T H E DEVELOPMENT O F THE H U M A N T Y P E I1 PNEUMOCYTE
JENNIFERMCDOUGALL AND J. F. SMITH Department of Morbid Anatomy, University College Hospital Medical School
PLATES CXLI-CXLVIII THERE have been many studies in animals of the development of the type I1 pneumocyte or great alveolar cell (Sorokin, 1967), viz., in foetal rats by Balk and Conen (1964), Sorokin (1967), O’Hare and Sheridan (1970), in foetal and newborn rabbits by Kikkawa, Motoyama and Gluck (1968) and in foetal sheep by Kikkawa and Spitzer (1969), but only a few on human material-Campiche ct al. (1963) and Balk and Conen (1964). In this paper observations on human foetal material are recorded, including in particular, five cases between 20 and 26 wk of gestational age, when the differentiation of type I1 cells with their inclusion bodies becomes manifest. The view that these are the source of pulmonary surfactant is supported and the mechanism of its elaboration and discharge discussed. MATERIAL AND METHODS Through the courtesy of gynaecological colleagues we were able to obtain 25 foetuses of 12-26 wk of gestational age of which 24 were removed by abdominal hysterotomy. An immediate dissection was done and portions of lung from representative peripheral and hilar regions taken for light and electron microscopy. The portions for ultrastructural studies were excised, placed in 4 per cent. gluteraldehyde in 0 . 1 2 5 ~phosphate buffer and trimmed. Blocks of approximately 1 mm cube were fixed by immersion in fresh cold fixative, washed and stored in fresh 0 . 1 2 5 ~phosphate buffer at 4°C. The tissue was postfixed in 1 per cent. OsO4 in 0 . 1 2 5 ~phosphate buffer for 2 hr, washed, dehydrated and embedded in epon. Sections of 1 pm were stained with toluidine blue; ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope. The age of each foetus was determined after consideration of the dates of the LMP of the mother, the CH and CR length of the foetus and its body weight.
RESULTS
Of the total number of 25 foetuses with an age range of 12-27 weeks’ gestation, 12 were between 20 and 27 wk. In this paper we shall be concerned principally with the peripheral lung studies of these 12 foetuses, in 10 of which type I pneumocytes were differentiated, and in particular the 5 in which type I1 pneumocytes with inclusion bodies were seen. Examination by light microscopy of tissues in the late pseudoglandular or early canalicular period between 16 and 20 wk, shows that the proportion of interstitial area to proliferating epithelium changes from approximately Received 16 Mar. 1974; accepted 9 May 1974. 1. PATH.-VOL
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JENNIFER McDOUGALL AND J. F. SMITH
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50 : 50 to 25 : 75. The developing respiratory bronchioles, with columnar epithelium become lined in some regions by uniform cuboidal cells (fig. 1). Ultrastructural study of these cells in their early development shows a central nucleus surrounded by a large area of sparse glycogen. A few small mitochondria and short cisternae of rough endopla:\mic reticulum (RER) lie around the periphery, especially near the luminal surfsce. As the cells mature the glycogen increases and small to medium dense bodies are seen more frequently, sometimes associated with a Golgi region (fig. 2). As development proceeds after 20 wk, the proportion of interstitial area to epithelium further decreases. The pseudoglandular appearance of the lung is completely lost and the expanded bronchioles no longer appear readily distinct from the surrounding cellular stroma. Moreover, capillaries are seen in obvious contiguity with the epithelium and some cells of the latter are attenuated (fig. 3). These type I pneumocytes are identifiable at 22 wk. The ultrastructural characteristics of these cells include a diminution in glycogen and an extension along the air-spaces of their cytoplasmic processes with sparse distribution of mitochondria, ribosomes, RER and occasional dense bodies (fig. 4). Between 22 and 24 wk a few of the other cuboidal epithelial cells, destined to become type I1 pneumocytes, show membrane-enclosed glycogen-free spaces in the cytoplasm (fig. 5). Sometimes dense bodies are in contiguity with these membranes. At about 24 wk the cytoplasmic differentiation of type I1 cells is dominated by the appearance of multiple membrane bound lamellar inclusion bodies (figs. 6 and 7 ) . The changes preceding and associated with this differentiation include : (i) (ii) (iii) (iv) (v) (vi)
Changes in glycogen. Lamellae and lattice structures in dense bodies. Opaque material in dilated cisternae of RER. Multivesicular bodies (MVBs) and increase in Golgi vesicles. Microtubules in the cytoplasm. Development and extension of microvilli at the luminal surface.
The changes in glycogen occur within focal areas of the cytoplasm, usually bound by a unit membrane. From 22 wk, glycogen-free spaces appear (fig. 5). In already differentiated type I1 cells areas of cytoplasm, devoid of organelles other than sparse glycogen are associated with a few lamellar membranes (fig. 8). Also from 22 wk, membrane limited areas may include both glycogen and some electron-dense material (fig. 9). Multiple membranes may also develop in association with these (fig. 10). Different stages of development of the inclusion bodies can be found in a single cell (fig. 11). Moreover, there is a progressive decrease in glycogen as the IBs are formed. During this time the membrane bound dense bodies become more numerous and some enlarge. Their internal structure is granular and in certain areas multiple parallel membranes may be seen (fig. 12). These are in close arrayeach unit membrane separated from the next by 10 nM and eight such membranes are seen forming within a segment of the dense body (fig. 12). The inner two membranes have a more complicated structure, viz., alternate light and
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DEVELOPMENT OF PNEUMOCYTE
FIG. 1.-18 wk. The bronchioles which are lined by columnar or cuboidal cells are separated by a wide interstitial area. Two mitotic figures are seen in the primitive bud. 1 pm Toluidine blue. x 640.
