Isolated rat alveolar type II cells protrude intracellular lamellar bodies by forming bubble-like structures during surfactant secretion

Pulmonary surfactant is synthesized and secreted by pulmonary alveolar type II epithelial cells (type II cells). It passes through the alveolar lining fluid and adsorbs to the air-liquid interface. The process from secretion to adsorption is not yet entirely understood. To acquire a detailed understanding of this process, we used multiple observations of type II cells isolated from rat lungs under electron microscopy (EM) and confocal laser scanning microscopy (CLSM). Transmission EM observation demonstrated a loosening process of the intracellular lamellar bodies from the inside to the outside of the cell. Scanning EM observation revealed bubble-like protrusions from the cell surface, and differential interference contrast microscopy illustrated the protrusions expanding with time. CLSM observation with FM 1–43, a fluorescent membrane probe, revealed that the bubble-like protrusions were composed of phospholipids. Thus, we have demonstrated that isolated rat type II cells protrude intracellular lamellar bodies by forming bubble-like structures, possibly enabling them to adsorb to the air-liquid interface directly. These observations suggest a new mechanism for surfactant secretion from type II cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells

Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monoclonal, LK2H10) and chromogranins A and B (polyclonal, LKZM1U). The chromogranins or chromogranin-like proteins were identified in cells in lung tissue sections and isolated Type II cells at the light and electron microscopic levels. We used post-embedding immunoelectron microscopy, with immunogold, to detect the proteins' immunoreactivity in osmicated tissues. Gold particles were distributed over the phospholipid lamellae within the lamellar bodies of alveolar Type II cells and over the lattice structure of tubular myelin. Quantitative analysis of gold labeling densities in the various cell compartments indicated that only the latter two structures were specifically labeled. Controls, which included pre-absorption of both anti-chromogranin antibodies with excess chromogranin A or with native surfactant, resulted in a greater than 60% decrease in gold labeling. A possible role of chromogranins or chromogranin-like proteins as Ca2+ binding proteins in alveolar Type II cells is discussed.     

A quantitative study of the development of type II pneumocytes in fetal lung

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II pneumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine-3R and by their intensity of low-angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2-d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low-angle light scatter and phosphine-3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24-d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P less than .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.     

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-alpha (CCT-alpha), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-alpha. Energy-filtering EM demonstrated that CCT-alpha bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.     

Binding and uptake of surfactant protein D by freshly isolated rat alveolar type II cells

Alveolar type II cells secrete, internalize, and recycle pulmonary surfactant, a lipid and protein complex that increases alveolar compliance and participates in pulmonary host defense. Surfactant protein (SP) D, a collagenous C-type lectin, has recently been described as a modulator of surfactant homeostasis. Mice lacking SP-D accumulate surfactant in their alveoli and type II cell lamellar bodies, organelles adapted for recycling and secretion of surfactant. The goal of current study was to characterize the interaction of SP-D with rat type II cells. Type II cells bound SP-D in a concentration-, time-, temperature-, and calcium-dependent manner. However, SP-D binding did not alter type II cell surfactant lipid uptake. Type II cells internalized SP-D into lamellar bodies and degraded a fraction of the SP-D pool. Our results also indicated that SP-D binding sites on type II cells may differ from those on alveolar macrophages. We conclude that, in vitro, type II cells bind and recycle SP-D to lamellar bodies, but SP-D may not directly modulate surfactant uptake by type II cells.     

Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues.

Immunoreactive surfactant protein-D (SP-D) was assessed in human fetal, newborn, and adult tissues. In the fetal lung, SP-D was detected on airway surfaces by 10 weeks' gestation, staining increasing in the distal airways, decreasing in the proximal conducting airways with advancing gestation. In lungs from near-term infants and adults, SP-D was detected in Type II cells, serous cells of tracheobronchial glands, and subsets of cells lining peripheral airways. Immunostaining was decreased or absent in areas of lungs of neonates after injury to Type II cells, infection, or hemorrhage and was decreased in collapsed or unseptated airways from older infants with bronchopulmonary dysplasia. SP-D was also detected in many organs at all ages. SP-D was readily detected in epithelial cells and luminal material in lacrimal glands, salivary glands, pancreas, bile ducts, renal tubules, esophageal muscle and glands, parietal cells of the stomach, crypts of Lieberkuhn, sebaceous and eccrine sweat glands, Von Ebner's glands, endocervical glands, seminal vesicles, adrenal cortex, myocardium, and anterior pituitary gland. SP-D is a widely distributed member of the "collectin" family of polypeptides secreted onto luminal surfaces by epithelial cells lining ducts of many organs, where it likely plays a role in innate host defense.     

Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids.

