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.

Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

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.

Regulated gene expression in cultured type II cells of adult human lung.

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

The great big alveolar TI cell: evolving concepts and paradigms.

Pulmonary alveolar type I cells (TI cell) are very large (approximately 5400 microm(2) in surface area) squamous cells that cover more than 98% of the internal surface area of rodent lungs. In the past, TI cells were believed to serve only passive barrier functions, with no active functional properties in the lung. The fairly recent development of methods to isolate TI cells has permitted investigation of functions of this cell type for the first time. Resolvable by electron microscopy, TI cells contain microvilli and organelles typically associated with metabolic functions, such as mitochondria, abundant smooth and rough endoplasmic reticulum and Golgi apparatus. TI cells contain the molecular machinery necessary for ion transport and take up Na(+), K(+), and Cl(-), from which one can infer that it is likely that they play a role in ion and fluid transport in vivo. Because the abundance/microm(2) of highly selective Na(+) channels (HSC channels, consisting of all three ENaC subunits) is the same in TI and TII cells and because TI cells cover the majority of the lung internal surface, TI cells may play the major role in bulk transport of Na(+). In vitro, TI cells can proliferate and exhibit phenotypic plasticity, raising the question of whether this cell type may play a role in development and lung repair after injury. From gene expression analysis of TI cells, one can infer a variety of other possible functions for TI cells. The development of techniques to administer transgenes specifically to TI cells will permit direct study of this cell type in vivo.

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.

Characteristics of Cl- uptake in rat alveolar type I cells.

Although Cl- transport in fetal lung is important for fluid secretion and normal lung development, the role of Cl- transport in adult lung is not well understood. In physiological studies, the cystic fibrosis transmembrane regulator (CFTR) plays a role in fluid absorption in the distal air spaces of adult lung, and alveolar type II cells cultured for 5 days have the capacity to transport Cl-. Although both alveolar type I and type II cells express CFTR, it has previously not been known whether type I cells transport Cl-. We studied Cl- uptake in isolated type I cells directly, using either radioisotopic tracers or halide-sensitive fluorescent indicators. By both methods, type I cells take up Cl-. In the presence of beta-adrenergic agonist stimulation, Cl- uptake can be inhibited by CFTR antagonists. Type I cells express both the Cl-/HCO3- anion exchanger AE2 and the voltage-gated Cl- channels CLC5 and CLC2. Inhibitors of AE2 also block Cl- uptake in type I cells. Together, these results demonstrate that type I cells are capable of Cl- uptake and suggest that the effects seen in whole lung studies establishing the importance of Cl- movement in alveolar fluid clearance may be, in part, the result of Cl- transport across type I cells.

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.

Surfactant proteins A and D enhance pulmonary clearance of Pseudomonas aeruginosa.

Surfactant protein (SP)-A and SP-D, members of the collectin family, are involved in innate host defenses against various bacterial and viral pathogens. In this study, we asked whether SP-A and SP-D enhance clearance of a nonmucoid strain of Pseudomonas aeruginosa from the lungs. We infected mice deficient in SP-A (SP-A-/-), SP-D (SP-D-/-) and both pulmonary collectins (SP-AD-/-) by intratracheal administration of P. aeruginosa. Six hours after infection, bacterial counts were significantly higher in SP-A-/-, SP-D-/-, and SP-AD-/- compared with wild-type (WT) mice. Forty-eight hours after infection, bacterial counts were significantly higher in SP-A-/- mice compared with WT mice and in SP-AD-/- mice compared with WT, SP-A-/-, and SP-D-/- mice. Phagocytosis of the bacteria by alveolar macrophages was decreased in SP-A-/- and SP-D-/- mice. Levels of macrophage inflammatory peptide-2 and IL-6 were more elevated in the lungs of SP-D and SP-AD-/- mice compared with WT mice. There was more infiltration by neutrophils in the lungs of SP-D-/- compared with WT and SP-A-/- mice 48 h after infection. This study shows that SP-A and SP-D enhance pulmonary clearance of P. aeruginosa by stimulating phagocytosis by alveolar macrophages and by modulating the inflammatory response in the lungs. These findings also show that the functions of SP-A and SP-D are not completely redundant in vivo.

Polyethylene glycol-surfactant for lavage lung injury in rats.

