSPHATIDYLINOSITOL: † 2058

SP-D, a recently described surfactant apoprotein structurally closely related to SP-A and other members of the collectin protein family, has been implicated in the host defense of the lung. Its potential role in alveolar surfactant homeostasis is unknown, however. SP-D interacts with and aggregates phosphatidylinositol (PI)-containing liposomes (Biochemistry, 31:12183-89, 1992). We previously reported the cloning and heterologous expression of mouse SP-D cDNA (rSP-D; Pediatric Research, 37:346A, 1995) and showed that it assembles as oligomers of trimers, binds the sugar maltose and aggregates PI-containing liposomes in a calcium-dependent manner.

Regulation of alveolar type II cell differentiation and proliferation in adult rat lung explants.

Alveolar type II cells produce pulmonary surfactant and serve as the stem cell of the alveolar epithelium by proliferating and transforming into type I cells. The study of the differentiated function and proliferative capacity of type II cells in response to injury in vivo has been hindered by the complexity of the systemic response to injury. In vitro studies have in turn been limited by the impaired proliferative potential and loss of markers of differentiation in isolated type II cells maintained in culture. We describe an in vitro system in which type II cells proliferate spontaneously and simultaneously maintain differentiated characteristics. Other investigators have maintained slices of adult lung in culture after agarose infusion for up to 9 wk. To further develop this model for the study of epithelial cell differentiation and proliferation, we assessed epithelial differentiation, proliferative capacity, and regulation of cell-specific gene expression in slice explants of agarose-infused rat lungs. We prepared 1-mm-thick explants and maintained them in culture for up to 2 wk. Maintenance of differentiation was confirmed morphologically by light and electron microscopy, by the accumulation of epithelial cell-specific surfactant proteins, and by phospholipid analysis. Proliferative capacity was assessed by measuring [3H]thymidine incorporation in alveolar and small airway cells at baseline and in response to growth stimuli. Type II cell proliferation was inhibited in a dose-dependent manner by glucocorticoids. Glucocorticoids regulated RNA levels in explants in a manner similar to that seen in vivo and in fetal lung explants. The alveolar epithelium in adult lung slice explants maintains differentiated function and the ability to proliferate, thereby providing a useful system for the study of distal airway and alveolar cell homeostasis and response to injury.

Stimulation of net active ion transport across alveolar type II cell monolayers

The active transcellular transport of electrolytes across the alveolar epithelium probably plays an important role in alveolar fluid homeostasis by helping to maintain the alveolus relatively free of fluid. To better understand the factors regulating active ion transport across alveolar epithelial cells, we examined the effect of a number of pharmacologically active agents on the bioelectric properties of alveolar type II cells in primary culture. Alveolar type II cells were isolated from adult male rats and cultured on collagen-coated Millipore filters for 6-14 days. The bioelectric properties of these monolayers were determined in Ussing-type chambers. The addition of 10(-3) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) increased the short-circuit current (Isc) from 2.9 +/- 0.75 to 6.9 +/- 0.73 microA/cm2 (means +/- SE; n = 8) and decreased the transepithelial resistance. Cholera toxin, 3-isobutyl-1-methylxanthine, and terbutaline sulfate produced similar increases in Isc and decreases in resistance. The Isc stimulated by 8-BrcAMP was Na but not Cl dependent and could be blocked by amiloride but not by furosemide. Thus 8-BrcAMP and agents that increase intracellular cAMP can stimulate a Na-dependent net active ion transport across alveolar type II cell monolayers. Similar regulatory mechanisms may be involved in controlling solute and fluid movement across the alveolar epithelium in vivo.