Ultrastructure of In Vitro Type 2 Epithelial Cell Cysts Derived from Adult Rabbit Lung Cells R. M . ROSENBAUM, P. PICCIANO, Y. KRESS AND M. WITTNER Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461
ABSTRACT Fragments of adult rabbit lung, composed chiefly of terminal airway obtained by a trypsin digestion technique were maintained on collagencoated cellulose sponges i n Ham's F12 medium. Cell-sponge associations were examined with light microscopy, scanning and transmission electron microscopy over a period from 6 to 28 days. After a n initial 24- to 48-hour period of cell migration from the airway fragment, sponge matrices became lined with cells suggestive of alveolar macrophages. After one week in culture, cysts appeared to be composed entirely of type 2 epithelial cells. These were characterized by a microvillous apical border and a n elaborate junctional complex. The lumen of these cysts contained both myelin-like lamellar configurations and tubular myelin structures such as have been described from pulmonary washings. Consistent with the age of the sponge cultures, one or more cyst types described as young, middle and late could be found simultaneously. Middle aged cysts showed signs of active secretion into the lumen. Late cysts showed changes in the epithelium comprising the cyst wall suggestive of a cell type intermediate between type 1 and type 2 epithelial cells.
The type 2 epithelial cell of the mammalian lung is the important secretory cell of the terminal airway (Askin and Kuhn, '71; Chevalier and Collet, '72; Adamson and Bowden, '73). In addition to its role as a site for synthesis and secretion of surface active phospholipids, the type 2 cell is assumed to play a major role in repair of alveolar injury (Kapanci et al., '69) and may contribute significantly to rendering animals tolerant to otherwise injurious agents (Rosenbaum et al., '69, '73; Yamamot0 et al., '70). There is also evidence that the type 2 cell can serve as precursor for the other major alveolar lining celltype, the squamous, type 1 epithelial cell (Adamson and Bowden, '74; Evans et al., '75). To date, studies such as the above have been done with the cell types in question present in intact lung, but it would be of great value to repeat and expand such studies using cultured lung type 2 cells still retaining structural and functional characteristics indicative of in situ activity. These include presence of the lamellar cytosome with its membrane-bound stacked or whorled osmiophilic contents as well as visualization of extracellular ANAT. Rxc., 188: 241-262.
secretory products such as accumulate in the lung alveoli (Harrison and Weibel, '68; Weibel et al.,'66; Gil and Reiss, '73). Reaggregation of primary isolated cell types frequently form histotypic structures still capable of maintaining the differentiated state. This has been achieved largely using monodispersed fetal or embryonic cells while aggregation of cell types from adult organs has been attempted relatively infrequently. Adult lung has been described as being composed of some 40 cell types (Sorokin, '70), so that maintenance of the differentiated state may depend on interactions between one or more of these cells. The purpose of the present investigation was to ascertain whether any cells of the diverse lung cell population possess capacity for self-recognition and ability to sustain the differentiated state in vitro. This paper describes the ultrastructure of type 2 cell aggregates formed from cells derived from isolated terminal airways of adult rabbit lung cultured on three dimenReceived June 8, '76. Accepted Nov. 19, '76. 'Supported by a Program Project HL-16137 and a Contract HR5-2952 from the National Heart and Lung Institute.
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sional sponge matrices. Of particular interest to us were the changes in these aggregates with age correlated with structural evidence suggestive of secretion. MATERIALS AND METHODS
Preparation of terminal airway fragments Young adult male New Zealand white rabbits (0.5-1.5 kg) were lightly heparinized (100 units/kg) and anesthetized with Nembutal (100 mg/kg). The trachea was cannulated and together with the heart and lungs, removed en bloc. The pulmonary trunk was catheterised with a No, 15 polyethylene catheter via the right ventricle; the heart itself was dissected away from the pulmonary vein and that portion from the level of the cross-section of both auricles down to the apex removed. This preparation, with the trachea open to room air, was placed in a sealed lucite perfusion chamber in which a slight vacuum was produced and released rhythmically, expanding and releasing the lungs. Approximately 50 ml of sterile, phosphate buffered Puck’s saline A, pH 8.0 (Kruse and Patterson, ’73) supplemented with 1% fetal calf serum and 300 units of heparin were perfused into the catheter at a rate of 4 ml/ min using a Harvard syringe pump. This was followed by 400 ml of 0.10% trypsin (Difco 1:250) in sterile buffered saline (pH 8.0) at room temperature infused over 30 minutes. At the conclusion of this step, the lungs appeared translucent and were exceedingly fragile. The lungs were carefully removed from the perfusion chamber and the well-perfused pure white regions about the hila and at the base of both lungs were removed and cut into 2.0 x 2.0 mm pieces. These fragments were then placed in 30 ml of sterile buffered saline, poured into the barrel of a 20 ml syringe and passed twice through a No. 16 Wintrobe needle. This was followed by an additional two passes through a No. 18 hypodermic needle. This procedure yielded several dozen fragments suitable for culture. Preparation of cellulose sponges Fine pore, dry cellulose sponge was cut into fragments 1.0 X 0.5 X 0.5 mm and placed in triple distilled water for 30 minutes. Slices with a maximum of 2.0 mm
thickness were boiled in glass distilled water for one hour, immersed in acetone, ethyl ether and finally, in 100% ethyl alcohol for 30 minutes each. After repeating this washing procedure, the sponges were air dried and kept under sterile conditions. Just prior to use, dried sponge fragments were impregnated with dispersed collagen after the method of Leighton (‘51; see also Kruse and Patterson, ’73). Culture conditions Between 24 to 36 terminal airway fragments, one to two per sponge, were placed onto the surface of collagen-coated sponges contained in 60 mm plastic Falcon petri dishes. The sponges were immersed in Ham’s F12 medium to which antibiotics had been added as follows: 100 pglml Kantrex, 7.5 pg/ml Fungizone; 100 units/ ml penicillin G ; 100 pg/ml streptomycin sulfate. Cultures were maintained under 5% COz at 36°C with high relative humidity to minimize evaporation. Electron microscopy Sponges were examined by light microscopy daily and removed from the culture medium at selected intervals beginning with six days, up to 28 days. They were washed in buffered saline (pH 7.2) and fixed for three hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Following a one hour rinse in buffer, sponges were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.2. Fixed fragments of terminal airway as well as sectioned sponge fragments containing cells were critically point dehydrated, gold coated and examined with an IS1 SuperMiniscan scanning electron microscope; other sponges were dehydrated, embedded in Araldite-Epon and sectioned for both light microscopy at 0.2 p and transmission electron microscopy. Grids were stained in uranyl acetate and lead, and examined with a Siemens 102 electron microscope. Quantitative examination We examined 320 cysts for this study, approximately equally divided amongst all three age groups. Initial screening of cystic structures developing in sponge was with 0.2 p sections using the light microscope
24 3
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG TABLE 1
Age characteristics of adult rabbit type 2 lung epithelial cysts in vitro Time of maximal appearance in culture 1
Characteristics
1
Frequency of appearance relative to all stages (early :middle :late)
Early
Cyst fully formed but little or no lumenal contents present; cyst wall cells low to medium height with poorly developed microvillous borders and few lamellar cytosomes.
