Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and clara cells

The goal of this study was to compare the functions of Clara and type II cells during alveolar clearance and recycling of surfactant protein (SP) A, a secretory product of both cell types. We examined the incorporation of instilled biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a measure of clearance and recycling of the protein. Ultrastructural localization of bSP-A was accomplished by an electron-microscopic immunogold technique at 7, 30, and 120 min after intratracheal instillation. Localization of bSP-A was quantitatively evaluated within extracellular surfactant components (lipid-rich forms: myelin figures, vesicles, and tubular myelin; and lipid-poor hypophase) and in compartments of type II and Clara cells. bSP-A was incorporated into myelinic and vesicular forms of extracellular surfactant, but tubular myelin and hypophase had little bSP-A. Lamellar bodies of type II cells demonstrated a significant time-dependent increase in their incorporation of bSP-A. There was a concentration of bSP-A in the secretory granules and mitochondria of Clara cells, but no Clara cell compartment showed a pattern of time-dependent change in immunolabeling. Our immunolabeling data demonstrated a time-dependent movement of exogenous SP-A from extracellular components into type II cells and their secretory granules. Clara cells did not demonstrate a time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must reach a steady state very quickly or very slowly.     

Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung.

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.     

Loss of Rab27 function results in abnormal lung epithelium structure in mice

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.     

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.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

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.     

Fetal mouse lung in circumfusion system cultures.

Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderatley dense secretory granule in the center of the whorl of the endoplasmic reticulum.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.     

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.     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.     

The three-dimensional aspect of the mammalian lung surfactant myelin figure.

Rodent and primate lung surfactant was studied at the ultrastructural level utilizing procedures that retained most of the carbohydrates and lipids in thin section. The three-dimensional aspect of tubular myelin surfactant was observed to be four, lipid bilayer membranes oriented at right angles so that in cross-section it was square. In longitudinal section it appeared as two parallel lipid bilayers. Inside the tubular myelin was a homogeneous matrix material that completely filled the tubule except for a small, central area. A single multilamellar body, after it expanded and rearranged lamellae to form tubular myelin surfactant, still retained its basic morphology so that it was possible to determine the number and orientation of bodies that comprised a given surfactant area. This enabled quantification of surfactant by serial sectioning. Each transformed multilamellar body was observed to contain from 2 to 13 groups of tubular myelin, oriented at angles within the transformed body. With three-dimensional understanding, many of the areas previously reported to be homogeneous were determined to actually be oblique cross or longitudinal sections through tubular myelin surfactant.Five distinct layers characterized tubular myelin surfactant: (1) Unexpanded layer—up to 63 recently secreted multilamellar bodies. (2) Formation layerp?aired lamellae expanding and rearranging to form tubules. (3) Mature layer—tubular myelin surfactant. (4) Air-surfactant interface layer—usually a single lipid bilayer which was the outermost layer of tubular myelin of from 1 to 12 transformed multilamellar bodies. (5) Degraded surfactant layer—lipid bilayer spheres were formed at the interface and degraded in the alveolar space.     

Respiratory portion of the lungs of intact BALB strain mice

Four male mice (1-month-old) of BALB strain have been studied. Three types of cells participate in formation of cellular lining of the terminal branchioles: ciliated, Clara cells and villose cells which are included in the neuroepithelial bodies composition. Clara cells are numerous and they are similar in their ultrastructural organization, they contain a great number of polymorphous secretory granules, single mitochondria and a poorly developed endoplasmic reticulum. A comparatively large number of pores of Khone are revealed: there are 4-8 pores per one alveole, and one pore is situated on 197.4 mcm2 of the alveolar surface. Alveolocytes of the second type of make the main actively secreting cell population of the alveolar passages and alveoles. Simultaneously, at the state of secretion there are 76% of the cells. The secretion of alveolar surfactant is realized by means of merocrine exocytosis. Relative volumetric fraction of mitochondria in alveolocytes of the II type comprises 12% and that of "cytophospholiposomes" (osmiophilic laminar bodies)--11%.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins.     

Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism

The influence of clofibrate (ethyl-alpha-p-chlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

ABCA3 as a lipid transporter in pulmonary surfactant biogenesis

ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in alveolar type II cells, and mutations in the ABCA3 gene cause lethal respiratory distress in newborn infants. To investigate the function of ABCA3 protein, we generated Abca3-deficient mice by targeting Abca3. Full-term Abca3(-/-) newborn pups died within an hour after birth because of acute respiratory failure. Ultrastructural analysis revealed abnormally dense lamellar body-like organelles and no normal lamellar bodies in Abca3(-/-) alveolar type II cells. TLC and electrospray ionization mass spectrometry analyses of lipids in the pulmonary interstitium showed that phosphatidylcholine and phosphatidylglycerol, which contain palmitic acid and are abundant in normal surfactant lipids, were dramatically decreased in Abca3(-/-) lung. These findings indicate that ABCA3 plays an essential role in pulmonary surfactant lipid metabolism and lamellar body biogenesis, probably by transporting these lipids as substrates.     

Endocrine cells and brush cells at the bronchiolo-alveolar junctions of neonatal syrian hamster lungs

Endocrine cells and brush cells at the bronchiolo-alveolar junctions of the lung of neonatal hamsters were studied by transmission electron microscopy. On both sides of the junctions (bronchiolar and alveolar), clusters of endocrine cells occur as neuroepithelial bodies (NEB). A few solitary endocrine cells are also present at the alveolar sides of the junctions. Some endocrine cells reach from the basement membrane to the air space but the area of apical cell membrane exposed to the airway is small as the cells are largely covered by Clara cells in the bronchioles and by thin attenuations of alveolar type 1 cells in the alveoli. Some Clara cells around NEB contain cytoplasmic lamellar bodies, similar to those characteristically associated with alveolar type 2 cells. A few brush cells are also seen at both sides of the junctions. Long wide microvilli with filamentous cores extend from the apices of the brush cells. Endoplasmic reticulum and Golgi apparatus are moderately developed. Well-developed bundles of intermediate filaments course throughout the cytoplasm of some of the brush cells. The functions of endocrine cells and brush cells are unknown. However, the presence of these cells at the bronchiolo-alveolar junctions of neonatal hamster lungs suggests a role in regulation of respiratory function.