FIG.2.-16 wk. Cuboidal cells lining a bronchiole have central nuclei surrounded by glycogen. Sparse mitochondria and cisternae of RER lie around the periphery and therc are a few dense bodies. EM. X 8400.
FIG. 3.-23 wk. Bronchioles with cuboidal lining cells have developing air spaces almost in contiguity with capillaries because of attenuation of some of the epithelial cells. 1pm. x 640.
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FIG.4.-Apical cytoplasm of cuboidal cells .probably de- FIG.5.-22 wk. The cytoplasm of a cuboidal cell is filled with glycogen, sparse mitochondria and cisveloping into type I1 pneumocytes and an attenuated ternae of RER lie peripherally. There are some cell in contiguity with a capillary. The microvilli are small dense bodies, a Golgi region and a few lost at the area of complete attenuation. EM. x 8000. smooth vesicles. A membrane is seen in the glycogen-free area. Cytoplasmic protruberances project into the lumen. EM. X 5600.
FIG.6.-24 wk. Type I1 pneumocytes with dense inclusion bodies mainly in the apical cytoplasm; a few have a vacuolar appearance. Several capillaries are overlaid by attenuated cells. 1 pm Toluidine blue. x 640.
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FIG.7.-24 wk. Type I1 pneumocytes with characteristic lamellated inclusion bodies. Mitochondria, RER and microvilli are clearly seen. EM. X 5000.
FIG.8.-24 wk. Part of the cytoplasm of a type I1 cell, showing part of a lamellated inclusion body and a membrane bound glycogen area with several membranes forming. EM. ~ 2 3 , 2 0 0 .
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MCDOUGALL AND SMITH DEVELOPMENT OF PNEUMOCYTE
FIG.9.-22 wk. Part of the cytoplasm of a differentiating cuboidal cell. Adjacent to the nucleus is an area of dense glycogen and electron-opaque material bound by a unit membrane. A smaller zone is also demarcated by a membrane which is in part triplicate ( t ). EM. x 19,000.
FIG.10.-22 wk. Part of the cytoplasm of a differentiating cuboidal cell showing an area of electrondense material and glycogen surrounded by a concentric series of membranes. EM. X 18,400.
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DEVELOPMENT OF PNEUMOCYTE
FIG.11.-24 wk. In a type I1 cell with formed lamellated inclusion bodies there are dense bodies associated with lamellar membranes, and a membrane-bound glycogen zone with a few concentric lamellations. Several intercellular spaces with microvilli separate adjoining cells.
EM. ~18,400.
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MCDOUGALL AND SMITH DEVELOPMENT OF PNEUMOCYTE
FIG.12a.
FIG.12b.
FIG.120 and b.-24 wks. A large, membrane bound body is filled with dense granular material. In one area a close array of parallel membranes, separated by 10 n M are seen (f). The innermost membranes have a more complicated structure reminiscent of that of myelin (tt). EM. (a) X 69,000. (b) X 118,000.
FIG.13.-24
wk. A large dense body with a lattice structure and some membranes surrounding it. The sides of the lattice are about 13 n M apart. EM. ~74,000.
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FIG.14.-24 wk. Part of the cytoplasm of a mature type I1 cell. Numerous microvilli protrude into the lumen on the left. There are many ribosomes and cisternae, some dilated, of RER and one inclusion body is closely apposed by a cisterna. There is a large MVB and some small Golgi vesicles. Glycogen is sparse. EM. x 30,400.
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FIG. 15.-24 wk. The intercellular connexions between two type I1 cells. Tight junctions persist at the luminal end and intercellular spaces with microvilli. The cytoplasm of the left cell, in which part of three inclusion bodies are seen, has numerous dilated cisternae of RER and microtubules. EM. x 20,900.
FIG. 16.-25
wk. An inclusion body partly extruded through the plasma membrane, between the microvilli. EM. x 30,400.
DEVELOPMENT OF PNEUMOCYTE
247
dark bands 4 nM wide within dense lines which are here 20 nM apart. This is similar in structure to myelin (12 nM between the thick dense lines and bands 3 nM wide). In three cases a lattice structure with sides approximately 13 nM was seen within a dense body (fig. 13). The RER with slightly dilated cisternae containing moderately electron opaque material (figs. 7 and 14) is seen extensively in five cases. At times the RER surrounds IBs but in no case is it in direct continuity with the lamellae of IBs as described in the anencephalic rat by Blackburn, Travers and Potter (1972). Rough-surfaced vesicles, often dilated, and containing granular material are seen in cells with numerous IBs (fig. 15). Mitochondria increase in number and elongate, but show no internal structural change, during this differentiation. They are distributed more generally through the cell and at this time the glycogen is diminished. MVBs are seen in differentiating cuboidal cells with membrane-bound glycogen-free or sparse areas and/or dense bodies. They are, however, more commonly seen in differentiated type I1 cells (figs. 8 and 14). The Golgi vesicles are also more common at 24 wk. The association of lamellar IBs with MVBs is seen infrequently, but sometimes vesicles surround the former. From about 20 wk some cuboidal cells have cytoplasmic protrusions into the lumen. In type I1 cells containing IBs, numerous well-formed microvilli are seen, presumably derived from the cytoplasmic extensions (fig. 14). They have an inner structure of microfilaments and microtubules. Microtubules are also seen beneath the cell membrane, at the luminal surface and deeper within the cytoplasm of cells containing numerous IBs (fig. 15). Occasionally IBs are seen partly extruded between the microvilli (fig. 16).