8-12 days
1:o:o
Middle
Cyst wall cells at maximal height with numerous lamellar cytosomes; rich lumenal contents with uniform myelin figures and tubular myelin forms present; osmiophilic granular material present in lumen to varying degree.
12-16 days
1 :6:O
Late
Cyst wall cells reduced in height and elongate with some lamellar cytosomes present and with vacuoles containing osmiophilic granular material; lumenal contents with few tubular myelin configurations but pleomorphic myelin figures predominate.
16-24 days
0:2:5
Calculated from time of initial placement of lung fragments on sponge.
where their frequency of appearance in each culture relative to age was determined by direct count (table 1). RESULTS
The general surface appearance of trypsinized lung fragments placed on sponges is shown in figure 1. Such fragments were able to provide a heterogeneous lung cell population so that within several hours, a variety of cell types, as judged by differences in cell shape and size, were seen to migrate out of the fragment. By 24 to 48 hours, large numbers of cells could be seen lining the surfaces of the collagenized sponge matrix (fig. 2). Transmission electron microscopy of these surfaces (fig. 3) revealed those to be irregularly shaped, elongate cells possessing extended cytoplasmic processes that interdigitated with other cells of the same type adhering to the walls of the cellulose sponge matrix. Cells migrated throughout the sponge matrix and were seen lining sponge surfaces (fig. 3). Virtually all these cells showed numerous osmiophilic inclusions and “empty” vacuoles, most of which were not membrane bound. Cystic structures began to appear by day 7 in culture. Their typical appearance
at this time is shown in a scanning electron micrograph (fig. 4). Clusters of cells, some rounded, others irregular in outline, could be seen within the recessess of the sponge matrix. Over the next seven days, more such cell clusters appeared. Examination of these cystic structures by both scanning and transmission electron microscopy took place over the next two weeks. Transmission electron microscopy revealed a structural sequence with respect to appearance and cellular makeup of cystic structures. Earliest formed cysts (fig. 5) varied in cross-section from 1630 and had lumina surrounded by somewhat elongate cells 2-3 p in height. The apical surface of these cells had few microvilli while their basal surfaces were in close contact with processes of the irregularly shaped extracystic cells. The cells comprising the walls of such young cysts had few lamellar cytosomes; the cyst lumen showed a sparse accumulation of extracellular material (fig. 5). In older cultures (12-16 days), many cysts appeared to be involved in active secretion as judged by their appearance (fig. 6A). In these, which can be regarded as middle aged cysts, cells forming the cyst wall were increased in height averaging 4.5 p and
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their apical borders showed numerous, well developed, microvilli (fig. 7A). The lumina of these cysts (figs. 6, 7A) were filled with two kinds of structures: a fine granular, electron dense osmiophilic material and numerous myelin figures and tubular myelin (fig. 6B). Measurements of tubular myelin within the cyst lumen showed transverse dimensions averaging 45 nm with a dense core of 15-18 nm across. In most cases, the osmiophilic membranes were oriented close to or at a 90” angle to each other. The concentric lamellar myelin structures in the cyst lumen were never seen to be membrane-bounded. The cells constituting the cyst wall could be judged to be type 2 epithelial cells by several criteria. All cyst wall cells contained varying numbers of membrane bound cytosomes frequently containing an osmiophilic dense body (fig. 7A). As is typical with rabbit type 2 epithelial cells, the contents of these cytosomes appeared weakly osmiophilic and homogeneous rather than of a darkly staining lamellar configuration as seen in other species (Littman et al., ’74) Mitochondria were slightly reduced in size from those seen in rabbit pulmonary type 2 epithelial cells in situ but retained their overall shape as seen in intact lung sections. Junctional complexes (fig. 7B) consisting of occluding and adhering zonules and accompanying desmosomes were evident in all cysts (fig. 7A). Many middle aged secreting cysts were large and cuboidal, often having crosssections of 20 p or more across and many assumed an irregular outline (fig. 8A). In these, the lumina were filled with large numbers of uniformly sized myelin figures as well as an osmiophilic fine granular extracellular material. We could not detect evidence of “tubular” myelin. With day 16 to 24 in culture, the cells forming the wall of late cysts were reduced in height (1-2 p ) in comparison with those forming the walls of young or middle aged cysts (fig. 9). While some of these older cells still had lamellar cytosomes in their cytoplasm, most did not. However, osmiophilic inclusions were generally present as well as vacuoles containing fine granular material (fig. 10). Large sized late cysts formed of elongate cells had their lumen filled with myelin figures of varying size and shape (figs. 9, lo), while the granular os-
miophilic material was nearly absent. Tubular myelin configurations, on the other hand, could not be detected in these lumina in significant amounts. Also, extracystic cells appeared to contain many more vacuoles than were evident in the younger cultures (fig. 9). A survey of all cultures from the seventh to twenty-fourth day revealed a distribution of the above described cysts consistent with age of the culture with some degree of overlapping. These are outlined in table 1. Thus, younger cultures (day 8-12), only contained early cyst stages. Cysts showing signs of secretion and regarded as “middle aged”, appeared predominately in cultures 12 to 16 days old. Such middle aged cysts were seen together with early cysts in a frequency of six of the former to one of the latter. No late cysts could be seen at this time. Late cysts were detected in cultures 16 to 24 days old where they appeared in a ratio of five late for every two middle aged cysts. DISCUSSION
The recombination of dispersed mammalian cells to form what are frequently referred to as histotypic aggregates has mostly been undertaken using cells from fetal or new-born animals (Pessiac and Defendi, ’72a,b; Douglas and Teel, ’75). Adult mammalian cell aggregation has been described in the case of liver cells maintained in vitro for up to 96 hours (Alwen and Lawn, ’74) and thyrotropin-stimulated thyroid epithelium (Kalderon and Wittner, ’67; Girard et al., ’74). The present report is, therefore, among the few describing adult mammalian cells aggregated in vitro to constitute a unit whose cells still maintain in situ characteristics. In the present paper we describe type 2 epithelial cell cysts formed from cells originating from tissue fragments of the terminal airway of adult rabbit lung. Since these fragments were derived from lung perfused with trypsin via the vascular route, it is reasonable to assume they were composed of loosely adherent, viable cells that were seen to migrate into a 3-dimensional sponge matrix. Of these, only two specific cell types formed a close relationship to the matrix. The initial migratory cell population lined the surface of the collagenized sponge matrix within 24 to 48