This review article attempts to present an overview of the occurrence and function of lipid storage and secretory organelles: the lamellar bodies. Morphologically these organelles vary considerably in size (100 nm to 2400 nm); they are surrounded by a membrane and contain multilamellar lipid membranes. Lamellar bodies may also contain apolipoproteins and lytic enzymes and have an acidic pH, which confers on them a lysosomal character. Under normal physiological conditions, the main function of lamellar bodies is the supply of extracellular domains with specialized lipid components related to a specialized function. The lamellar bodies of the lung epithelium are best investigated in their functional and structural features and are the storage form of the lung surfactant. They provide a monomolecular lipid film of dipalmitoyl phosphatidylcholine (DPPC) on the surface of lung alveoli to lower surface tension necessary for optimal gas exchange and a hydrophobic protective lining against environmental influences. Additional cells of the respiratory system such as the mucosa of the human nose and the bronchi contain lamellar bodies. Lamellar bodies are also found in the gastrointestinal tract, in tongue papillae, oral epithelium, and mucosa cells of the stomach. The major phospholipid of lamellar bodies in mucosa cells of the stomach is DPPC, providing a hydrophobic protective lipid film against the tissue-damaging activities of gastric juice. The hydrophobic water-protective barrier of the skin, which consists mainly of neutral lipids, however, also originates from lamellar bodies secreted by epithelial cells. Lamellar bodies, mainly consisting of DPPC, also occur in mesodermal cell layers of sliding surfaces to provide the lubrication of joints, of the peritoneum, pericardium, and pleural mesothelium. In certain pathological conditions, such as atherosclerosis, Niemann-Pick disease, lecithin:cholesterol acyltransferase (LCAT) deficiency, cholestasis, degeneration of nerves and brain, and regeneration of nerves and wound healing, lipid-containing lamellar bodies have been observed in various cells, the function of which still remains to be elucidated. In early and late lesions of atherosclerotic plaques, lamellar bodies, consisting of unesterified cholesterol and phospholipids, are associated with the extracellular matrix of the intima. During regression of fatty streaks, lamellar bodies are seen intracellularly in macrophages and smooth muscle cells. Inherited metabolic disorders, such as Niemann-Pick disease type I and type II, result in the excessive accumulation of lamellar body-containing cells, for example in bone marrow, spleen, and lymphoid tissue. Type I is a deficiency in sphingomyelinase and type II is a defect in intracellular trafficking of lipoprotein-derived cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.     

Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes.

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.     

Ultrastructure of the lung of the rattlesnake,Crotalus viridis oreganus

Scanning and transmission electron microscopic observations were made on the rattlesnake lung, which has the form of a cigar-shaped bag enclosing a large axial air chamber. The lungs were fixed by tracheal instillation of fixative to preserve the structural features of inflated lungs. An open tracheal groove along the ventral aspect of the lung is the only structural “airway” present. The wall of the lung has two histologically distinct regions: anteriorly, a respiratory portion, where up to three generations of septa subdivide the wall into cup-shaped gas-exchange chambers, termed faveoli; and posteriorly, a simple, thin-walled saccular portion. The epithelium lining the internal surface of the lung is composed of several cell types: (1) ciliated cells; (2) type I pneumonocytes; (3) type II pneumonocytes, secretory cells characterized by the presence of lamellar bodies; and (4) serous epithelial cells, secretory cells characterized by the presence of homogeneous, densely staining secretory granules. However, the distinctiveness of the secretory cell types in the snake lung is blurred because intermediate-appearing cells have both the lamellar body and homogenous type of secretory granule. The nonepithelial components of the pulmonary wall and septa consist of blood vessels and lymphatics, smooth muscle cells and fibroblasts, embedded in a matrix of extracellular connective tissue fibers. Tubular myelin figures were observed in the faveolar lining layer.     

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.     

The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a three-dimensional substrate.

Enzymatically dissociated lungs from rat fetuses at 19-days gestation yield single cells which reaggregate to form alveolar-like structures when cultured on gelatin sponge discs. These structures form within 2 days and have been maintained in vitro for as long as 6 weeks. They are composed primarily of type II pneumonocytes as characterized by large, lightly stained nuclei and cytoplasmic inclusion bodies. The lamellar structure of these inclusion bodies has been confirmed by electron microscopy. The dynamic formation of inclusion bodies is suggested by the presence of lamellar bodies in the extra-cellular space and the appearance of new inclusions in the cytoplasm of the type II pneumonocytes. The formation and long-term maintenance of histotypic lung structures in vitro provides a model system for the study of lung development and synthesis of surfactant by type II alveolar pneumonocytes.     

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.     

The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a three-dimensional substrate

Summary Enzymatically dissociated lungs from rat fetuses at 19-days gestation yield single cells which reaggregate to form alveolar-like structures when cultured on gelatin sponge discs. These structures form within 2 days and have been maintained in vitro for as long as 6 weeks. They are composed primarily of type II pneumonocytes as characterized by large, lightly stained nuclei and cytoplasmic inclusion bodies. The lamellar structure of these inclusion bodies has been confirmed by electron microscopy. The dynamic formation of inclusion bodies is suggested by the presence of lamellar bodies in the extra-cellular space and the appearance of new inclusions in the cytoplasm of the type II pneumonocytes. The formation and long-term maintenance of histotypic lung structures in vitro provides a model system for the study of lung development and synthesis of surfactant by type II alveolar pneumonocytes. This work was supported by funds from the American Lung Association, National Heart and Lung Institute (grant HL-17110-01) and the W. Alton Jones Foundation.     

Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells

A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.     

Differentiation of the pulmonary surfactant system. Disaturated phosphatidylcholine accumulation in fetal rat lung in vivo and in vitro

Fetal rat lung was placed in organ culture at 15 days gestation (22 days total gestation period), before biochemical and morphological development of the pulmonary surfactant system. At the fifth day of culture numerous Type II cells containing lamellar bodies were present as determined by electron micrography. Phospholipid accumulation in the cultures increased abruptly beginning at 6 days in culture. The phospholipid which accumulated between the sixth and twelfth culture days was composed of 21--27% disaturated phosphatidylcholines. Both the percent of disaturated phosphatidylcholines in the phospholipid fraction and the qualitative pattern of accumulation as a function of time were similar to observations for fetal rat lung developing in vivo. The data presented provide evidence for development of the pulmonary surfactant system in organ culture in vitro.     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1alpha and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.     

Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.     

Swim bladder gas gland cells produce surfactant: in vivo and in culture

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.