Increased lung expansion alters lung growth but not alveolar epithelial cell differentiation in newborn lambs

Although increased lung expansion markedly alters lung growth and epithelial cell differentiation during fetal life, the effect of increasing lung expansion after birth is unknown. We hypothesized that increased basal lung expansion, caused by ventilating newborn lambs with a positive end-expiratory pressure (PEEP), would stimulate lung growth and alter alveolar epithelial cell (AEC) proportions and decrease surfactant protein mRNA levels. Two groups of lambs were sedated and ventilated with either 0 cmH(2)O PEEP (controls, n = 5) or 10 cmH(2)O PEEP (n = 5) for 48 h beginning at 15 +/- 1 days after normal term birth. A further group of nonventilated 2-wk-old lambs was used for comparison. We determined wet and dry lung weights, DNA and protein content, a labeling index for proliferating cells, surfactant protein mRNA expression, and proportions of AECs using electron microscopy. Although ventilating lambs for 48 h with 10 cmH(2)O PEEP did not affect total lung DNA or protein, it significantly increased the proportion of proliferating cells in the lung when compared with nonventilated 2-wk-old controls and lambs ventilated with 0 cmH(2)O PEEP (control: 2.6 +/- 0.5%; 0 PEEP: 1.9 +/- 0.3%; 10 PEEP: 3.5 +/- 0.3%). In contrast, no differences were observed in AEC proportions or surfactant protein mRNA levels between either of the ventilated groups. This study demonstrates that increases in end-expiratory lung volumes, induced by the application of PEEP, lead to increased lung growth in mechanically ventilated 2-wk-old lambs but do not alter the proportions of AECs.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Volume flow across the alveolar epithelium of adult rat lung

We separated the solute and water flow across the alveolar epithelium from flow across airway epithelia of the adult rat. Small volumes (0.5-1.0 ml) of Krebs-Ringer bicarbonate (KRB) were trapped in the distal air space of the isolated vascular-perfused left lung lobes while the airways were blocked by immiscible O2-carrying fluorocarbon. Lobe weight was lost or gained in response to colloid gradients and was raised by metabolic inhibitors but did not change with only fluorocarbon in the air space or in response to modifiers of epithelial ion transport. When serum was added to the KRB-colloid perfusion, weight loss occurred in the absence of a colloid gradient (3.4 ml/min) and was Na+ dependent (inhibited by luminal Na(+)-free KRB). The change in the concentration of blue dextran in liquid sampled by micropuncture from subpleural alveoli was smaller than expected from lobe weight under basal conditions or with a colloid gradient, even though the volume marker accurately detected edema formation (weight gain) induced by metabolic inhibitors. We conclude that 1) weight changes represent volume absorption from the air spaces, 2) serum stimulates a Na+ absorptive process, and 3) by exclusion, small airways and/or other subpopulations of alveoli are the site of this absorption.     

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (beta-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1alpha) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (alpha-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

 To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells. Accepted: 22 January 1997     

Characterization of rat colonic cell surface glycoconjugates by fluoresceinated lectins

Summary Cryostat and paraffin embedded sections from cecum, proximal and distal colonic segments of male Sherman rats were examined by fluorescence microscopy after labeling with six fluorescein-conjugated lectins. These FITC-conjugated lectins were used as specific probes to define the labeling pattern of carbohydrate containing components of the lumenal and basolateral surfaces of epithelial cells, goblet cell mucin and lumenal mucin at all three sites. Marked regional differences in labeling were detected, indicating that the various carbohydrate components of these cells differ significantly along the length of the colon. Furthermore, the patterns of labeling components with each lectin appeared to vary depending on the fixation technique employed. Cryostat preparations generally resulted in a broader distribution of label and more intense staining with these lectins than fixed paraffin sections. While the reason(s) for these variations remain unclear at this time and will require further studies, the present data emphasize the importance of the fixation method when interpreting results obtained utilizing FITC-conjugated lectins.     

Heterogeneity in the immunolocalization of cytokeratin-specific monoclonal antibodies in the rat lung: evaluation of three different alveolar epithelial cell types

The distribution of individual cytokeratin polypeptides in the adult rat lung parenchyma was investigated by immunohistochemistry with 44 monoclonal and 2 polyclonal antibodies. Simple epithelial cytokeratins 7, 8, 18 and 19 were found to be expressed differently in alveolar and bronchial epithelial cells. Three distinct types of alveolar cells were detected according to their pattern of immunoreactivity: type II cells strongly expressing cytokeratins 8 and 18 and weakly expressing cytokeratins 7 and 19 in the cell periphery; type I cells predominantly positive for cytokeratins 7 and 19 and weakly for cytokeratin 8; and a newly defined third cell type III (alveolar brush cell) with cytokeratin 18 abundantly expressed but organized in an unusual intracellular (globular) structure. The latter cell type failed to bind the type II specific Maclura pomifera lectin, and contained no surfactant proteins. Bronchial epithelial cells exhibited a more or less uniform staining pattern for cytokeratins 8, 18 and 19 and focally for cytokeratins 4 and 7.This work was supported by Bundesminister für Forschung und Technologie (07NBL03) and Dakopatts (Glostrup, Denmark)     

Tracheal epithelium: cell kinetics and differentiation in normal rat tissue

Abstract. The fate of [³H]thymidine ([³H]Tdr) pulse-labelled cells was followed in tracheal epithelium of young male rats. The time course for cell differentiation, and the relation of events to tissue composition were studied. In vivo labelling and light microscope autoradiography of epoxy embedded sections were used. Labelled and total nuclei for each cell type, and combinations of labelled cells which were adjacent to one another, were tallied. Hierarchical analyses of variance were performed on the several data sets. All cell types, except ciliated, were labelled at 1 hr. A few labelled ciliated cells were seen 24 hr post-label. The frequency of labelled intermediate cells peaked at day 2; goblet and ciliated cells at day 3. No significant changes occurred in the labelling index, but at 24 hr the frequency of adjacent labelled cells (ALC) had increased > 5-fold, and changes had occurred in patterns of ALC combinations. The labelled ciliated cells which were seen at 24 hr were adjacent to labelled intermediate cells. No labelled basal-ciliated cell combinations were seen at any time. Data indicated that ciliated cells can develop from S-phase intermediate cells within 24 hr, and neither basal nor superficial goblet cells are progenitors of ciliated cells. It is proposed that both superficial goblet cell and ciliated cell development is preceded by two divisions: a basal cell division followed by an intermediate cell division.     