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG
hours. On the basis of aspects of their ultrastructure, their irregular shape and the presence of numerous extended processes, these initial cells may be alveolar macrophages containing large numbers of osmiophilic digestive vacuoles and neutral lipid. The life span of macrophages in culture has been thought to be limited although some evidence of relatively long survival of pulmonary macrophages in culture has been presented (Sonderland and Naum, ’73). We have seen no evidence of division in these extracystic cells by direct observation; nor have we seen evidence of the presence of other cell types such as fibroblasts which could also serve to provide a supportive framework for the cystic structures. We do not know whether or not formation of type 2 cell cysts depends on the initial layer established by these extracystic cells. Within a week, cysts consisting of cells with structural characteristics clearly marking them as type 2 epithelial cells were present in our cultures. From this point on, it became possible to establish a frequency of distribution between young, active and aged cysts. The fact that such differently aged cysts could exist side by side in the same culture was presumably caused by a sustained rate of release of appropriate cells by the lung fragment into the sponge. Evidence that the cells forming the cysts we described are type 2 epithelial cells rests foremost with the presence of lamellar cytosomes which represent specialized lysosomes in these cells (Goldfischer et al., ’68). As with secondary lysosomes, these are membrane bound structures (Gil and Fkiss, ’73), frequently having a dense core, presumably a remnant of the multivesicular body associated with genesis of these structures (Kikkawa and Spitzer, ’69; Yamamot0 et al., ’70). As has been noted in situ and in isolated lamellar bodies with rabbit lung (Littman et al., ’74), the cytoplasmic inclusions we describe in our cultured type 2 rabbit cells remained more homogeneous than lamellar in form throughout our experiments. The luminal contents of type 2 cysts as described in this study and that of Douglas and Teel (‘75), are similar in appearance to whorls representing “membranous” components of alveolar exudate (Harrison and Weibel, ’68) and from fluids
245
of rat lung (Weibel et al., ’66). The presence of these configurations has been taken to represent water crystals of surface-active phospholipid (Schaffner et al., ’67; Weibel et al., ’66), as well as products of cell breakdown (Weibel et al., ’68; Sun, ’66). Both the dimensions and orientation of the osmiophilic components of tubular myelin agree with the observations of Weibe1 et al. (’66) on pulmonary washings. In addition, these authors also suggest continuity between lamellar structures and tubular myelin configurations. Of particular interest in mature type 2 cell cysts is the presence of elaborate junctional complexes, not unlike those seen in situ in lung (Schneeberger-Keeley and Karnovsky, ’68). Douglas and Teel (‘75) reported similar elaborate junctional complexes. Their presence is confined to epithelium restricting exchange of large molecules between the lumen they surround and the intercellular space (Friend and Gilula, ’72). Synthesis of desmosomes at sites where they do not usually occur is frequently encountered among aggregating cells of the same type (Overton, ’74). The amount and appearance of extracellular material in the lumen of cultured type 2 cell cysts as well as morphological differences in the cells forming the cyst walls suggest a sequence of secretory activity in culture. Such changes appear to encompass early aggregation of type 2 epithelium into young cysts, activation of a secretory mechanism and secretion into the cyst lumen with, finally, loss of secretory activity leading to eventual cyst breakdown. It is not unreasonable to assume that such events could account for many of the morphological features we describe such as increase in height of cyst wall cells, increase in the number of lamellar cytosomes and extracellular myelin configurations as the cyst assumes secretory activity. As cysts passed into the “aged” category, the cells comprising their walls became elongate, lost most of their lamellar cytosomes and developed both osmiophilic inclusions and cytoplasmic vacuoles containing fine granular material similar in appearance to that seen earlier in the lumen. Superficially, at least, type 2 cells constituting aged cyst walls assumed the appearance of a cell type described as intermediate between types 1 and 2 (Evans
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et al., ‘75; Adamson and Bowden, ’74). This change could account for the loss of the granular osmiophilic material from the lumen of aged cysts and the pleomorphic appearance of what remains of the myelin figures in the lumen as synthesis becomes reduced. Also, these “aged” cells may represent non-specific dedifferentiation common in long-term cultures of primary cells. LITERATURE CITED Adamson, I. Y. R., and D. H. Bowden 1973 The intracellular site of surfactant synthesis. Autoradiographic studies on murine and avian lung explants. Exp. Mol. Path., 18: 112-124. 1974 The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Inv., 30: 35-42. Alwen, J., and A. M. Lawn 1974 The reaggregation of adult liver cells maintained in uitro. Exp. Cell Res., 89:197-205. Askin, F., and C. Kuhn 1971 The cellular origin of pulmonary surfactant. Lab. Inv., 25:260-268. Chevalier, G., and A. J. Collet 1972 In uiuo incorporation of choline-H3, leucine-HS and galactose-Ha in alveolar type I1 pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy. Anat. Rec., 174:289-310. Douglas, W. H. J., and R. W. Tee1 1975 A n organotypic in uitro model system for studying pulmonary surfactant production by type I1 alveolar pneumocytes. Am. Rev. Resp. Dis., 113: 17-23. Evans, M. J., L. J. Cabral, R. J. Stephens and G. Freeman 1975 Transformation of alveolar type I1 to type I cells following exposure to NO2. Fxp. Mol. Path., 22: 142-150. Friend, D. S.,and N. B. Gilula 1972 Variations in tight and gap junctions in mammalian tissues. J. Cell Biol., 52: 758-776. Gil, J., and 0. Reiss 1973 Isolation and characterization and lamellar bodies and tubular myelin from rat lung homogenates. J. Cell Biol., 5 : 152-171 .8_ _ . - -~ Girard, A., G. Fayet and S. Lissitsky 1974 Thyrotropin-induced aggregation promoting factors of adult cultured thyroid cells. Fxp. Cell Res., 87:359-364. Goldfischer, S . , Y. Kikkawa and L. Hoffman 1968 The demonstration of acid hydrolase activities in the inclusion bodies of type I1 alveolar cells and other lysosomes i n the rabbit lung. J. Histochem. Cytochem., 16: 102-109. Harrison, G. A., and J. Weibel 1968 The membranous component of alveolar exudate. J. U1trastr. Res., 24: 334442. Kalderon, A. E., and M. Wittner 1967 Histochemical studies of thyroid cells i n long-term tissue culture. Ehdocrinol., 80: 797-807.