Summary of diferentiation of type II cells The type I1 pneumocyte develops from a cuboidal cell with cytoplasm largely filled with glycogen. The process of differentiation involves cytoplasmic changes which indicate active synthesis. The progressive decrease in glycogen with these changes and the appearance of the inclusion bodies may indicate its breakdown, partly for energy requirement and partly for either the participation or the incorporation of simpler molecules in the inclusion body formation. The increase in number and size of mitochondria suggests increased aerobic metabolism, while the increase and dilatation of the RER probably indicates protein synthesis. Extension of the Golgi system, the MVBs and the increase in number and size of the membrane-bound dense bodies with a complex internal structure precede the appearance of the lamellated inclusion bodies. These are extruded through the luminal plasma membrane. DISCUSSION The essential point to be considered is the relevance of the ultrastructural changes to the function of the cell. It is convenient to consider the biological function of the cell separately from its biochemical function in the first instance. The point at issue in the
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literature is whether the cell's primary function is synthesis or phagocytosis. The great majority of investigators of (animal) lung have thought that the type I1 pneumocyte makes and secretes pulmonary surfactant (Macklin, 1954) which is essential for the lowering of surface tension to enable normal respiration to continue. A few have suggested that the surfactant is made by the non-ciliated cell, the Clara cell (Niden, 1967; Etherton et al., 1973) of the bronchiole and that the inclusion bodies in type I1 pneumocytes indicate phagocytosis and breakdown of excess surfactant. The investigation by Etherton et al. depends on the earlier incorporation of labelled surfactant precursor (palmitic acid) into dipalmitoyl lecithin in the Clara cells of the terminal bronchioles. However, the bulk of the experimental evidence-summarised by Heppleston and Young (1973) is against any marked phagocytic role for the type I1 pneumocyte. This does not exclude the possibility of the Clara cell having a role in surfactant synthesis after birth. The sequence of events in hyaline membrane disease suggest that it is not the main source in foetal and early neonatal life. Taghizadeh and Reynolds (1975) have shown that in the initial stages of this disease the surface tension rises and the inclusion bodies in type I1 pneumocytes are absent. After a few days the inclusion bodies reappear and the surface tension falls, indicating an association of surfactant secretion with the bodies at a time when the epithelium of the terminal airways with its Clara cells is still destroyed and that of the bronchi is damaged. Furthermore, we think that the ultrastructural changes summarised above point to a synthetic role for the cell, while freely admitting that the endproduct of the synthesis-the lamellated inclusion bodies-resemble in some ways cytosegresomes (Ericsson, 1969) and the membranous cytoplasmic bodies of GM2 gangliosidosis (Tay Sachs disease) and other storage diseases in which there is degradation of excessive unwanted material by lysosomes. We would suggest that the close association of the dense bodies with the developing inclusion bodies (figs. 9-12) is significant; their role may be to provide some of the enzymes necessary for the construction of the inclusion bodies as well as for their discharge from the cells in the process of reversed endocytosis rather than phagocytosis. This does not exclude a role for the intracytoplasmic microtubules in the discharge process also (fig. 15). The changes in glycogen are the earliest in the series of ultrastructural events. One obvious explanation is that glycogen is utilised to a considerable degree for anaerobic glycolysis before the increase in mitochondria provides a greater source of aerobic metabolism. However, the earliest membranes of the inclusion bodies appear in unit-membrane-enclosed glycogen-free or glycogen rich spaces (figs. 5, 8 , 9 and 10). In the rabbit Kikkawa et al. (1971) also found striking changes in glycogen and suggested that these were associated with the synthesis of lecithins through the coupling of diglyceride to CDP-choline, the former being provided by glycogenolysis. However, in the human foetus the predominant pathway of lecithin synthesis is the methyl transferase (Gluck and Kulovich, 1973) and their explanation would need modification. Whether these early membranes sediment with the microscomal fraction in which Gluck
DEVELOPMENT OF PNEUMOC YTE
249
and his associates (1967a and b) found both pathways of lecithin synthesis or the mitochondria is uncertain. The methyl transferase pathway appears about 22-24 wk in the human-at the same time as the changes in glycogen begin. There remains to be considered the role of protein synthesis as indicated by the active endoplasmic reticulum (figs. 7, 14 and 15). This was first emphasised by Sorokin (1967) in marsupials, bats and rodents and thought by him to be concerned with synthesis of the enzymes required for the complex metabolic processes involved in the inclusion body formation. This is probably part of the story, but the lattice and lamellar structures seen in developing inclusion bodies (figs. 12 and 13) suggest that protein is built into the bodies. Indeed, the assembly of the bodies may well be a later event than lecithin and protein synthesis. If the bodies are lipoprotein complexes this would fit with the work of Mingins and Taylor (1973) who suggest that a lipoprotein rather than a monolayer of dipalmitoyl lecithin is necessary to provide adequate hysteresis for lung distensibility. We have not, however, in our material seen the intimate association of inclusion bodies with rough endoplasmic reticulum described by Blackburn, Travers and Potter (1972) in the anencephalic