Reactive alveolar epithelium in chondroid hamartoma of the lung

OBJECTIVE: To determine the morphologic characteristics of the nonciliated epithelium found in chondroid hamartoma of the lung. STUDY DESIGN: The morphologic characteristics and immunohistochemical reaction for surfactant protein A of the nonciliated epithelium in chondroid hamartoma of the lung was studied by immunohistochemistry. Alveolar epithelium in normal lung tissue and lung tissue surrounding primary lung cancer or metastatic lung lesions was used as a control. RESULTS: In all cases, the nonciliated epithelium in chondroid hamartoma showed the morphologic criteria of hyperplastic alveolar type II cells and a very strong positive surfactant protein A reaction in the cytoplasm when compared with alveolar epithelium of the normal lung. Similar hyperplastic type II cells were also found in the alveolar lung around metastatic or primary lung tumors. CONCLUSION: These findings may indicate that the nonciliated cells found in chondroid hamartoma of the lung are hyperplastic type II cells. This suggests that the alveolar epithelium found in chondroid hamartoma of the lung is a secondary reaction around the hamartoma and not a primary component of the lesion.     

Evidence for active sodium transport across alveolar epithelium of isolated rat lung

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, a process that can be inhibited by sodium transport blockers and stimulated by beta-agonists. In this study, the isolated perfused rat lung was utilized in order to investigate the presence of active sodium transport by intact adult mammalian alveolar epithelium. Radioactive tracers (22Na and [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs whose weight was continuously monitored. The appearance of isotopes in the recirculated perfusate was measured, and fluxes and apparent permeability-surface area products were determined. A pharmacological agent (amiloride, ouabain, or terbutaline) was added to the perfusate during each experiment after a suitable control period. Amiloride and ouabain resulted in decreased 22Na fluxes and a faster rate of lung weight gain. Terbutaline resulted in increased 22Na flux and a more rapid rate of lung weight loss. [14C]sucrose fluxes were unchanged by the presence of these pharmacological agents. These data are most consistent with the presence of a regulable active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space, with anions and water following passively. Regulation of the rate of sodium and fluid removal from the alveolar space may play an important role in the prevention and/or resolution of alveolar pulmonary edema.     

Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture

We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development.     

Phenotype-dependent synthesis of transferrin receptor in rat alveolar epithelial cell monolayers

The iron carrier protein transferrin plays a prominent antioxidant and anti-bacterial role in the lower respiratory tract and is present at elevated concentrations in lung epithelial lining fluid relative to plasma. The level of transferrin receptor synthesis in primary cultures of rat alveolar epithelial cells (AECs) was investigated. Transferrin receptor was found to be synthesized early in AEC cultures with the alveolar type II cell-like phenotype. Cell-surface receptor localization was attenuated upon apparent transdifferentiation to the alveolar type I cell-like phenotype later in culture. Binding of (125)I-labeled transferrin to the receptor indicated that surface and total cellular transferrin receptor levels were decreased in the type I-like cells. Inclusion of keratinocyte growth factor (KGF) in culture media (10 ng/ml) resulted in retention of transferrin receptor localized to the basolateral surface. Transferrin-receptor-specific internalization of (59)Fe-transferrin was also limited to the basolateral surface of KGF-treated monolayers. These data suggest that alveolar type II (but not type I) cells express functional transferrin receptor in adult rat alveolar epithelium.     

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.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: IV. Cell surface proteins

There are stem cells in RT4 neurotumor that undergo spontaneous differentiation into three distinct cell types in culture (cell type conversion). Stem cells (RT4-AC) and one of the differentiated cell types (RT4-D) are tumorigenic and synthesize glial-specific S100 protein, while the others (RT4-B and RT4-E) are nontumorigenic and demonstrate some neuronal function. Interrelationships between cell surface proteins and differentiation of RT4 cells were analyzed. The surface proteins are radioiodinated by a lactoperoxidase method, separated by gel electrophoresis in two dimensions, visualized by autoradiography, and quantitated according to the relative radioactivity associated with each protein species. Integral surface proteins are compared in the present study which remain associated with plasma membrane after the extraction of radioiodinated cells with 0.1 N NaOH. The extraction also helps to remove serum proteins in the medium which are absorbed on cell surfaces and may be artifactually recognized as surface proteins. All RT4 cell types are complex in cell surface protein composition and nearly 100 integral surface proteins have been identified when all RT4 cell types are combined. Many unique proteins as well as common proteins have been identified. Considerable similarity exists between RT4-AC and RT4-D and between RT4-B and RT4-E. The two cell types of each pair are distinct yet share some common neurological and tumorigenic characteristics. In contrast, little similarity exists in other combinations of the two cell types, e.g., RT4-AC and RT4-E, etc. The results support the notion that the pattern of cell surface protein expression is a stable differentiation property and a characteristic set of proteins corresponds to each stage of differentiation.     

Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.