Kapanci, Y., E. R. Weibel, H. P. Kaplan and F. R. Robinson 1969 Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. 11. Ultrastructural and morphometric studies. Lab. Invest., 20: 101-118. Kikkawa, Y., and R. Spitzer 1969 Inclusion bodies of type I1 alveolar cells: species differences and morphogenesis. Anat. Rec., 163: 525541. Kruse, P. F., Jr. and M. K. Patterson, Jr. 1973 Tissue Culture: Methods and Applications. Academic Press, New York. Leighton, J. 1951 A sponge matrix method for tissue culture. Formation of organized aggregates of cells in vitro. J. Nat. Cancer Inst., 12: 545561. Littman, J., Y. Kress, M. F. Frosolono, R. M. Rosenbaum, G. Colacicco and E. M. Scarpelli 1974 A morphological and biochemical characterization of the lamellar inclusion bodies from rabbit alveolar pneumocytes. Fed. Proc., 33: 345. Overton, J. 1974 Cell junctions and their development. In: Progress i n Surface Science and Membrane Science. Vol. 8 . Academic Press, N.Y., pp. 161-208. Pessiac, B., and V. Defendi 1972a Evidence for distinct aggregation factors and receptors in cells. Nature (London), 238: 13-15. 1972b Cell aggregation: role of acid mucopolysaccharides. Science, 175: 898-900. Rosenbaum, R. M., M. Wittner and M. Lenger 1969 Mitochondria1 and other ultrastructural changes in great alveolar cells of oxygen-adapted and poisoned rats. Lab. Inv., 20: 516-528. Rosenbaum, R. M.,M. Wittner and E. Scarpelli 1973 Parameters in the pathophysiology and pathobiology of oxygen tolerance i n the rat lung. Amer. J. Path., 70:57. Schaffner, F., P. Felig and E. Trachtenberg 1967 Structure of rat lung after protracted oxygen breathing. Arch. Path., 83:99-107. Schneeberger-Keeley, E. E., and M. J. Karnovsky 1968 The ultrastructural basis of alveolarcapillary membrane permeability to peroxidase as a tracer. J. Cell Biol., 37: 781-793. Sonderland, S. C., and Y. Naum 1973 Growth of pulmonary alveolar macrophages in uitro. Nature (London), 245: 150-151. Sorokin, S. P. 1970 The cells of the lungs. In: Morphology of Experimental Respiratory Carcinogenesis. P. Nettlebaum, M. G. Hanna, Jr. and J. W. Deatherage, eds. U. S. Atomic Energy Symposium Series No. 21,Oak Ridge, Tennessee. Sun, C. N. 1966 Lattice structures and osmiophilic bodies in the developing respiratory tissue of rats. J. Ultrastructural Res., 15: 380388. Weibel, E.R., G. S. Kistler and G. Tondury 1966 A sterologic electron microscopic study of “tubular myelin figures” in alveolar fluids of rat lungs. Zeitschr. Zellforsch., 69: 418427. Yamamoto, E., M. Wittner and R. M. Rosenbaum 1970 Resistance and susceptibility to oxygen toxicity by cells of the gas-blood barrier of the rat lung. Am. J. Path., 59:409436.
PLATES
PLATE 1 EXPLANATION OF FIGURES
248
1
Scanning electron micrograph of a terminal airway fragment from trypsinized rabbit lung. Such fragments were placed on sponges and provided the source of cells for culture as described i n the text. Several lobular units with alveolar ducts and a respiratory bronchiole are visible. ac, alveolar epithelial cells; ad, alveolar duct; rb, respiratory bronchiole; bv, bronchiolar blood vessel; int, enzymatically released interstitum. X 3,000.
2
Light micrograph of a segment of cellulose sponge 24 to 48 hours following placement of lung fragments. The early accumulation of cells along the margin of collagenized sponge (arrow) can be seen. X 480.
3
Transmission electron micrograph of cells shown i n figure 2 lining collagenized sponge matrix between 24 to 48 hours following placement of lung fragment onto sponges. Note numerous osmiophilic inclusions and “empty” appearing vacuoles. Sponge material (SM) is frequently surrounded by cell processes (arrows). X 3,000.
4
Scanning electron micrograph of sponge exposed to rabbit lung fragments for one week. Clusters of cells forming early cysts (arrow) are seen i n the cavity of a sectioned sponge. X 4,800.
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P. Picciano, Y. Kress and M . Wittner
PLATE 1
249
PLATE 2 EXPLANATION OF FIGURE
5
250
Cross-section of a young type 2 epithelial cell cyst derived from lung fragments in contact with sponge for approximately ten days. Epithelial cells forming the cyst contain a number of lamellar cytosomes (small arrows) and have microvilli a t their apical border. The cyst lumen contains fine granular osmiophilic material as well as occasional myelin figures (arrow). Cells such as are present during early stages of tissue-sponge association appear outside the cyst. These continue to show large numbers of osmiophilic inclusions and contain “empty” vacuoles. X 9,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT L U N G R. M. Rosenbaum, P . Picciano, Y . Kress and M . Wittner
PLATE 2
25 1
PLATE 3 EXPLANATION OF FIGURES
6A,B
252
Actively secreting type 2 epithelial cell cyst from a 12-day culture. In the lumen, several osmiophilic lamellar structures are present a s well as lattice forms representing tubular myelin configurations. x 9,000. There are sites (6B, arrows), suggesting the derivation of the tubular myelin from the surface of the lamellar structures. X 45,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 3
253
PLATE 4 EXPLANATION O F FIGURES
254
7A
A group of four type 2 epithelial cells forming the wall of a middle aged cyst developing within a sponge matrix at day 14. Note the numerous membrane bound lamellar cytosomes, the contents of which frequently appear homogeneous in rabbit lung cells. Several cytosomes (Ic) show osmiophilic dense bodies. Some osmiophilic inclusions not related to lamellar cytosomes (small arrow) are also evident. The apical surface of all cells have numerous microvilli. All cells share a junctional complex (circle and 7B). The cyst lumen is filled with osmiophilic fine granular material, lamellar structures and lattices of tubular myelin configurations (large arrow). x 12,000.
B
Detail of a junctional complex between adjacent type 2 cells of a cyst wall. zo, zonular occludens; za, zonula adherens; ma, macula ad. herens. X 75,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 4
255
PLATE 5 E X P L A N A T I O N OF F I G U R E S
8A
A large irregularly shaped middle aged type 2 epithelial cell cyst derived from adult rabbit lung fragments on sponge for 14 days. The two arrows mark opposite cyst walls where these come close to each other at w h a t appears to be the isthmus of a bilobed cyst. Nearly all type 2 cells contain lamellar cytosomes (lc). The cyst lumen i s filled with numerous lamellar structures, myelin-like strands (ms) and a highly osmiophilic fine granular material. es, extracystic space. X 3,000.
B
Higher magnification of the luminal contents seen i n figure 8A.
x 9,000.
256
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 5
PLATE 6 EXPLANATION
9
258
O F FIGURE
General relationships between middle aged and old type 2 cell cysts a n d extracystic cell elements i n a n 18-day-old sponge culture. Two middle aged cysts (B) may be recognized by the height of the cuboidal epithelial cells forming the wall, a n d by the presence of lamellar cytosomes within these cells. The lower cyst is not sectioned through its lumen but the irregular, centrally-located l u m e n of the upper cyst reveals a n osmiophilic granular component with a few myelin figures. An old cyst (A) is marked by the greatly thinned out appearance of cells (arrows) forming the wall. There are small amounts of osmiophilic granular material in the lumen a n d numerous myelin figures of varying size. A number of extracystic cells, many with greatly extended processes, appear, between the cysts. These cells have numerous empty vacuoles presumably originally containing lipid and large numbers of osmiophilic dense bodies. X 3,000.