foetal rat. Many authors (Balis and Conen, 1964; Sorokin, 1967; Kikkawa et al., 1968; Kikkawa and Spitzer, 1969; Meyrick and Reid, 1973) have stressed the importance of the MVB in the formation of the inclusion bodies. Kikkawa and Spitzer especially observed fusion of MVB with dense bodies. We have occasionally observed such a phenomen, and IBs surrounded by vesicles. As these organelles, together with an increased Golgi zone are common in the differentiating type I1 pneumocytes we suggest that they may act in transporting enzymes or the building proteins to the inclusion bodies. The earlier suggestion that the inclusion bodies are transformed mitochondria (Kisch, 1955) received no support from our observations. We saw no structural alterations within them and would attribute the increase in their number, size and distribution in the developing type I1 cell to increase in aerobic metabolism as the glycogen diminished. However, Kikkawa et al. (1968) saw no increase in mitochondria at a comparable stage in the differentiation of type I1 cells in the foetal rabbit. Our conclusion is therefore that the formation of lamellated inclusion bodies in the type I1 pneumocytes is a synthetic process. Changes in glycogen and participation of the dense bodies are striking. Lattice and lamellar formations within dense bodies are thought to indicate an assembly of protein and lipid in the formation of the inclusion bodies which are then extruded into the lumen of air sacs. In a recent paper Di Augustine (1974) has found a phospholipid to protein ratio of 12 : 1 in inclusion bodies isolated from adult rabbit lung, and demonstrated that they contain hydrolases similar to those in lysosomes. He has discussed a possible regulating function for these bodies which are unique in mammalian physiology. SUMMARY
Portions of lung from 25 human foetuses between 12 and 26 wk of gestational age were examined for ultrastructural changes during maturation of the
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epithelial cells. In 10 cases type I pneumocytes and in five cases type I1 cells were differentiated. The changes associated with the formation of lamellar inclusion bodies in type I1 cells are described. It is concluded that this is a synthetic process in which protein and lipid assemblyare important. Changes in glycogen and participation of dense bodies are striking. REFERENCES P. E. 1964. The role of alveolar inclusion bodies in the developing BALIS,J. V., AND CONEN, lung. Lab. Invest., 13 (LO), 1215. BJARNSTAD, P., AND BREMER, J. 1966. " In vivo " studies on pathways for the bio synthesis of lecithins in the rat. J. Lipid Res., 7 , 38. BLACKBURN, W. R., TRAVERS, H., AND POTTER,M. 1972. The role of the pituitary-adrenalthyroid axes in lung differentiation. Lab. Invest., 26 (3), 306. CAMPICHE, M. A., GAUTIER,A., HERMANDEZ, E. I., AND REYMOND, A. 1963. An electron microscope study of the foetal development of human lung. Ped., 32. 976. DI AUGUSTINE, R. P. 1974. Lung concentric laminar organelle. J. Biol. Chem., 249, 584. EHICSSON, J. 1969. Lysosomes in biology and pathology, edited by J. T. Dingle and H. B. Fell, North Holland Publ. Co., Amsrerdam, vol. 2, p. 354. ETHERTON, J. E., CONNING, D. M., A m CORRIN,B. 1973. Autoradiographical and morphological evidence for apocrine secretion of dipalmitoyl lecithin in the terminal bronchiole of mouse lung. Am. Jour. Anat., 138 (l), 11. GLUCK, L., AND KULOVICH, M. V. 1973. The pediatric clinics of North America, edited by L. Cluck, Saunders, Philadelphia, vol. 20 (2), p. 370. GLUCK, L., KULOVICH, M. V., EIDELMAN, A. I., CORDERO, L., AND KHAZIN,A. F. 1972. Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in human foetus and newborn and etiology of the respiratory distress syndrome. Ped. Res., 6, 81-99. CLUCK, L., MOTOYAMA, E. K., SMITS,H., AND KULOVICH,M. V. 1967. Biochemical development of surface activity in mammalian lung. I. The surface active phospholipids; the separation and distribution of surface active lecithin in the lung of the developing rabbit foetus. Ped. Res., 1, 237. GLUCK, L., SRIBNEY,M., AND KULOVICH,M. V. 1967. The biochemical development of surface activity in mammalian lung. 11. The biosynthesis of phospholipids in the lung of the developing rabbit fetus and newborn. Ped. Res., 1, 247. HEPPLESTON, A. G., AND YOUNG,A. E. 1973. Uptake of inert particulate matter by alveolar cells: an ultrastructural study. J. Pafh., 111, 159. KIKKAwA, Y., KNBARA,M., MOTOYAMA, E. K.,ORZALESI, M. M., AND COOK, c. D. 1971. Morphologic development of foetal rabbit lung and its acceleration with cortisol. Am. Jour. Path., 64 (2), 423. KIKKAWA, Y., MOTOYAMA, E. K., AND CLUCK, L. 1968. Study of the lungs of foetal and newborn rabbits. Am. Jour. Path., 52, 177. KIKKAWA,Y.,AND SPITZER,R. 1969. Inclusion bodies of type I1 alveolar cells: Species differences and morphogenesis. Anat. Rec., 163, 525. KISCH,B. 1955. Electron microscopic investigation of the lungs (capillaries and specific cells). Exp. Med. Surg., 13, 101. MACKLIN, C. C. 1954. The pulmonary alveolar mucoid film and the pneumocytes. Lancet, i, 1099. MEYRICK, B., AND REID,L. 1973. Electron microscopic aspects of surfactant secretion. Proc. Roy. SOC.Med., 66, 6. MINGINS,J., AND TAYLOR, J. A. G. 1973. Physicochemical properties of phospholipid " monomolecular layers ". Proc. Roy. SOC. Med., 66, 383. NIDEN, A. H. 1967. Bronchiolar and large alveolar cell in pulmonary phospholipid metabolism. Science, 158, 1323.
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O’HARE,K. H., AND SHERIDAN, M. N. 1970. Electron microscopic observations on the morphogenesis of the albino rat lung, with special reference to pulmonary epithelial cells. Am. Jour. Anat., 127, 181. SOROKIN, S. P. 1967. A morphologic and cytochemical study on the Great alveolar cell. Jour. Histochem. and Cytochem., Id (12), 884. TAGHIZADEH, A., AND REYNOLDS, E. 0. R. 1975. The pathogenesis of bronchopulmonary dysplasia following hyaline membrane disease. In course of preparation.