Addition of ionic and nonionic water-soluble polymers to pulmonary surfactants in the presence of inactivating substances prevents surfactant inactivation in vitro and improves lung function in several models of lung injury. However, a recent report found opposite effects when surfactant plus polyethylene glycol (PEG) was used to treat lung injury caused by saline lung lavage. Therefore, we examined the reasons why the polymer effect is less evident in the saline lung lavage lung injury model. We treated rats with lavage lung injury with a commercial lung surfactant extract derived from bovine lung (Survanta) with or without addition of PEG. Groups treated with Survanta + PEG had significantly higher static post mortem lung volumes than groups treated with Survanta. However, groups treated with Survanta + PEG had more tracheal fluid and no significant benefit in arterial oxygenation compared with the group treated with Survanta, despite our use of measures to reduce pulmonary edema. Measurements after intravascular injections of (125)I-labeled albumin confirmed that addition of PEG increased extravascular lung water and that this effect is mitigated by furosemide. We conclude that surfactant + PEG mixtures are less effective in lavage injury than in other forms of lung injury because of increased extravascular lung water.

Design-based stereological analysis of the lung parenchymal architecture and alveolar type II cells in surfactant protein A and D double deficient mice.

Alveolar epithelial type II cells synthesize and secrete surfactant. The surfactant-associated proteins A and D (SP-A and SP-D), members of the collectin protein family, participate in pulmonary immune defense, modulation of inflammation, and surfactant metabolism. Both proteins are known to have overlapping as well as distinct functions. The present study provides a design-based stereological analysis of adult mice deficient in both SP-A and SP-D (A(-)D(-)) with special emphasis on parameters characterizing alveolar architecture and surfactant-producing type II cells. Compared to wild-type, A(-)D(-) mice have fewer and larger alveoli, an increase in the number and size of type II cells, as well as more numerous and larger alveolar macrophages. More surfactant-storing lamellar bodies are seen in type II cells, leading to a threefold increase in the total volume of lamellar bodies per lung, but the mean volume of a single lamellar body remains constant. These results demonstrate that chronic deficiency of SP-A and SP-D in mice leads to parenchymal remodeling, type II cell hyperplasia and hypertrophy, and disturbed intracellular surfactant metabolism. The design-based stereological approach presented here provides a framework for the quantitative lung structure analysis in gene-manipulated mice as well as in human lung disease.

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes.

We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice.

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.

Pulmonary collectins modulate strain-specific influenza a virus infection and host responses.

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A(-/-) mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.

Identification of genes differentially expressed in rat alveolar type I cells.

Although approximately 98% of the internal surface area of the lung is lined by alveolar type I cells, little is known about the functions of this cell type. Using freshly isolated rat type I and type II cells, we created a subtraction library by suppression subtractive hybridization to identify genes differentially expressed by type I cells. We identified twelve genes of known function that are differentially expressed by type I cells. Differential expression of all 12 genes was confirmed by Northern blotting; we confirmed differential expression by immunocytochemistry for 3 genes for which suitable antibodies were available. Most of the genes code for proteins that are multifunctional. From the known functions of these genes, we infer that type I cells may play a role in the maintenance of normal alveolar homeostasis and protection from injury, lung development and remodeling, host defense, tumor/growth suppression, and surfactant metabolism, among other functions.

CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury.

The proinflammatory CXC chemokines GRO, CINC-2alpha, and macrophage inflammatory protein (MIP)-2 are a closely related family of neutrophil chemoattractants. Here, we report that freshly isolated alveolar Type II (TII) cells express these chemokine mRNAs at much higher levels than do freshly isolated Type I cells or alveolar macrophages (AM). TII cells also express CXCR2, the receptor for these chemokines. Lung injury caused by acid or Pseudomonas aeruginosa (Pa) caused an increase in TII cell expression of chemokine mRNAs and GRO protein. We compared the time courses of chemokine mRNA expression in cultured TII cells and AM. In TII cells, GRO mRNA levels were stable over 4 h, but decreased to undetectable levels by 24 h. CINC-2alpha and MIP-2 mRNA levels were low in freshly isolated cells, increased over 2-4 h in culture, and by 24 h dropped to undetectable levels. In contrast, none of these chemokine mRNAs were detected in freshly isolated AM, but expression was induced by tissue culture. In summary, we have shown that TII alveolar epithelial cells produce three of the major proinflammatory CXC chemokines (GRO, CINC-2alpha, and MIP-2) and their cognate receptor CXCR2. Chemokine expression is upregulated in response to lung injury. These observations support a central role for the TII cell as an immunologic effector cell in the alveolus and raise intriguing questions about how CXC chemokines and receptors modulate diverse normal and pathologic cellular responses in the alveoli.