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG
PLATE 6
R. M. Rosenbaum, P. Picciano, Y. Kress and M. Wittiier
259
PLATE 7 EXPLANATION
10
260
OF FIGURE
Portion of the wall of a n aged cyst maintained in sponge culture for 18 days. Two type 2 epithelial cells containing lamellar cytosomes show a reduced number of microvilli. These cells also contain vacuoles filled with fine granular osmiophilic material (small arrows), some evident in the cyst lumen. The large amount of darkly staining osmiophilic granular material seen in younger cysts is gone from the lumen of such older cysts while the myelin figures appear irregular i n shape and are less numerous. Tubular myelin configurations are not present in great numbers. x 9,000,
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R . M . Rosenbaum, P . Picciano, Y. Kress and M . Wittner
PLATE '7
261
ABSTRACT Fragments of adult rabbit lung, composed chiefly of terminal airway obtained by a trypsin digestion technique were maintained on collagencoated cellulose sponges i n Ham's F12 medium. Cell-sponge associations were examined with light microscopy, scanning and transmission electron microscopy over a period from 6 to 28 days. After a n initial 24- to 48-hour period of cell migration from the airway fragment, sponge matrices became lined with cells suggestive of alveolar macrophages. After one week in culture, cysts appeared to be composed entirely of type 2 epithelial cells. These were characterized by a microvillous apical border and a n elaborate junctional complex. The lumen of these cysts contained both myelin-like lamellar configurations and tubular myelin structures such as have been described from pulmonary washings. Consistent with the age of the sponge cultures, one or more cyst types described as young, middle and late could be found simultaneously. Middle aged cysts showed signs of active secretion into the lumen. Late cysts showed changes in the epithelium comprising the cyst wall suggestive of a cell type intermediate between type 1 and type 2 epithelial cells.
The type 2 epithelial cell of the mammalian lung is the important secretory cell of the terminal airway (Askin and Kuhn, '71; Chevalier and Collet, '72; Adamson and Bowden, '73). In addition to its role as a site for synthesis and secretion of surface active phospholipids, the type 2 cell is assumed to play a major role in repair of alveolar injury (Kapanci et al., '69) and may contribute significantly to rendering animals tolerant to otherwise injurious agents (Rosenbaum et al., '69, '73; Yamamot0 et al., '70). There is also evidence that the type 2 cell can serve as precursor for the other major alveolar lining celltype, the squamous, type 1 epithelial cell (Adamson and Bowden, '74; Evans et al., '75). To date, studies such as the above have been done with the cell types in question present in intact lung, but it would be of great value to repeat and expand such studies using cultured lung type 2 cells still retaining structural and functional characteristics indicative of in situ activity. These include presence of the lamellar cytosome with its membrane-bound stacked or whorled osmiophilic contents as well as visualization of extracellular ANAT. Rxc., 188: 241-262.
secretory products such as accumulate in the lung alveoli (Harrison and Weibel, '68; Weibel et al.,'66; Gil and Reiss, '73). Reaggregation of primary isolated cell types frequently form histotypic structures still capable of maintaining the differentiated state. This has been achieved largely using monodispersed fetal or embryonic cells while aggregation of cell types from adult organs has been attempted relatively infrequently. Adult lung has been described as being composed of some 40 cell types (Sorokin, '70), so that maintenance of the differentiated state may depend on interactions between one or more of these cells. The purpose of the present investigation was to ascertain whether any cells of the diverse lung cell population possess capacity for self-recognition and ability to sustain the differentiated state in vitro. This paper describes the ultrastructure of type 2 cell aggregates formed from cells derived from isolated terminal airways of adult rabbit lung cultured on three dimenReceived June 8, '76. Accepted Nov. 19, '76. 'Supported by a Program Project HL-16137 and a Contract HR5-2952 from the National Heart and Lung Institute.
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sional sponge matrices. Of particular interest to us were the changes in these aggregates with age correlated with structural evidence suggestive of secretion. MATERIALS AND METHODS
Preparation of terminal airway fragments Young adult male New Zealand white rabbits (0.5-1.5 kg) were lightly heparinized (100 units/kg) and anesthetized with Nembutal (100 mg/kg). The trachea was cannulated and together with the heart and lungs, removed en bloc. The pulmonary trunk was catheterised with a No, 15 polyethylene catheter via the right ventricle; the heart itself was dissected away from the pulmonary vein and that portion from the level of the cross-section of both auricles down to the apex removed. This preparation, with the trachea open to room air, was placed in a sealed lucite perfusion chamber in which a slight vacuum was produced and released rhythmically, expanding and releasing the lungs. Approximately 50 ml of sterile, phosphate buffered Puck’s saline A, pH 8.0 (Kruse and Patterson, ’73) supplemented with 1% fetal calf serum and 300 units of heparin were perfused into the catheter at a rate of 4 ml/ min using a Harvard syringe pump. This was followed by 400 ml of 0.10% trypsin (Difco 1:250) in sterile buffered saline (pH 8.0) at room temperature infused over 30 minutes. At the conclusion of this step, the lungs appeared translucent and were exceedingly fragile. The lungs were carefully removed from the perfusion chamber and the well-perfused pure white regions about the hila and at the base of both lungs were removed and cut into 2.0 x 2.0 mm pieces. These fragments were then placed in 30 ml of sterile buffered saline, poured into the barrel of a 20 ml syringe and passed twice through a No. 16 Wintrobe needle. This was followed by an additional two passes through a No. 18 hypodermic needle. This procedure yielded several dozen fragments suitable for culture. Preparation of cellulose sponges Fine pore, dry cellulose sponge was cut into fragments 1.0 X 0.5 X 0.5 mm and placed in triple distilled water for 30 minutes. Slices with a maximum of 2.0 mm
thickness were boiled in glass distilled water for one hour, immersed in acetone, ethyl ether and finally, in 100% ethyl alcohol for 30 minutes each. After repeating this washing procedure, the sponges were air dried and kept under sterile conditions. Just prior to use, dried sponge fragments were impregnated with dispersed collagen after the method of Leighton (‘51; see also Kruse and Patterson, ’73). Culture conditions Between 24 to 36 terminal airway fragments, one to two per sponge, were placed onto the surface of collagen-coated sponges contained in 60 mm plastic Falcon petri dishes. The sponges were immersed in Ham’s F12 medium to which antibiotics had been added as follows: 100 pglml Kantrex, 7.5 pg/ml Fungizone; 100 units/ ml penicillin G ; 100 pg/ml streptomycin sulfate. Cultures were maintained under 5% COz at 36°C with high relative humidity to minimize evaporation. Electron microscopy Sponges were examined by light microscopy daily and removed from the culture medium at selected intervals beginning with six days, up to 28 days. They were washed in buffered saline (pH 7.2) and fixed for three hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Following a one hour rinse in buffer, sponges were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.2. Fixed fragments of terminal airway as well as sectioned sponge fragments containing cells were critically point dehydrated, gold coated and examined with an IS1 SuperMiniscan scanning electron microscope; other sponges were dehydrated, embedded in Araldite-Epon and sectioned for both light microscopy at 0.2 p and transmission electron microscopy. Grids were stained in uranyl acetate and lead, and examined with a Siemens 102 electron microscope. Quantitative examination We examined 320 cysts for this study, approximately equally divided amongst all three age groups. Initial screening of cystic structures developing in sponge was with 0.2 p sections using the light microscope
24 3
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG TABLE 1
Age characteristics of adult rabbit type 2 lung epithelial cysts in vitro Time of maximal appearance in culture 1
Characteristics
1
Frequency of appearance relative to all stages (early :middle :late)
Early
Cyst fully formed but little or no lumenal contents present; cyst wall cells low to medium height with poorly developed microvillous borders and few lamellar cytosomes.