JENNIFERMCDOUGALL AND J. F. SMITH Department of Morbid Anatomy, University College Hospital Medical School
PLATES CXLI-CXLVIII THERE have been many studies in animals of the development of the type I1 pneumocyte or great alveolar cell (Sorokin, 1967), viz., in foetal rats by Balk and Conen (1964), Sorokin (1967), O’Hare and Sheridan (1970), in foetal and newborn rabbits by Kikkawa, Motoyama and Gluck (1968) and in foetal sheep by Kikkawa and Spitzer (1969), but only a few on human material-Campiche ct al. (1963) and Balk and Conen (1964). In this paper observations on human foetal material are recorded, including in particular, five cases between 20 and 26 wk of gestational age, when the differentiation of type I1 cells with their inclusion bodies becomes manifest. The view that these are the source of pulmonary surfactant is supported and the mechanism of its elaboration and discharge discussed. MATERIAL AND METHODS Through the courtesy of gynaecological colleagues we were able to obtain 25 foetuses of 12-26 wk of gestational age of which 24 were removed by abdominal hysterotomy. An immediate dissection was done and portions of lung from representative peripheral and hilar regions taken for light and electron microscopy. The portions for ultrastructural studies were excised, placed in 4 per cent. gluteraldehyde in 0 . 1 2 5 ~phosphate buffer and trimmed. Blocks of approximately 1 mm cube were fixed by immersion in fresh cold fixative, washed and stored in fresh 0 . 1 2 5 ~phosphate buffer at 4°C. The tissue was postfixed in 1 per cent. OsO4 in 0 . 1 2 5 ~phosphate buffer for 2 hr, washed, dehydrated and embedded in epon. Sections of 1 pm were stained with toluidine blue; ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope. The age of each foetus was determined after consideration of the dates of the LMP of the mother, the CH and CR length of the foetus and its body weight.
RESULTS
Of the total number of 25 foetuses with an age range of 12-27 weeks’ gestation, 12 were between 20 and 27 wk. In this paper we shall be concerned principally with the peripheral lung studies of these 12 foetuses, in 10 of which type I pneumocytes were differentiated, and in particular the 5 in which type I1 pneumocytes with inclusion bodies were seen. Examination by light microscopy of tissues in the late pseudoglandular or early canalicular period between 16 and 20 wk, shows that the proportion of interstitial area to proliferating epithelium changes from approximately Received 16 Mar. 1974; accepted 9 May 1974. 1. PATH.-VOL
115 (19751
245
JENNIFER McDOUGALL AND J. F. SMITH
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50 : 50 to 25 : 75. The developing respiratory bronchioles, with columnar epithelium become lined in some regions by uniform cuboidal cells (fig. 1). Ultrastructural study of these cells in their early development shows a central nucleus surrounded by a large area of sparse glycogen. A few small mitochondria and short cisternae of rough endopla:\mic reticulum (RER) lie around the periphery, especially near the luminal surfsce. As the cells mature the glycogen increases and small to medium dense bodies are seen more frequently, sometimes associated with a Golgi region (fig. 2). As development proceeds after 20 wk, the proportion of interstitial area to epithelium further decreases. The pseudoglandular appearance of the lung is completely lost and the expanded bronchioles no longer appear readily distinct from the surrounding cellular stroma. Moreover, capillaries are seen in obvious contiguity with the epithelium and some cells of the latter are attenuated (fig. 3). These type I pneumocytes are identifiable at 22 wk. The ultrastructural characteristics of these cells include a diminution in glycogen and an extension along the air-spaces of their cytoplasmic processes with sparse distribution of mitochondria, ribosomes, RER and occasional dense bodies (fig. 4). Between 22 and 24 wk a few of the other cuboidal epithelial cells, destined to become type I1 pneumocytes, show membrane-enclosed glycogen-free spaces in the cytoplasm (fig. 5). Sometimes dense bodies are in contiguity with these membranes. At about 24 wk the cytoplasmic differentiation of type I1 cells is dominated by the appearance of multiple membrane bound lamellar inclusion bodies (figs. 6 and 7 ) . The changes preceding and associated with this differentiation include : (i) (ii) (iii) (iv) (v) (vi)
Changes in glycogen. Lamellae and lattice structures in dense bodies. Opaque material in dilated cisternae of RER. Multivesicular bodies (MVBs) and increase in Golgi vesicles. Microtubules in the cytoplasm. Development and extension of microvilli at the luminal surface.
The changes in glycogen occur within focal areas of the cytoplasm, usually bound by a unit membrane. From 22 wk, glycogen-free spaces appear (fig. 5). In already differentiated type I1 cells areas of cytoplasm, devoid of organelles other than sparse glycogen are associated with a few lamellar membranes (fig. 8). Also from 22 wk, membrane limited areas may include both glycogen and some electron-dense material (fig. 9). Multiple membranes may also develop in association with these (fig. 10). Different stages of development of the inclusion bodies can be found in a single cell (fig. 11). Moreover, there is a progressive decrease in glycogen as the IBs are formed. During this time the membrane bound dense bodies become more numerous and some enlarge. Their internal structure is granular and in certain areas multiple parallel membranes may be seen (fig. 12). These are in close arrayeach unit membrane separated from the next by 10 nM and eight such membranes are seen forming within a segment of the dense body (fig. 12). The inner two membranes have a more complicated structure, viz., alternate light and
MCDOUGALL AND SMITH
PLATECXLI
DEVELOPMENT OF PNEUMOCYTE
FIG. 1.-18 wk. The bronchioles which are lined by columnar or cuboidal cells are separated by a wide interstitial area. Two mitotic figures are seen in the primitive bud. 1 pm Toluidine blue. x 640.
