Distribution of E-cadherin and Ep-CAM in the human lung during development and after injury

Paraffin sections were obtained of human fetal, adult, and pathological lung (pulmonary fibrosis after radiotherapy or chemotherapy). The localization of epithelial adhesion molecules E-cadherin and Ep-CAM (former epithelial surface 40 kDa glycoprotein) was investigated by immunoperoxidase and/or immunofluorescence techniques with monoclonal antibodies. During development, the epithelia of the primary pulmonary primordium, the secondary bronchi and the adult bronchial epithelium retained immunoreactivity for E-cadherin and Ep-CAM with lateral immunostaining of cell membranes. In normal adult lungs, Ep-CAM was detected in type I and II alveolar epithelial cells, whereas E-cadherin was confined to the basolateral domain of type II cells. In pulmonary fibrosis, Ep-CAM could be further detected on the cell surface of epithelial remnants. In contrast, E-cadherin expression was characterized by a change of the membrane localization to a spotty, cytoplasmic pattern in the alveolar epithelium, possibly indicating functional inactivation of the protein during fibrogenesis.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ～80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ～50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.     

CTP:phosphocholine cytidylyltransferase alpha is a cytosolic protein in pulmonary epithelial cells and tissues

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in de novo synthesis of phosphatidylcholine (PC). The lung requires a steady synthesis of PC for lung surfactant of which disaturated PC is the essential active agent. Surfactant synthesis occurs in alveolar type II cells. Studies with non-pulmonary cells have suggested that CCT is both a nuclear and cytoplasmic protein. The unusual requirements of the lung for PC synthesis and, therefore, CCT activity suggest a unique mechanism of regulation and possibly localization of CCT. The localization of CCT alpha in lung epithelial cells and, of greater consequence, lung tissues are yet unknown. Three isoforms of CCT have been identified. Herein we investigated the localization of the ubiquitously expressed CCT alpha isoform. To ascertain CCT alpha localization in lungs and lung-related epithelial cells, we employed a number of localization methods. Immunogold electron microscopy using polyclonal antibodies raised to either the carboxyl terminus, catalytic domain, or amino terminus of CCT alpha localized CCT alpha mostly to the exterior plasma membrane or regions of the endoplasmic reticulum (ER) in both A549 and MLE-15 epithelial lung cell lines and primary cultures of fetal rat lung epithelial cells. In contrast to other studies, little or no nuclear labeling was observed. Indirect immunofluorescence of these cells with anti-CCT alpha antibodies resulted in a similar distribution. Indirect visualization of both hemagglutinin- and FLAG-tagged CCT alpha as well as direct visualization of enhanced green fluorescence protein-CCT alpha fusion protein corroborated a cytoplasmic localization of CCT alpha in pulmonary cells. Moreover, analysis of lung tissue from fetal and adult mouse by either immunogold electron microscopy or indirect immunofluorescence yielded a strong cytoplasmic CCT alpha signal with virtually no nuclear localization in epithelial cells lining the airways. The cytoplasmic localization of CCT alpha in type II cells was further substantiated with transgenic mice overexpressing FLAG-tagged CCT alpha using the lung-specific human surfactant protein C (SP-C) promoter. We conclude that CCT alpha does not localize to the nucleus in pulmonary tissues, and, therefore, nuclear localization of CCT alpha is not a universal event.     

Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung

Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung’s ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro–SP-B and pro–SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry.     

Developmental regulation of re-uptake of phosphatidylcholine by type II alveolar epithelium

Type II alveolar epithelia produce, store and secrete pulmonary surfactant, a phospholipid and protein mixture which stabilizes alveoli at low lung volumes and, thereby, prevents alveolar collapse. We determined the developmental changes in the uptake, metabolism and reutilization of surfactant-related phospholipid in primary cultures of type II cells derived from fetal rat lung. Primary cultures of fetal and neonatal type II cells were incubated in media containing labelled liposomes. After the incubation phospholipids were extracted from the cells and uptake of label was analyzed. Re-uptake of radiolabelled dipalmitoyl phosphatidylcholine (DPPC) was concentration-dependent in undifferentiated fetal cells, differentiated fetal cells and neonatal cells. Re-uptake of DPPC by undifferentiated fetal cells was lower than re-uptake by both differentiated fetal and neonatal cells at 15 and 75 μM PC. Binding of DPPC to the cell surface involved a protein interaction, since trypsin was able to dissociate this trypsin-releasable fraction from internalized label. Undifferentiated fetal, differentiated fetal and neonatal cells all exhibited approx. 50% metabolic degradation of internalized phospholipid. Degraded lipids were reutilized in the synthesis of phosphatidylglycerol, but neonatal cells resynthesized twice as much phosphatidylglycerol as did undifferentiated fetal cells. These are the first studies which show that morphologically undifferentiated fetal type II cells are capable of the uptake of surfactant phospholipid as well as the degradation and reutilization of internalized phospholipid. Re-uptake, degradation and reutilization of internalized phospholipid appear to be under developmental control.     