8-12 days
1:o:o
Middle
Cyst wall cells at maximal height with numerous lamellar cytosomes; rich lumenal contents with uniform myelin figures and tubular myelin forms present; osmiophilic granular material present in lumen to varying degree.
12-16 days
1 :6:O
Late
Cyst wall cells reduced in height and elongate with some lamellar cytosomes present and with vacuoles containing osmiophilic granular material; lumenal contents with few tubular myelin configurations but pleomorphic myelin figures predominate.
16-24 days
0:2:5
Calculated from time of initial placement of lung fragments on sponge.
where their frequency of appearance in each culture relative to age was determined by direct count (table 1). RESULTS
The general surface appearance of trypsinized lung fragments placed on sponges is shown in figure 1. Such fragments were able to provide a heterogeneous lung cell population so that within several hours, a variety of cell types, as judged by differences in cell shape and size, were seen to migrate out of the fragment. By 24 to 48 hours, large numbers of cells could be seen lining the surfaces of the collagenized sponge matrix (fig. 2). Transmission electron microscopy of these surfaces (fig. 3) revealed those to be irregularly shaped, elongate cells possessing extended cytoplasmic processes that interdigitated with other cells of the same type adhering to the walls of the cellulose sponge matrix. Cells migrated throughout the sponge matrix and were seen lining sponge surfaces (fig. 3). Virtually all these cells showed numerous osmiophilic inclusions and “empty” vacuoles, most of which were not membrane bound. Cystic structures began to appear by day 7 in culture. Their typical appearance
at this time is shown in a scanning electron micrograph (fig. 4). Clusters of cells, some rounded, others irregular in outline, could be seen within the recessess of the sponge matrix. Over the next seven days, more such cell clusters appeared. Examination of these cystic structures by both scanning and transmission electron microscopy took place over the next two weeks. Transmission electron microscopy revealed a structural sequence with respect to appearance and cellular makeup of cystic structures. Earliest formed cysts (fig. 5) varied in cross-section from 1630 and had lumina surrounded by somewhat elongate cells 2-3 p in height. The apical surface of these cells had few microvilli while their basal surfaces were in close contact with processes of the irregularly shaped extracystic cells. The cells comprising the walls of such young cysts had few lamellar cytosomes; the cyst lumen showed a sparse accumulation of extracellular material (fig. 5). In older cultures (12-16 days), many cysts appeared to be involved in active secretion as judged by their appearance (fig. 6A). In these, which can be regarded as middle aged cysts, cells forming the cyst wall were increased in height averaging 4.5 p and
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their apical borders showed numerous, well developed, microvilli (fig. 7A). The lumina of these cysts (figs. 6, 7A) were filled with two kinds of structures: a fine granular, electron dense osmiophilic material and numerous myelin figures and tubular myelin (fig. 6B). Measurements of tubular myelin within the cyst lumen showed transverse dimensions averaging 45 nm with a dense core of 15-18 nm across. In most cases, the osmiophilic membranes were oriented close to or at a 90” angle to each other. The concentric lamellar myelin structures in the cyst lumen were never seen to be membrane-bounded. The cells constituting the cyst wall could be judged to be type 2 epithelial cells by several criteria. All cyst wall cells contained varying numbers of membrane bound cytosomes frequently containing an osmiophilic dense body (fig. 7A). As is typical with rabbit type 2 epithelial cells, the contents of these cytosomes appeared weakly osmiophilic and homogeneous rather than of a darkly staining lamellar configuration as seen in other species (Littman et al., ’74) Mitochondria were slightly reduced in size from those seen in rabbit pulmonary type 2 epithelial cells in situ but retained their overall shape as seen in intact lung sections. Junctional complexes (fig. 7B) consisting of occluding and adhering zonules and accompanying desmosomes were evident in all cysts (fig. 7A). Many middle aged secreting cysts were large and cuboidal, often having crosssections of 20 p or more across and many assumed an irregular outline (fig. 8A). In these, the lumina were filled with large numbers of uniformly sized myelin figures as well as an osmiophilic fine granular extracellular material. We could not detect evidence of “tubular” myelin. With day 16 to 24 in culture, the cells forming the wall of late cysts were reduced in height (1-2 p ) in comparison with those forming the walls of young or middle aged cysts (fig. 9). While some of these older cells still had lamellar cytosomes in their cytoplasm, most did not. However, osmiophilic inclusions were generally present as well as vacuoles containing fine granular material (fig. 10). Large sized late cysts formed of elongate cells had their lumen filled with myelin figures of varying size and shape (figs. 9, lo), while the granular os-
miophilic material was nearly absent. Tubular myelin configurations, on the other hand, could not be detected in these lumina in significant amounts. Also, extracystic cells appeared to contain many more vacuoles than were evident in the younger cultures (fig. 9). A survey of all cultures from the seventh to twenty-fourth day revealed a distribution of the above described cysts consistent with age of the culture with some degree of overlapping. These are outlined in table 1. Thus, younger cultures (day 8-12), only contained early cyst stages. Cysts showing signs of secretion and regarded as “middle aged”, appeared predominately in cultures 12 to 16 days old. Such middle aged cysts were seen together with early cysts in a frequency of six of the former to one of the latter. No late cysts could be seen at this time. Late cysts were detected in cultures 16 to 24 days old where they appeared in a ratio of five late for every two middle aged cysts. DISCUSSION
The recombination of dispersed mammalian cells to form what are frequently referred to as histotypic aggregates has mostly been undertaken using cells from fetal or new-born animals (Pessiac and Defendi, ’72a,b; Douglas and Teel, ’75). Adult mammalian cell aggregation has been described in the case of liver cells maintained in vitro for up to 96 hours (Alwen and Lawn, ’74) and thyrotropin-stimulated thyroid epithelium (Kalderon and Wittner, ’67; Girard et al., ’74). The present report is, therefore, among the few describing adult mammalian cells aggregated in vitro to constitute a unit whose cells still maintain in situ characteristics. In the present paper we describe type 2 epithelial cell cysts formed from cells originating from tissue fragments of the terminal airway of adult rabbit lung. Since these fragments were derived from lung perfused with trypsin via the vascular route, it is reasonable to assume they were composed of loosely adherent, viable cells that were seen to migrate into a 3-dimensional sponge matrix. Of these, only two specific cell types formed a close relationship to the matrix. The initial migratory cell population lined the surface of the collagenized sponge matrix within 24 to 48