FIG.2.-16 wk. Cuboidal cells lining a bronchiole have central nuclei surrounded by glycogen. Sparse mitochondria and cisternae of RER lie around the periphery and therc are a few dense bodies. EM. X 8400.
FIG. 3.-23 wk. Bronchioles with cuboidal lining cells have developing air spaces almost in contiguity with capillaries because of attenuation of some of the epithelial cells. 1pm. x 640.
PLATECXLII
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DEVELOPMENT OF PNEUMOCYTE
FIG.4.-Apical cytoplasm of cuboidal cells .probably de- FIG.5.-22 wk. The cytoplasm of a cuboidal cell is filled with glycogen, sparse mitochondria and cisveloping into type I1 pneumocytes and an attenuated ternae of RER lie peripherally. There are some cell in contiguity with a capillary. The microvilli are small dense bodies, a Golgi region and a few lost at the area of complete attenuation. EM. x 8000. smooth vesicles. A membrane is seen in the glycogen-free area. Cytoplasmic protruberances project into the lumen. EM. X 5600.
FIG.6.-24 wk. Type I1 pneumocytes with dense inclusion bodies mainly in the apical cytoplasm; a few have a vacuolar appearance. Several capillaries are overlaid by attenuated cells. 1 pm Toluidine blue. x 640.
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FIG.7.-24 wk. Type I1 pneumocytes with characteristic lamellated inclusion bodies. Mitochondria, RER and microvilli are clearly seen. EM. X 5000.
FIG.8.-24 wk. Part of the cytoplasm of a type I1 cell, showing part of a lamellated inclusion body and a membrane bound glycogen area with several membranes forming. EM. ~ 2 3 , 2 0 0 .
PLATE CXLlV
MCDOUGALL AND SMITH DEVELOPMENT OF PNEUMOCYTE
FIG.9.-22 wk. Part of the cytoplasm of a differentiating cuboidal cell. Adjacent to the nucleus is an area of dense glycogen and electron-opaque material bound by a unit membrane. A smaller zone is also demarcated by a membrane which is in part triplicate ( t ). EM. x 19,000.
FIG.10.-22 wk. Part of the cytoplasm of a differentiating cuboidal cell showing an area of electrondense material and glycogen surrounded by a concentric series of membranes. EM. X 18,400.
MCDOUGALL AND SMITH
DEVELOPMENT OF PNEUMOCYTE
FIG.11.-24 wk. In a type I1 cell with formed lamellated inclusion bodies there are dense bodies associated with lamellar membranes, and a membrane-bound glycogen zone with a few concentric lamellations. Several intercellular spaces with microvilli separate adjoining cells.
EM. ~18,400.
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MCDOUGALL AND SMITH DEVELOPMENT OF PNEUMOCYTE
FIG.12a.
FIG.12b.
FIG.120 and b.-24 wks. A large, membrane bound body is filled with dense granular material. In one area a close array of parallel membranes, separated by 10 n M are seen (f). The innermost membranes have a more complicated structure reminiscent of that of myelin (tt). EM. (a) X 69,000. (b) X 118,000.
FIG.13.-24
wk. A large dense body with a lattice structure and some membranes surrounding it. The sides of the lattice are about 13 n M apart. EM. ~74,000.
MCDOUGALL AND SMITH
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DEVELOPMENT OF PNEUMOCYTE
FIG.14.-24 wk. Part of the cytoplasm of a mature type I1 cell. Numerous microvilli protrude into the lumen on the left. There are many ribosomes and cisternae, some dilated, of RER and one inclusion body is closely apposed by a cisterna. There is a large MVB and some small Golgi vesicles. Glycogen is sparse. EM. x 30,400.
MCDOUGALL AND SMITH
PLATECXLVIII DEVELOPMENT OF PNEUMOCYTE
FIG. 15.-24 wk. The intercellular connexions between two type I1 cells. Tight junctions persist at the luminal end and intercellular spaces with microvilli. The cytoplasm of the left cell, in which part of three inclusion bodies are seen, has numerous dilated cisternae of RER and microtubules. EM. x 20,900.
FIG. 16.-25
wk. An inclusion body partly extruded through the plasma membrane, between the microvilli. EM. x 30,400.
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dark bands 4 nM wide within dense lines which are here 20 nM apart. This is similar in structure to myelin (12 nM between the thick dense lines and bands 3 nM wide). In three cases a lattice structure with sides approximately 13 nM was seen within a dense body (fig. 13). The RER with slightly dilated cisternae containing moderately electron opaque material (figs. 7 and 14) is seen extensively in five cases. At times the RER surrounds IBs but in no case is it in direct continuity with the lamellae of IBs as described in the anencephalic rat by Blackburn, Travers and Potter (1972). Rough-surfaced vesicles, often dilated, and containing granular material are seen in cells with numerous IBs (fig. 15). Mitochondria increase in number and elongate, but show no internal structural change, during this differentiation. They are distributed more generally through the cell and at this time the glycogen is diminished. MVBs are seen in differentiating cuboidal cells with membrane-bound glycogen-free or sparse areas and/or dense bodies. They are, however, more commonly seen in differentiated type I1 cells (figs. 8 and 14). The Golgi vesicles are also more common at 24 wk. The association of lamellar IBs with MVBs is seen infrequently, but sometimes vesicles surround the former. From about 20 wk some cuboidal cells have cytoplasmic protrusions into the lumen. In type I1 cells containing IBs, numerous well-formed microvilli are seen, presumably derived from the cytoplasmic extensions (fig. 14). They have an inner structure of microfilaments and microtubules. Microtubules are also seen beneath the cell membrane, at the luminal surface and deeper within the cytoplasm of cells containing numerous IBs (fig. 15). Occasionally IBs are seen partly extruded between the microvilli (fig. 16).