Impaired Surfactant Production by Alveolar Epithelial Cells in a SCID-hu Lung Mouse Model of Congenital Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.     

Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development

Antibodies directed against the major apoprotein associated with rabbit lung surfactant were used to characterize the induction and cellular localization of this protein during rabbit fetal lung development. In lung tissues from rabbits of 26 days gestational age and older, discrete epithelial type II cells were stained positively using the peroxidase antiperoxidase technique. The content of the major protein in homogenates of fetal lung tissue was analyzed using an immunoblotting technique. A protein of about 29 kDa, pI less than or equal to 5.6, was first detectable in fetal lung tissue on day 24 of gestation. The 29-36 kDa, mature form of the surfactant apoprotein was first detectable in lung homogenates from 30-day gestational age fetal rabbits. Treatment of homogenates of day 26 and 31 fetal lung tissues with endoglycosidase F, yielded, in both cases, an immunoreactive triplet with more neutral isoelectric points than the proteins in the untreated homogenates. By immunoblot analysis, we found that only the 29-36 kDa, mature form of the surfactant apoprotein was present in lamellar bodies purified from lung tissues of fetuses of 28 and 31 days and from day 2 neonates. These findings are suggestive that only the mature, 29-36 kDa form of the surfactant apoprotein is associated with lamellar bodies during fetal lung type II cell differentiation in vivo.     

Pepsinogen C: a type 2 cell-specific protease

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.     

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1alpha and -beta isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1alpha localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1alpha is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.     

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.     

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung‐targeted VEGF gene inactivation, and alterations in VEGF‐dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF‐deficient lungs increased PAO1 levels and pro‐inflammatory cytokines, TNFα and IL‐6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung‐targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP‐D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)‐B and ‐D, and the lipid transporters, ABCA1 and Rab3D. TPA‐induced DSPC secretion and apoptosis were elevated in VEGF‐deficient type II cells. These results suggest a potential protective role for type II cell‐expressed VEGF against bacterial initiated infection. J. Cell. Physiol. 228: 371–379, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Synthesis of surfactant-associated protein, 35,000 daltons, in fetal lung

The major human pulmonary surfactant-associated protein of 35,000 daltons (Da) (SAP-35), consists of a group of related proteins of 27,000-36,000 Da, with isoelectric points ranging from pH 4.6 to 5.2. SAP-35 precursors were identified by immunoprecipitation of protein products of in vitro translation of normal adult human poly(A)+ mRNA with human SAP-35 antiserum. The translation products nearly comigrated with the most basic components of alveolar SAP-35 (mol mass = 24,500-27,000 Da). Processing of the primary translation products by canine pancreatic microsomal membranes increased their apparent molecular weight to 29,000-30,000-Da forms, which were sensitive to endoglycosidase F, suggesting the addition of asparagine-linked oligosaccharides to the molecules. A smaller protein of 24,500 Da was generated during treatment with canine microsomal membranes likely representing cleavage of a signal peptide. SAP-35 was not detected in explants of [35S]methionine-labeled fetal lung (20-24 wk gestation) after 1 day of culture or immunoprecipitates of in vitro translated poly(A)+ mRNA isolated from fetal human lung. However, after 3-5 days of organ culture, synthesis of SAP-35 was readily detected by immunoprecipitation of [35S] methionine-labeled tissue. Fully sialylated (neuraminidase-sensitive forms) comigrated with fully glycosylated SAP-35 isolated from human surfactant. High mannose (endoglycosidase H-sensitive precursors) were also synthesized by the organ cultures and were distinct from the secreted form in surfactant. Synthesis of surfactant-associated SAP-35 and its precursors was induced in association with morphological maturation of the type II epithelial cell during organ culture of human fetal lung.     

cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal)

In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of Mr = 5,000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. We have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNA encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated an Mr = 22,000 precursor protein, the active hydrophobic peptide being produced by proteolytic processing to Mr = 5,000-6,000. Two classes of cDNAs encoding SPL(pVal) were identified. mRNA of approximately 900 bases was identified on Northern analysis of fetal and adult RNA. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.