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG
hours. On the basis of aspects of their ultrastructure, their irregular shape and the presence of numerous extended processes, these initial cells may be alveolar macrophages containing large numbers of osmiophilic digestive vacuoles and neutral lipid. The life span of macrophages in culture has been thought to be limited although some evidence of relatively long survival of pulmonary macrophages in culture has been presented (Sonderland and Naum, ’73). We have seen no evidence of division in these extracystic cells by direct observation; nor have we seen evidence of the presence of other cell types such as fibroblasts which could also serve to provide a supportive framework for the cystic structures. We do not know whether or not formation of type 2 cell cysts depends on the initial layer established by these extracystic cells. Within a week, cysts consisting of cells with structural characteristics clearly marking them as type 2 epithelial cells were present in our cultures. From this point on, it became possible to establish a frequency of distribution between young, active and aged cysts. The fact that such differently aged cysts could exist side by side in the same culture was presumably caused by a sustained rate of release of appropriate cells by the lung fragment into the sponge. Evidence that the cells forming the cysts we described are type 2 epithelial cells rests foremost with the presence of lamellar cytosomes which represent specialized lysosomes in these cells (Goldfischer et al., ’68). As with secondary lysosomes, these are membrane bound structures (Gil and Fkiss, ’73), frequently having a dense core, presumably a remnant of the multivesicular body associated with genesis of these structures (Kikkawa and Spitzer, ’69; Yamamot0 et al., ’70). As has been noted in situ and in isolated lamellar bodies with rabbit lung (Littman et al., ’74), the cytoplasmic inclusions we describe in our cultured type 2 rabbit cells remained more homogeneous than lamellar in form throughout our experiments. The luminal contents of type 2 cysts as described in this study and that of Douglas and Teel (‘75), are similar in appearance to whorls representing “membranous” components of alveolar exudate (Harrison and Weibel, ’68) and from fluids
245
of rat lung (Weibel et al., ’66). The presence of these configurations has been taken to represent water crystals of surface-active phospholipid (Schaffner et al., ’67; Weibel et al., ’66), as well as products of cell breakdown (Weibel et al., ’68; Sun, ’66). Both the dimensions and orientation of the osmiophilic components of tubular myelin agree with the observations of Weibe1 et al. (’66) on pulmonary washings. In addition, these authors also suggest continuity between lamellar structures and tubular myelin configurations. Of particular interest in mature type 2 cell cysts is the presence of elaborate junctional complexes, not unlike those seen in situ in lung (Schneeberger-Keeley and Karnovsky, ’68). Douglas and Teel (‘75) reported similar elaborate junctional complexes. Their presence is confined to epithelium restricting exchange of large molecules between the lumen they surround and the intercellular space (Friend and Gilula, ’72). Synthesis of desmosomes at sites where they do not usually occur is frequently encountered among aggregating cells of the same type (Overton, ’74). The amount and appearance of extracellular material in the lumen of cultured type 2 cell cysts as well as morphological differences in the cells forming the cyst walls suggest a sequence of secretory activity in culture. Such changes appear to encompass early aggregation of type 2 epithelium into young cysts, activation of a secretory mechanism and secretion into the cyst lumen with, finally, loss of secretory activity leading to eventual cyst breakdown. It is not unreasonable to assume that such events could account for many of the morphological features we describe such as increase in height of cyst wall cells, increase in the number of lamellar cytosomes and extracellular myelin configurations as the cyst assumes secretory activity. As cysts passed into the “aged” category, the cells comprising their walls became elongate, lost most of their lamellar cytosomes and developed both osmiophilic inclusions and cytoplasmic vacuoles containing fine granular material similar in appearance to that seen earlier in the lumen. Superficially, at least, type 2 cells constituting aged cyst walls assumed the appearance of a cell type described as intermediate between types 1 and 2 (Evans
246
R. M. ROSENBAUM, P. PICCIANO, Y. KRESS AND M. WITTNER
et al., ‘75; Adamson and Bowden, ’74). This change could account for the loss of the granular osmiophilic material from the lumen of aged cysts and the pleomorphic appearance of what remains of the myelin figures in the lumen as synthesis becomes reduced. Also, these “aged” cells may represent non-specific dedifferentiation common in long-term cultures of primary cells. LITERATURE CITED Adamson, I. Y. R., and D. H. Bowden 1973 The intracellular site of surfactant synthesis. Autoradiographic studies on murine and avian lung explants. Exp. Mol. Path., 18: 112-124. 1974 The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Inv., 30: 35-42. Alwen, J., and A. M. Lawn 1974 The reaggregation of adult liver cells maintained in uitro. Exp. Cell Res., 89:197-205. Askin, F., and C. Kuhn 1971 The cellular origin of pulmonary surfactant. Lab. Inv., 25:260-268. Chevalier, G., and A. J. Collet 1972 In uiuo incorporation of choline-H3, leucine-HS and galactose-Ha in alveolar type I1 pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy. Anat. Rec., 174:289-310. Douglas, W. H. J., and R. W. Tee1 1975 A n organotypic in uitro model system for studying pulmonary surfactant production by type I1 alveolar pneumocytes. Am. Rev. Resp. Dis., 113: 17-23. Evans, M. J., L. J. Cabral, R. J. Stephens and G. Freeman 1975 Transformation of alveolar type I1 to type I cells following exposure to NO2. Fxp. Mol. Path., 22: 142-150. Friend, D. S.,and N. B. Gilula 1972 Variations in tight and gap junctions in mammalian tissues. J. Cell Biol., 52: 758-776. Gil, J., and 0. Reiss 1973 Isolation and characterization and lamellar bodies and tubular myelin from rat lung homogenates. J. Cell Biol., 5 : 152-171 .8_ _ . - -~ Girard, A., G. Fayet and S. Lissitsky 1974 Thyrotropin-induced aggregation promoting factors of adult cultured thyroid cells. Fxp. Cell Res., 87:359-364. Goldfischer, S . , Y. Kikkawa and L. Hoffman 1968 The demonstration of acid hydrolase activities in the inclusion bodies of type I1 alveolar cells and other lysosomes i n the rabbit lung. J. Histochem. Cytochem., 16: 102-109. Harrison, G. A., and J. Weibel 1968 The membranous component of alveolar exudate. J. U1trastr. Res., 24: 334442. Kalderon, A. E., and M. Wittner 1967 Histochemical studies of thyroid cells i n long-term tissue culture. Ehdocrinol., 80: 797-807.