Summary of diferentiation of type II cells The type I1 pneumocyte develops from a cuboidal cell with cytoplasm largely filled with glycogen. The process of differentiation involves cytoplasmic changes which indicate active synthesis. The progressive decrease in glycogen with these changes and the appearance of the inclusion bodies may indicate its breakdown, partly for energy requirement and partly for either the participation or the incorporation of simpler molecules in the inclusion body formation. The increase in number and size of mitochondria suggests increased aerobic metabolism, while the increase and dilatation of the RER probably indicates protein synthesis. Extension of the Golgi system, the MVBs and the increase in number and size of the membrane-bound dense bodies with a complex internal structure precede the appearance of the lamellated inclusion bodies. These are extruded through the luminal plasma membrane. DISCUSSION The essential point to be considered is the relevance of the ultrastructural changes to the function of the cell. It is convenient to consider the biological function of the cell separately from its biochemical function in the first instance. The point at issue in the
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literature is whether the cell's primary function is synthesis or phagocytosis. The great majority of investigators of (animal) lung have thought that the type I1 pneumocyte makes and secretes pulmonary surfactant (Macklin, 1954) which is essential for the lowering of surface tension to enable normal respiration to continue. A few have suggested that the surfactant is made by the non-ciliated cell, the Clara cell (Niden, 1967; Etherton et al., 1973) of the bronchiole and that the inclusion bodies in type I1 pneumocytes indicate phagocytosis and breakdown of excess surfactant. The investigation by Etherton et al. depends on the earlier incorporation of labelled surfactant precursor (palmitic acid) into dipalmitoyl lecithin in the Clara cells of the terminal bronchioles. However, the bulk of the experimental evidence-summarised by Heppleston and Young (1973) is against any marked phagocytic role for the type I1 pneumocyte. This does not exclude the possibility of the Clara cell having a role in surfactant synthesis after birth. The sequence of events in hyaline membrane disease suggest that it is not the main source in foetal and early neonatal life. Taghizadeh and Reynolds (1975) have shown that in the initial stages of this disease the surface tension rises and the inclusion bodies in type I1 pneumocytes are absent. After a few days the inclusion bodies reappear and the surface tension falls, indicating an association of surfactant secretion with the bodies at a time when the epithelium of the terminal airways with its Clara cells is still destroyed and that of the bronchi is damaged. Furthermore, we think that the ultrastructural changes summarised above point to a synthetic role for the cell, while freely admitting that the endproduct of the synthesis-the lamellated inclusion bodies-resemble in some ways cytosegresomes (Ericsson, 1969) and the membranous cytoplasmic bodies of GM2 gangliosidosis (Tay Sachs disease) and other storage diseases in which there is degradation of excessive unwanted material by lysosomes. We would suggest that the close association of the dense bodies with the developing inclusion bodies (figs. 9-12) is significant; their role may be to provide some of the enzymes necessary for the construction of the inclusion bodies as well as for their discharge from the cells in the process of reversed endocytosis rather than phagocytosis. This does not exclude a role for the intracytoplasmic microtubules in the discharge process also (fig. 15). The changes in glycogen are the earliest in the series of ultrastructural events. One obvious explanation is that glycogen is utilised to a considerable degree for anaerobic glycolysis before the increase in mitochondria provides a greater source of aerobic metabolism. However, the earliest membranes of the inclusion bodies appear in unit-membrane-enclosed glycogen-free or glycogen rich spaces (figs. 5, 8 , 9 and 10). In the rabbit Kikkawa et al. (1971) also found striking changes in glycogen and suggested that these were associated with the synthesis of lecithins through the coupling of diglyceride to CDP-choline, the former being provided by glycogenolysis. However, in the human foetus the predominant pathway of lecithin synthesis is the methyl transferase (Gluck and Kulovich, 1973) and their explanation would need modification. Whether these early membranes sediment with the microscomal fraction in which Gluck
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and his associates (1967a and b) found both pathways of lecithin synthesis or the mitochondria is uncertain. The methyl transferase pathway appears about 22-24 wk in the human-at the same time as the changes in glycogen begin. There remains to be considered the role of protein synthesis as indicated by the active endoplasmic reticulum (figs. 7, 14 and 15). This was first emphasised by Sorokin (1967) in marsupials, bats and rodents and thought by him to be concerned with synthesis of the enzymes required for the complex metabolic processes involved in the inclusion body formation. This is probably part of the story, but the lattice and lamellar structures seen in developing inclusion bodies (figs. 12 and 13) suggest that protein is built into the bodies. Indeed, the assembly of the bodies may well be a later event than lecithin and protein synthesis. If the bodies are lipoprotein complexes this would fit with the work of Mingins and Taylor (1973) who suggest that a lipoprotein rather than a monolayer of dipalmitoyl lecithin is necessary to provide adequate hysteresis for lung distensibility. We have not, however, in our material seen the intimate association of inclusion bodies with rough endoplasmic reticulum described by Blackburn, Travers and Potter (1972) in the