scholarly journals Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung.

1986 ◽  
Vol 34 (9) ◽  
pp. 1137-1148 ◽  
Author(s):  
S R Walker ◽  
M C Williams ◽  
B Benson
Keyword(s):  
Alveolar Macrophages ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Multivesicular Bodies ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Lamellar Bodies ◽  
Thin Sections

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.

Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane

1998 ◽  
Vol 275 (1) ◽  
pp. L172-L183 ◽  
Author(s):  
K. Zen ◽  
K. Notarfrancesco ◽  
V. Oorschot ◽  
J. W. Slot ◽  
A. B. Fisher ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cell ◽  
Type Ii Cell

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic βHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.

Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions

1999 ◽  
Vol 276 (3) ◽  
pp. L452-L458 ◽  
Author(s):  
Howard Clark ◽  
Lennell Allen ◽  
Erin Collins ◽  
Frederick Barr ◽  
Leland Dobbs ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Alveolar Macrophages ◽  
Pulmonary Surfactant ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Protein Distribution ◽  
Serine Hydrolase ◽  
Surfactant Metabolism

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222–230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L404–L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

Pulmonary surfactant protein A-mediated uptake of phosphatidylcholine by alveolar type II cells

1993 ◽  
Vol 265 (2) ◽  
pp. L193-L199 ◽  
Author(s):  
A. Tsuzuki ◽  
Y. Kuroki ◽  
T. Akino
Keyword(s):  
Pulmonary Surfactant ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Pulmonary Surfactant Protein

Pulmonary surfactant protein A (SP-A)-mediated uptake of phosphatidylcholine (PC) by alveolar type II cells was investigated. SP-A enhanced the uptake of liposomes containing dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), or 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether), a diether analogue of DPPC, but about twice as much DPPC was taken up by type II cells as PLPC or DPPC-ether. When subcellular distribution was analyzed, 51.3 +/- 2.9% (mean +/- SD, n = 3) of cell-associated radiolabeled DPPC was recovered in the lamellar body-rich fraction in the presence of SP-A, whereas only 19.3 +/- 1.9% (mean +/- SD, n = 3) was found to this fraction in the absence of SP-A. When type II cells were incubated either with DPPC at 0 degree C or with DPPC-ether at 37 degrees C, or no cells were included, low proportions of the cell-associated lipids were present in the fractions corresponding to lamellar bodies even in the presence of SP-A. Anti-SP-A antibody significantly reduced the radioactivity incorporated into the lamellar body fraction. Phosphatidylcholine that had been incorporated into lamellar bodies remained largely intact when SP-A was present. Subcellular fractionations of type II cells with radiolabeled SP-A and DPPC revealed that the sedimentation characteristics of cell-associated SP-A are different from those of DPPC, although a small broad peak of radiolabeled SP-A was found in the lamellar body fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory granule calcium loss after isolation of rat alveolar type II cells

1991 ◽  
Vol 260 (2) ◽  
pp. L129-L135 ◽  
Author(s):  
R. G. Eckenhoff ◽  
S. R. Rannels ◽  
A. B. Fisher
Keyword(s):  
Primary Culture ◽  
Sulfur Content ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Isolated Cells ◽  
Alveolar Type Ii Cells ◽  
Type Ii Cell

Morphological change and lamellar body loss suggests that alveolar type II cells rapidly de- or redifferentiate after several days of primary culture. To determine whether type II cells or lamellar body compositional changes precede these obvious morphological changes, we examined the in situ elemental composition of lamellar bodies and type II cells from intact lung and at different times after isolation using electron probe microanalysis (EPMA). Isolated cells were prepared by standard methods and plated on either tissue culture plastic or kept in suspension with stirrer flasks. Cell pellets obtained at 0, 3, 24, and 48 h after isolation were rapidly frozen, and thin freeze-dried cryosections were prepared and examined cold in a transmission electron microscope equipped for EPMA. Eight to ten type II cells from each of three to four different preparations for each time period were analyzed. A rapid, progressive, and sustained fall in lamellar body calcium and sulfur content occurred by 48 h of primary culture, suggesting rapid alteration in calcium and protein metabolism by type II cells and/or lamellar bodies after isolation. Also, marked changes in type II cell cytoplasmic Na and K occurred in freshly isolated cells, with incomplete normalization by 48 h. Culture on laminin-enriched Matrigel for 1 wk increased both lamellar body calcium or sulfur content, but 100 nM dexamethasone had no effect. Lamellar body calcium accumulation appears to be a very sensitive index of differentiated type II cell function.

Immunocytochemical localization and identification of the major surfactant protein in adult rat lung.

