Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is expressed in the epithelium of various primate tissues, including lung airway and alveoli. In human lung, CEACAM6 is developmentally and hormonally regulated, protects surfactant function, has anti-apoptotic activity and is dysregulated in cancers. We hypothesized that alveolar CEACAM6 expression increases in lung injury and promotes cell proliferation during repair. Studies were performed in CEABAC transgenic mice-containing human CEACAM genes. The level of CEACAM6 in adult CEABAC lung was comparable to that in human infants; expression occurred in epithelium of airways and of some alveoli but rarely co-localized with markers of type I or type II cells. Ten days after bleomycin instillation, both the number of CEACAM6(+) cells and immunostaining intensity were elevated in injured lung areas, and there was increased co-localization with type I and II cell markers. To specifically address type II cells, we crossed CEABAC mice with animals expressing EGFP driven by the SP-C promoter. After bleomycin injury, partially flattened, elongated epithelial cells were observed that expressed type I cell markers and were primarily either EGFP(+) or CEACAM6(+). In cell cycle studies, mitosis was greater in CEACAM6(+) non-type II cells versus CEACAM6(+)/EGFP(+) cells. CEACAM6 epithelial expression was also increased after hyperoxic exposure and LPS instillation, suggesting a generalized response to acute lung injuries. We conclude that CEACAM6 expression is comparable in human lung and the CEABAC mouse. CEACAM6 in this model appears to be a marker of a progenitor cell population that contributes to alveolar epithelial cell replenishment after lung injury.

High-efficiency type II cell-enhanced green fluorescent protein expression facilitates cellular identification, tracking, and isolation.

We have developed a transgenic mouse expressing enhanced green fluorescent protein (EGFP) in virtually all type II (TII) alveolar epithelial cells. The CBG mouse (SPC-BAC-EGFP) contains a bacterial artificial chromosome modified to express EGFP within the mouse surfactant protein (SP)-C gene 3' untranslated region. EGFP mRNA expression is limited to the lung. EGFP fluorescence is both limited to and exhibited by all cells expressing pro-SP-C; fluorescence is uniform throughout all lobes of the lung and does not change as mice age. EGFP(+) cells also express SP-B but do not express podoplanin, a type I (TI) cell marker. CBG mice show no evidence of lung disease with aging. In 3 hours, TII cells can be isolated in >99% purity from CBG mice by FACS; the yield of 3.7 ± 0.6 × 10(6) cells represents approximately 25 to 60% of the TII cells in the lung. By FACS analysis, approximately 0.9% of TII cells are in mitosis in uninjured lungs; after bleomycin injury, 4.1% are in mitosis. Because EGFP fluorescence can be detected for >14 days in culture, at a time that SP-C mRNA expression is essentially nil, this line may be useful for tracking TII cells in culture and in vivo. When CBG mice are crossed to transgenic mice expressing rat podoplanin, TI and TII cells can be easily simultaneously identified and isolated. When bred to other strains of mice, EGFP expression can be used to identify TII cells without the need for immunostaining for SP-C. These mice should be useful in models of mouse pulmonary disease and in studies of TII cell biology, biochemistry, and genetics.

Isolation and culture of alveolar epithelial Type I and Type II cells from rat lungs.

The pulmonary alveolar epithelium, comprised of alveolar Type I (TI) and Type II (TII) cells, covers more than 99% of the internal surface area of the lungs. The study of isolated and cultured alveolar epithelial TI and TII cells has provided a large amount of information about the functions of both cell types. This chapter provides information about methods for isolating and culturing both of these cell types from rat lungs.

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.

HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells.

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors.

Rat alveolar type I cells proliferate, express OCT-4, and exhibit phenotypic plasticity in vitro.

