Alveolar Epithelial Type I Research Articles

The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment

Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (≙ stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (≙ stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.

Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31-CD45-tdTomatoSPC-T1α+ for ATI and EpCAM+CD31-CD45-tdTomatoSPC+T1α- for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

TRPV4 channels are essential for alveolar epithelial barrier function as protection from lung edema

Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4–/– ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.

Mechanical stretch activates piezo1 in caveolae of alveolar type I cells to trigger ATP release and paracrine stimulation of surfactant secretion from alveolar type II cells.

Secretion of pulmonary surfactant in the alveoli of the lungs is essential to maintain lung function. Stretching of alveoli during lung inflation is the main trigger for surfactant secretion. Yet, the molecular mechanisms how mechanical distension of alveoli results in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar epithelial type I (ATI) and surfactant secreting type II (ATII) cells. ATI, but not ATII cells, express caveolae, small plasma membrane invaginations that can respond to plasma membrane stresses and serve mechanotransductive roles. Within this study, we investigated the role of caveolae as mechanosensors in the alveolus. We generated a human caveolin-1 knockout ATI cell (hAELVicav-/- ) using CRISPR/Cas9. Wildtype (hAELViwt ) and hAELVicav-/- cells grown on flexible membranes responded to increasing stretch amplitudes with rises in intracellular Ca2+ . The response was less frequent and started at higher stretch amplitudes in hAELVicav-/- cells. Stretch-induced Ca2+ -signals depended on Ca2+ -entry via piezo1 channels, localized within caveolae in hAELViwt and primary ATI cells. Ca2+ -entry via piezo1 activated pannexin-1 hemichannels resulting in ATP release from ATI cells. ATP release was reduced in hAELVicav-/- cells. In co-cultures resembling the alveolar epithelium, released ATP stimulated Ca2+ signals and surfactant secretion from neighboring ATII cells when co-cultured with hAELViwt but not hAELVicav-/- cells. In summary, we propose that caveolae in ATI cells are mechanosensors within alveoli regulating stretch-induced surfactant secretion from ATII cells.

Identification of an alveolar type I epithelial cell-specific DNA nuclear import sequence for gene delivery.

The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, especially in the lung. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. We demonstrate, through microinjection of fluorescently labeled plasmids, that a DNA sequence within the rat T1α promoter was able to mediate ATI cell-specific plasmid DNA nuclear import due to the binding of ATI-enriched transcription factors. Promoter deletion analysis and site-directed mutagenesis of specific transcription-factor-binding sites within the +101 to -200 bp region of the T1α promoter identified HNF3 and TTF-1 as critical transcription factors for import. To test for nuclear import in vivo, plasmids expressing GFP from the CMV promoter were delivered into the lungs of mice by electroporation and evaluated immunohistochemically 48 h later. Plasmids carrying the 1.3 kbp T1α sequence resulted in GFP expression almost exclusively in ATI cells. This represents a new and highly efficient way to target a specific lung epithelial cell type both in vitro and in vivo based on the restriction of DNA nuclear import.

Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

Enolase 1 and protein disulfide isomerase associated 3 regulate Wnt/β-catenin driven alveolar epithelial cell trans-differentiation

ABSTRACTThe alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling

Alveolar epithelial type II (ATII) cells are essential for maintaining normal lung homeostasis because they produce surfactant, express innate immune proteins, and can function as progenitors for alveolar epithelial type I (ATI) cells. Although autocrine production of transforming growth factor (TGF)-β1 has been shown to promote the transdifferentiation of primary rat ATII to ATI cells in vitro, mechanisms controlling this process still remain poorly defined. Here, evidence is provided that Tgf-β1, -2, -3 mRNA and phosphorylated SMAD2 and SMAD3 significantly increase as primary cultures of mouse ATII cells transdifferentiate to ATI cells. Concomitantly, bone morphogenetic protein (Bmp)-2 and -4 mRNA, and phosphorylated SMAD1/5/8 expression decrease. Exogenously supplied recombinant human TGF-β1 inhibited BMP signaling and enhanced transdifferentiation by promoting the loss of ATII cell-specific gene expression and weakly stimulating ATI cell-specific gene expression. On the other hand, exogenously supplied recombinant human BMP-4 inhibited TGF-β signaling and delayed transdifferentiation by inhibiting the gain in ATI cell-specific gene expression and weakly delaying the loss of ATII cell-specific gene expression. In mouse lung epithelial (MLE15) cells, small-interfering RNA (siRNA) knockdown of TGF-β receptor type-1 enhanced basal expression of ATII genes while siRNA RNA knockdown of BMP receptors type-1a and -1b enhanced basal expression of ATI genes. Together, these results suggest that the rate of ATII cell transdifferentiation is controlled by the opposing actions of BMP and TGF-β signaling that switch during the process of transdifferentiation.

Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model

BackgroundOur aim was to differentiate human (h) embryonic stem (ES) cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI) and type II (AEII) cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF). A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB) differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.Methodology and Principal FindingsOmission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP) transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC−/− mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.ConclusionsThis study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.

Effects of atorvastatin on smoking-induced alveolar injury in rat lungs

Background: Smoking is one of the most serious health care issues worldwide, as one third to one half of all people who smoke eventually use tobacco habitually. Chronic smoke exposure causes airway and lung parenchymal inflammation and the destruction of alveolar cell walls. Statins may have anti-inflammatory effects that would play a role in preventing the cellular damage associated with smoking. Objective: The aim of this study was to investigate whether atorvastatin protects against smoking-induced inflammation in alveolar epithelial type I (ATI) and type II (ATII) cells in the lungs of rats. Methods: Adult male albino Wistar rats (200–250 g) were randomly divided into 3 groups and exposed to cigarette smoke 8 hours per day for 15 days. During that 15-day period, the 2 treatment groups received atorvastatin 0.5 or 1.0 mg/kg/d in 2 mL of methyl cellulose solution and the control group received 2 mL of methyl cellulose solution alone, all via nasogastric catheter. After the 15 days, the lungs were excised and the tissues were examined by transmission electron microscopy. Results: Thirty rats were divided into 3 groups of 10 rats each. All rats survived the 15 days. In the atorvastatin 0.5-mg group, no changes were found in the ATI cells or in the blood-air barrier. In the atorvastatin 1.0-mg group, we observed hyperplasia in the common basal membranes. Hypertrophy, mitochondrial crystolysis (MC), and intracytoplasmic edema (ICE) were detected in the ATI cells in the 1.0-mg group, while chromatin condensation, atrophic appearance, cell shrinkage, and cyto-plasmic vacuolization were observed in the ATII cells. The rough endoplasmic reticulum (rER) tubules of the ATII cells appeared spiral-shaped. In the control group, minimal ICE was detected in the ATI cells. However, microvillus deformation, pseu-dopod formation, edema, mitochondrial swelling, and MC were observed in the ATII cells. We also observed MC, several pinocytic vesicles, and normal rER tubules in the endothelial cells of the control group. Conclusions: The administration of atorvastatin 0.5 mg/kg/d was associated with some attenuation of lung injury caused by smoke inhalation in these rat lungs. However, atorvastatin 1.0 mg/kg/d was associated with lung damage. Future studies are needed to evaluate the dose-response relationship of atorvastatin to smoking-induced alveolar damage.

Implication of receptor for advanced glycation end product (RAGE) in pulmonary health and pathophysiology

Receptor for advanced glycation end products (RAGE) is a membrane bound receptor and member of the immunoglobulin super family and is normally present in a highly abundant basal level expression in lung. This high expression of RAGE in lung alveolar epithelial type I (ATI) cells is presumably involved in the proliferation and differentiation of pulmonary epithelial cells. However, typically higher than basal level expression of RAGE may indicate the existence of severe pathophysiological condition in lung, e.g. acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). During pulmonary tissue injury an endogenous secretory isoform of RAGE called EsRAGE is noticed at high levels in broncho-alveolar lavage (BAL) and plasma. Recently, a soluble form of RAGE (sRAGE) produced by recombinant gene technology was shown to exhibit a therapeutic potential in experimental animal models. Detailed study of RAGE in the pulmonary tissues will facilitate the understanding of the importance of RAGE signaling in the pulmonary health and pathophysiology.

Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection

Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis.

Alveolar Epithelial Ion and Fluid Transport

Studies of epithelial ion and fluid transport across the distal pulmonary epithelia have provided important new concepts regarding the resolution of pulmonary edema, specifically the removal of edema from the distal airspaces of the lung. Overall, there is convincing evidence that vectorial ion transport across the alveolar and distal airway epithelia is the primary determinant of alveolar fluid clearance (AFC). The general paradigm is that active Na and Cl transport drives net alveolar fluid clearance, as demonstrated in several different species, including the human lung. The objective of this article is to consider some areas of recent progress in the field of alveolar fluid transport under normal and pathologic conditions. More detailed reviews of this field including studies of the immature and the newborn lung are available (1–10). In the lung, as in other epithelia, ion transporters and other membrane proteins are asymmetrically distributed on opposing cell surfaces, conferring vectorial transport properties to the polarized epithelial cells (Figure 1). There are epithelial cells in the distal airway epithelia, such as Clara cells, that are capable of vectorial ion transport. The vast majority of the surface area available for transport in the distal lung is occupied by the alveolar epithelial type I (ATI) and type II cells (ATII). Tight junctions populate these epithelial cells near their apical surfaces, thereby sustaining apical and basolateral cell polarity (11). The permeability of tight junctions is dynamic and regulated, in part, by cytoskeletal proteins and intracellular Ca concentrations and possibly by ion channels (11). Figure 1. A section of an ARDS lung. Note the fluid-filled alveolar spaces with significant red blood cell infiltration. Insert demonstrates apical ENaC staining and basolateral Na,K-ATPase staining in lung epithelia. (Reprinted with permission from Ref. 3) Because ATII cells can be isolated from the lung and studied in vitro, they have been studied extensively. ATII cells are responsible for surfactant secretion (12) as well as vectorial Na and Cl transport. Na uptake occurs on the apical surface, partly through amiloride-sensitive and amiloride-insensitive channels. Subsequently, Na is pumped actively from the basolateral surface into the lung interstitium by the Na,K-ATPase. An epithelial Na channel (ENaC) participating in Na movement across the apical cell membrane has been cloned and well characterized (13, 14). Recent evidence indicates that the CFTR is expressed in ATII cells and plays a role in cAMP-mediated fluid transport (15–18). The role of ATI cells for AFC is less known, although several studies have established a possible contribution of ATI cells to vectorial fluid transport (19–22). ATI cells express aquaporin 5 (23), Na,K-ATPase (21), and ENaC (21, 22). The presence of Na,K-ATPase is consistent with a role for ATI cells in AFC, but is not conclusive since Na,K-ATPases are needed to maintain cell volume. However, one study using pharmacologic methods to inhibit the α2-subunit of the Na,K-ATPase suggested a role for Na,K-ATPase in driving fluid clearance across type I cells in vivo (24). Another study reported a role for the α2-Na,K-ATPase under cAMP-stimulated conditions, suggesting that type I cells may be involved, as the α2-subunit seems to be expressed only in type I cells (25). Detailed studies of type I cells have been limited to date because of difficulty maintaining them in cell culture although recent work has demonstrated functional ion channels in freshly isolated ATI cells with electrophysiologic evidence for Na channels (ENaC), K channels, and CFTR Cl channels (26). In addition, there is evidence for ENaC expression (21, 22) and a partial amiloride inhibition of 22Na-uptake in freshly isolated rat ATI cells (22). Evidence for β-adrenoceptor (βAR) expression in ATI cells has also been reported (20, 27). In addition, ATI cells may be involved in macromolecular transport due to the presence of vesicles and caveolin (28). The distal airway epithelium also actively transports Na (29–31).

Caveolin-1 Expression and Caveolae Biogenesis during Cell Transdifferentiation in Lung Alveolar Epithelial Primary Cultures

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-“like” phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.

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.