anencephalic foetal rat. Many authors (Balis and Conen, 1964; Sorokin, 1967; Kikkawa et al., 1968; Kikkawa and Spitzer, 1969; Meyrick and Reid, 1973) have stressed the importance of the MVB in the formation of the inclusion bodies. Kikkawa and Spitzer especially observed fusion of MVB with dense bodies. We have occasionally observed such a phenomen, and IBs surrounded by vesicles. As these organelles, together with an increased Golgi zone are common in the differentiating type I1 pneumocytes we suggest that they may act in transporting enzymes or the building proteins to the inclusion bodies. The earlier suggestion that the inclusion bodies are transformed mitochondria (Kisch, 1955) received no support from our observations. We saw no structural alterations within them and would attribute the increase in their number, size and distribution in the developing type I1 cell to increase in aerobic metabolism as the glycogen diminished. However, Kikkawa et al. (1968) saw no increase in mitochondria at a comparable stage in the differentiation of type I1 cells in the foetal rabbit. Our conclusion is therefore that the formation of lamellated inclusion bodies in the type I1 pneumocytes is a synthetic process. Changes in glycogen and participation of the dense bodies are striking. Lattice and lamellar formations within dense bodies are thought to indicate an assembly of protein and lipid in the formation of the inclusion bodies which are then extruded into the lumen of air sacs. In a recent paper Di Augustine (1974) has found a phospholipid to protein ratio of 12 : 1 in inclusion bodies isolated from adult rabbit lung, and demonstrated that they contain hydrolases similar to those in lysosomes. He has discussed a possible regulating function for these bodies which are unique in mammalian physiology. SUMMARY
Portions of lung from 25 human foetuses between 12 and 26 wk of gestational age were examined for ultrastructural changes during maturation of the
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epithelial cells. In 10 cases type I pneumocytes and in five cases type I1 cells were differentiated. The changes associated with the formation of lamellar inclusion bodies in type I1 cells are described. It is concluded that this is a synthetic process in which protein and lipid assemblyare important. Changes in glycogen and participation of dense bodies are striking. REFERENCES P. E. 1964. The role of alveolar inclusion bodies in the developing BALIS,J. V., AND CONEN, lung. Lab. Invest., 13 (LO), 1215. BJARNSTAD, P., AND BREMER, J. 1966. " In vivo " studies on pathways for the bio synthesis of lecithins in the rat. J. Lipid Res., 7 , 38. BLACKBURN, W. R., TRAVERS, H., AND POTTER,M. 1972. The role of the pituitary-adrenalthyroid axes in lung differentiation. Lab. Invest., 26 (3), 306. CAMPICHE, M. A., GAUTIER,A., HERMANDEZ, E. I., AND REYMOND, A. 1963. An electron microscope study of the foetal development of human lung. Ped., 32. 976. DI AUGUSTINE, R. P. 1974. Lung concentric laminar organelle. J. Biol. Chem., 249, 584. EHICSSON, J. 1969. Lysosomes in biology and pathology, edited by J. T. Dingle and H. B. Fell, North Holland Publ. Co., Amsrerdam, vol. 2, p. 354. ETHERTON, J. E., CONNING, D. M., A m CORRIN,B. 1973. Autoradiographical and morphological evidence for apocrine secretion of dipalmitoyl lecithin in the terminal bronchiole of mouse lung. Am. Jour. Anat., 138 (l), 11. GLUCK, L., AND KULOVICH, M. V. 1973. The pediatric clinics of North America, edited by L. Cluck, Saunders, Philadelphia, vol. 20 (2), p. 370. GLUCK, L., KULOVICH, M. V., EIDELMAN, A. I., CORDERO, L., AND KHAZIN,A. F. 1972. Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in human foetus and newborn and etiology of the respiratory distress syndrome. Ped. Res., 6, 81-99. CLUCK, L., MOTOYAMA, E. K., SMITS,H., AND KULOVICH,M. V. 1967. Biochemical development of surface activity in mammalian lung. I. The surface active phospholipids; the separation and distribution of surface active lecithin in the lung of the developing rabbit foetus. Ped. Res., 1, 237. GLUCK, L., SRIBNEY,M., AND KULOVICH,M. V. 1967. The biochemical development of surface activity in mammalian lung. 11. The biosynthesis of phospholipids in the lung of the developing rabbit fetus and newborn. Ped. Res., 1, 247. HEPPLESTON, A. G., AND YOUNG,A. E. 1973. Uptake of inert particulate matter by alveolar cells: an ultrastructural study. J. Pafh., 111, 159. KIKKAwA, Y., KNBARA,M., MOTOYAMA, E. K.,ORZALESI, M. M., AND COOK, c. D. 1971. Morphologic development of foetal rabbit lung and its acceleration with cortisol. Am. Jour. Path., 64 (2), 423. KIKKAWA, Y., MOTOYAMA, E. K., AND CLUCK, L. 1968. Study of the lungs of foetal and newborn rabbits. Am. Jour. Path., 52, 177. KIKKAWA,Y.,AND SPITZER,R. 1969. Inclusion bodies of type I1 alveolar cells: Species differences and morphogenesis. Anat. Rec., 163, 525. KISCH,B. 1955. Electron microscopic investigation of the lungs (capillaries and specific cells). Exp. Med. Surg., 13, 101. MACKLIN, C. C. 1954. The pulmonary alveolar mucoid film and the pneumocytes. Lancet, i, 1099. MEYRICK, B., AND REID,L. 1973. Electron microscopic aspects of surfactant secretion. Proc. Roy. SOC.Med., 66, 6. MINGINS,J., AND TAYLOR, J. A. G. 1973. Physicochemical properties of phospholipid " monomolecular layers ". Proc. Roy. SOC. Med., 66, 383. NIDEN, A. H. 1967. Bronchiolar and large alveolar cell in pulmonary phospholipid metabolism. Science, 158, 1323.
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O’HARE,K. H., AND SHERIDAN, M. N. 1970. Electron microscopic observations on the morphogenesis of the albino rat lung, with special reference to pulmonary epithelial cells. Am. Jour. Anat., 127, 181. SOROKIN, S. P. 1967. A morphologic and cytochemical study on the Great alveolar cell. Jour. Histochem. and Cytochem., Id (12), 884. TAGHIZADEH, A., AND REYNOLDS, E. 0. R. 1975. The pathogenesis of bronchopulmonary dysplasia following hyaline membrane disease. In course of preparation.