Sequential targeted deficiency of SP-A and -D leads to progressive alveolar lipoproteinosis and emphysema.

Surfactant proteins-A and -D (SP-A and SP-D) are members of the collectin protein family. Mice singly deficient in SP-A and SP-D have distinct phenotypes. Both have altered inflammatory responses to microbial challenges. To further investigate the functions of SP-A and SP-D in vivo, we developed mice deficient in both proteins by sequentially targeting the closely linked genes in embryonic stem cells using graded resistance to G-418. There is a progressive increase in bronchoalveolar lavage phospholipid, protein, and macrophage content through 24 wk of age. The macrophages from doubly deficient mice express high levels of the matrix metalloproteinase MMP-12 and develop intense but patchy lung inflammation. Stereological analysis demonstrates significant air space enlargement and reduction in alveolar septal tissue per unit volume, consistent with emphysema. These changes qualitatively resemble the lung pathology seen in SP-D-deficient mice. These doubly deficient mice will be useful in dissecting the potential overlap in function between SP-A and SP-D in host defense.

Tissue distribution of surfactant proteins A and D in the mouse.

Surfactant proteins A and D, collagen-like lectins (collectins), were first isolated from the lung. In the lung, SP-A and SP-D have roles in surfactant homeostasis and innate immunity. In this study we show that SP-A and SP-D mRNA can be detected in a significant number of non-pulmonary tissues but the proteins have a more limited distribution. SP-D protein was detected in lung, uterus, ovary, and lacrimal gland, whereas SP-A protein was detected only in the lung. The results suggest that SP-D participates in mucosal immunity throughout the body.

Surfactant protein-A--deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza A virus.

Surfactant protein (SP)-A is a member of the collectin family of proteins. In vitro, SP-A binds influenza A virus (IAV), neutralizes infectivity, and enhances uptake by macrophages. SP-D also binds and neutralizes certain strains of IAV. To determine if SP-A has a role in protecting the intact animal against IAV infection, we inoculated gene-targeted SP-A-deficient mice (-/-) and littermate controls (+/+) with either saline or increasing doses of an IAV strain that binds SP-A but not SP-D. IAV was more virulent in SP-A-/- compared with +/+ mice, with a significantly lower mean lethal dose (LD(50)) and significantly greater weight loss during infection. SP-A-/- mice also had increased airway epithelial injury and more alveolar cellular infiltrates than +/+ mice. On Day 2, SP-A-/- mice had more neutrophils and higher MIP-2 levels in the lung than +/+ mice. We conclude the altered host response and increased susceptibility to X-79Delta167 infection in SP-A-/- mice reflects a protective role for SP-A in regulating the host response to IAV. Because the recovery of virus from lung homogenates on Days 2 and 6 after inoculation was comparable in -/- and +/+ mice, we speculate SP-A reduces IAV virulence independently of direct viral neutralization.

Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis.

Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells <5%. Fluid transport is regulated by ion transport, with water movement following passively. Current concepts are that TII cells are the main sites of ion transport in the lung. TI cells have been thought to provide only passive barrier, rather than active, functions. Because TI cells line most of the internal surface area of the lung, we hypothesized that TI cells could be important in the regulation of lung liquid homeostasis. We measured both Na(+) and K(+) (Rb(+)) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na(+) in an amiloride-inhibitable fashion, suggesting the presence of Na(+) channels; TI cell Na(+) uptake, per microgram of protein, is approximately 2.5 times that of TII cells. Rb(+) uptake in TI cells was approximately 3 times that in TII cells and was inhibited by 10(-4) M ouabain, the latter observation suggesting that TI cells exhibit Na(+)-, K(+)-ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (alpha, beta, and gamma) of the epithelial sodium channel ENaC and two subunits of Na(+)-, K(+)-ATPase. By Western blot analysis, TI cells contain approximately 3 times the amount of alphaENaC/microg protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.