1981 ◽  
Vol 29 (2) ◽  
pp. 291-305 ◽  
Author(s):  
M C Williams ◽  
B J Benson
Keyword(s):  
Alveolar Macrophages ◽  
Gradient Centrifugation ◽  
Surfactant Protein ◽  
Multivesicular Bodies ◽  
Type I ◽  
Type Ii Cells ◽  
Secretory Product ◽  
Type Ii ◽  
Rat Lung ◽  
Adult Rat

We investigated the cellular and subcellular sites of metabolism of the 72,000 dalton protein of pulmonary surfactant in order to provide insights into mechanisms of synthesis, intracellular assembly, and intraalveolar metabolism of this phospholipid-rich secretory product. Surfactant (approximately 90% lipid, 10% protein by weight) was purified by density gradient centrifugation of material obtained by lavaging rat lungs. The purified material was used to generate an antiserum from which a specific antibody was obtained by affinity chromatography. A horseradish peroxidase-labeled Fab was used to localize the antigen in rat lung. The antibody labeled the rough endoplasmic reticulum and Golgi apparatus of type II cells only. Some multivesicular bodies in type II cells were also labeled, but whether the antigen was present in lamellar bodies was uncertain. Phagosomes of alveolar macrophages were labeled as were similar inclusions in type I cells. Using indirect immunocytochemistry we determined that the labeling of alveolar cell surfaces does not represent the presence of a continuous layer of secreted surfactant. These results suggest that only the type II cell synthesizes surfactant protein and than mainly alveolar macrophages participate in its catabolism. The initial intracellular site of the association of protein with lipid may be multivesicular bodies as suggested previously by others.

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

1992 ◽  
Vol 40 (10) ◽  
pp. 1589-1597 ◽  
Author(s):  
W F Voorhout ◽  
T Veenendaal ◽  
Y Kuroki ◽  
Y Ogasawara ◽  
L M van Golde ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Alveolar Macrophages ◽  
Surfactant Protein ◽  
Surfactant Protein D ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Double Labeling

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.

Surfactant treatment effects on alveolar type II cell morphology in rabbit lungs

1993 ◽  
Vol 74 (3) ◽  
pp. 1240-1247 ◽  
Author(s):  
K. E. Pinkerton ◽  
J. Lewis ◽  
A. M. Mulder ◽  
M. Ikegami ◽  
A. H. Jobe
Keyword(s):  
Volume Fraction ◽  
Lamellar Body ◽  
Exogenous Surfactant ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells ◽  
Alveolar Type Ii Cell ◽  
Type Ii Cell

The effects of exogenous surfactant administration on alveolar type II cells and the lung parenchyma were examined in adult rabbits. Natural surfactant was instilled into the left lobe of New Zealand White rabbits while the right lobe served as the control. Four hours post-instillation, the lungs were fixed by vascular perfusion. Surfactant instillation did not change alveolar type II cell size but was associated with a significant reduction in the volume fraction of lamellar bodies in type II cells (20.4% in control lobes compared with 11.9% in surfactant-treated lobes). The size distribution of lamellar body profiles was different in surfactant-treated lobes compared with control lobes, with a significant decrease in lamellar bodies > 0.8 microns in diameter and a twofold increase in lamellar bodies 0.2–0.4 microns in diameter. Composite body profile number was also increased by 87% (P < 0.05) after instillation of surfactant compared with control. Saline instillation decreased lamellar body volume fraction in type II cells but three times less than surfactant instillation. These observations are consistent with a strong stimulus for secretion of endogenous surfactant 4 h after surfactant instillation in normal adult rabbit lungs, whereas the increase in composite bodies is consistent with new lamellar body formation, probably from both de novo synthesized and exogenous natural rabbit surfactant. These observations confirm that the secretory and synthetic processes of alveolar type II cells are significantly affected by exogenous surfactant instillation.

Lamellar body membrane turnover is stimulated by secretagogues

2000 ◽  
Vol 278 (3) ◽  
pp. L443-L452 ◽  
Author(s):  
Sandra R. Bates ◽  
Jian-Qin Tao ◽  
Susanne Schaller ◽  
Aron B. Fisher ◽  
Henry Shuman
Keyword(s):  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Membrane Turnover ◽  
Concentration Dependent Manner

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. 125I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

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

2012 ◽  
Vol 302 (9) ◽  
pp. L919-L932 ◽  
Author(s):  
Blair R. Roszell ◽  
Jian-Qin Tao ◽  
Kevin J. Yu ◽  
Shaohui Huang ◽  
Sandra R. Bates
Keyword(s):  
Lamellar Body ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells ◽  
Type Ii Pneumocytes ◽  
Niemann Pick

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.

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