Alveolar type I (TI) cells are large, squamous cells that cover 95-99% of the internal surface area of the lung. Although TI cells are believed to be terminally differentiated, incapable of either proliferation or phenotypic plasticity, TI cells in vitro both proliferate and express phenotypic markers of other differentiated cell types. Rat TI cells isolated in purities of >99% proliferate in culture, with a sixfold increase in cell number before the cells reach confluence; >50% of the cultured TI cells are Ki67+. At cell densities of 1-2 cells/well, approximately 50% of the cells had the capacity to form colonies. Under the same conditions, type II cells do not proliferate. Cultured TI cells express RTI40 and aquaporin 5, phenotypic markers of the TI cell phenotype. By immunofluorescence, Western blotting, and Q-PCR, TI cells express OCT-4A (POU5F1), a transcription factor associated with maintenance of the pluripotent state in stem cells. Based on the expression patterns of various marker proteins, TI cells are distinct from either of two recently described putative pulmonary multipotent cell populations, the bronchoalveolar stem cell or the OCT-4+ stem/progenitor cell. Although TI cells in adult rat lung tissue do not express either surfactant protein C (SP-C) or CC10, respective markers of the TII and Clara cell phenotypes, in culture TI cells can be induced to express both SP-C and CC10. Together, the findings that TI cells proliferate and exhibit phenotypic plasticity in vitro raise the possibility that TI cells may have similar properties in vivo.

Directed expression of transgenes to alveolar type I cells in the mouse.

Podoplanin (RTI40, aggrus, T1alpha, hT1alpha-2, E11, PA2.26, RANDAM-2, gp36, gp38, gp40, OTS8) is a type I cell marker in rat lung. We show that a bacterial artificial chromosome vector containing the rat podoplanin gene (RTIbac) delivers a pattern of transgene expression in lung that is more restricted to mouse type I cells than that of the endogenous mouse podoplanin gene. RTIbac-transgenic mice expressed rat podoplanin in type I cells; type II cells, airways, and vascular endothelium were negative. A modified bacterial artificial chromosome containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) sequences in the podoplanin 3'UTR expressed rat podoplanin and transgenic GFP in type I cells. RTIbac transgene expression was absent or reduced in pulmonary pleura, lymphatic endothelium, and putative lymphoid-associated stromal tissue, all of which contained abundant mouse podoplanin. Rat podoplanin mRNA levels in normal rat lung and RTIbac transgenic lung were 25-fold higher than in corresponding kidney and brain samples. On Western blots, transgenic rat and endogenous mouse podoplanin displayed very similar patterns of protein expression in various organs. Highest protein levels were observed in lung with 10- to 20-fold less in brain; there were low levels in thymus and kidney. Both GFP and rat podoplanin transgenes were expressed at extrapulmonary sites of endogenous mouse podoplanin gene expression, including choroid plexus, eye ciliary epithelium, and renal glomerulus. Because their pulmonary expression is more restricted than endogenous mouse podoplanin, RTIbac derivatives should be useful for mouse type I cell-specific transgene delivery.

Nitric oxide decreases surfactant protein gene expression in primary cultures of type II pneumocytes.

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator effective in treating persistent pulmonary hypertension in newborns and in infants following congenital heart disease surgery. Recently, multiple in vivo and in vitro studies have shown a negative effect of NO on surfactant activity as well as surfactant protein gene expression. Although the relationship between NO and surfactant has been studied previously, the data has been hard to interpret due to the model systems used. The objective of the current study was to characterize the effect of NO on surfactant protein gene expression in primary rat type II pneumocytes cultured on a substratum that promoted the maintenance of type II cell phenotype. Exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), decreased surfactant protein (SP)-A, (SP)-B, and (SP)-C mRNA levels in type II pneumocytes in a time- and dose-dependent manner. The effect was mediated in part by an increase in endothelin-1 secretion and a decrease in the intracellular messenger, phosphorylated ERK1/2 mitogen-activated protein kinases (MAPK). Exposing type II pneumocytes to endothelin-1 receptor antagonists PD-156707 or bosentan before exposure to SNAP partially prevented the decrease in surfactant protein gene expression. The results showed that NO mediated the decrease in surfactant protein gene expression at least in part through an increase in endothelin-1 secretion and a decrease in phosphorylated ERK1/2 MAPKs.