Kapanci, Y., E. R. Weibel, H. P. Kaplan and F. R. Robinson 1969 Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. 11. Ultrastructural and morphometric studies. Lab. Invest., 20: 101-118. Kikkawa, Y., and R. Spitzer 1969 Inclusion bodies of type I1 alveolar cells: species differences and morphogenesis. Anat. Rec., 163: 525541. Kruse, P. F., Jr. and M. K. Patterson, Jr. 1973 Tissue Culture: Methods and Applications. Academic Press, New York. Leighton, J. 1951 A sponge matrix method for tissue culture. Formation of organized aggregates of cells in vitro. J. Nat. Cancer Inst., 12: 545561. Littman, J., Y. Kress, M. F. Frosolono, R. M. Rosenbaum, G. Colacicco and E. M. Scarpelli 1974 A morphological and biochemical characterization of the lamellar inclusion bodies from rabbit alveolar pneumocytes. Fed. Proc., 33: 345. Overton, J. 1974 Cell junctions and their development. In: Progress i n Surface Science and Membrane Science. Vol. 8 . Academic Press, N.Y., pp. 161-208. Pessiac, B., and V. Defendi 1972a Evidence for distinct aggregation factors and receptors in cells. Nature (London), 238: 13-15. 1972b Cell aggregation: role of acid mucopolysaccharides. Science, 175: 898-900. Rosenbaum, R. M., M. Wittner and M. Lenger 1969 Mitochondria1 and other ultrastructural changes in great alveolar cells of oxygen-adapted and poisoned rats. Lab. Inv., 20: 516-528. Rosenbaum, R. M.,M. Wittner and E. Scarpelli 1973 Parameters in the pathophysiology and pathobiology of oxygen tolerance i n the rat lung. Amer. J. Path., 70:57. Schaffner, F., P. Felig and E. Trachtenberg 1967 Structure of rat lung after protracted oxygen breathing. Arch. Path., 83:99-107. Schneeberger-Keeley, E. E., and M. J. Karnovsky 1968 The ultrastructural basis of alveolarcapillary membrane permeability to peroxidase as a tracer. J. Cell Biol., 37: 781-793. Sonderland, S. C., and Y. Naum 1973 Growth of pulmonary alveolar macrophages in uitro. Nature (London), 245: 150-151. Sorokin, S. P. 1970 The cells of the lungs. In: Morphology of Experimental Respiratory Carcinogenesis. P. Nettlebaum, M. G. Hanna, Jr. and J. W. Deatherage, eds. U. S. Atomic Energy Symposium Series No. 21,Oak Ridge, Tennessee. Sun, C. N. 1966 Lattice structures and osmiophilic bodies in the developing respiratory tissue of rats. J. Ultrastructural Res., 15: 380388. Weibel, E.R., G. S. Kistler and G. Tondury 1966 A sterologic electron microscopic study of “tubular myelin figures” in alveolar fluids of rat lungs. Zeitschr. Zellforsch., 69: 418427. Yamamoto, E., M. Wittner and R. M. Rosenbaum 1970 Resistance and susceptibility to oxygen toxicity by cells of the gas-blood barrier of the rat lung. Am. J. Path., 59:409436.
PLATES
PLATE 1 EXPLANATION OF FIGURES
248
1
Scanning electron micrograph of a terminal airway fragment from trypsinized rabbit lung. Such fragments were placed on sponges and provided the source of cells for culture as described i n the text. Several lobular units with alveolar ducts and a respiratory bronchiole are visible. ac, alveolar epithelial cells; ad, alveolar duct; rb, respiratory bronchiole; bv, bronchiolar blood vessel; int, enzymatically released interstitum. X 3,000.
2
Light micrograph of a segment of cellulose sponge 24 to 48 hours following placement of lung fragments. The early accumulation of cells along the margin of collagenized sponge (arrow) can be seen. X 480.
3
Transmission electron micrograph of cells shown i n figure 2 lining collagenized sponge matrix between 24 to 48 hours following placement of lung fragment onto sponges. Note numerous osmiophilic inclusions and “empty” appearing vacuoles. Sponge material (SM) is frequently surrounded by cell processes (arrows). X 3,000.
4
Scanning electron micrograph of sponge exposed to rabbit lung fragments for one week. Clusters of cells forming early cysts (arrow) are seen i n the cavity of a sectioned sponge. X 4,800.
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P. Picciano, Y. Kress and M . Wittner
PLATE 1
249
PLATE 2 EXPLANATION OF FIGURE
5
250
Cross-section of a young type 2 epithelial cell cyst derived from lung fragments in contact with sponge for approximately ten days. Epithelial cells forming the cyst contain a number of lamellar cytosomes (small arrows) and have microvilli a t their apical border. The cyst lumen contains fine granular osmiophilic material as well as occasional myelin figures (arrow). Cells such as are present during early stages of tissue-sponge association appear outside the cyst. These continue to show large numbers of osmiophilic inclusions and contain “empty” vacuoles. X 9,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT L U N G R. M. Rosenbaum, P . Picciano, Y . Kress and M . Wittner
PLATE 2
25 1
PLATE 3 EXPLANATION OF FIGURES
6A,B
252
Actively secreting type 2 epithelial cell cyst from a 12-day culture. In the lumen, several osmiophilic lamellar structures are present a s well as lattice forms representing tubular myelin configurations. x 9,000. There are sites (6B, arrows), suggesting the derivation of the tubular myelin from the surface of the lamellar structures. X 45,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 3
253
PLATE 4 EXPLANATION O F FIGURES
254
7A
A group of four type 2 epithelial cells forming the wall of a middle aged cyst developing within a sponge matrix at day 14. Note the numerous membrane bound lamellar cytosomes, the contents of which frequently appear homogeneous in rabbit lung cells. Several cytosomes (Ic) show osmiophilic dense bodies. Some osmiophilic inclusions not related to lamellar cytosomes (small arrow) are also evident. The apical surface of all cells have numerous microvilli. All cells share a junctional complex (circle and 7B). The cyst lumen is filled with osmiophilic fine granular material, lamellar structures and lattices of tubular myelin configurations (large arrow). x 12,000.
B
Detail of a junctional complex between adjacent type 2 cells of a cyst wall. zo, zonular occludens; za, zonula adherens; ma, macula ad. herens. X 75,000.
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 4
255
PLATE 5 E X P L A N A T I O N OF F I G U R E S
8A
A large irregularly shaped middle aged type 2 epithelial cell cyst derived from adult rabbit lung fragments on sponge for 14 days. The two arrows mark opposite cyst walls where these come close to each other at w h a t appears to be the isthmus of a bilobed cyst. Nearly all type 2 cells contain lamellar cytosomes (lc). The cyst lumen i s filled with numerous lamellar structures, myelin-like strands (ms) and a highly osmiophilic fine granular material. es, extracystic space. X 3,000.
B
Higher magnification of the luminal contents seen i n figure 8A.
x 9,000.
256
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R. M. Rosenbaum, P . Picciano, Y. Kress and M. Wittner
PLATE 5
PLATE 6 EXPLANATION
9
258
O F FIGURE
General relationships between middle aged and old type 2 cell cysts a n d extracystic cell elements i n a n 18-day-old sponge culture. Two middle aged cysts (B) may be recognized by the height of the cuboidal epithelial cells forming the wall, a n d by the presence of lamellar cytosomes within these cells. The lower cyst is not sectioned through its lumen but the irregular, centrally-located l u m e n of the upper cyst reveals a n osmiophilic granular component with a few myelin figures. An old cyst (A) is marked by the greatly thinned out appearance of cells (arrows) forming the wall. There are small amounts of osmiophilic granular material in the lumen a n d numerous myelin figures of varying size. A number of extracystic cells, many with greatly extended processes, appear, between the cysts. These cells have numerous empty vacuoles presumably originally containing lipid and large numbers of osmiophilic dense bodies. X 3,000.
I N VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG
PLATE 6
R. M. Rosenbaum, P. Picciano, Y. Kress and M. Wittiier
259
PLATE 7 EXPLANATION
10
260
OF FIGURE
Portion of the wall of a n aged cyst maintained in sponge culture for 18 days. Two type 2 epithelial cells containing lamellar cytosomes show a reduced number of microvilli. These cells also contain vacuoles filled with fine granular osmiophilic material (small arrows), some evident in the cyst lumen. The large amount of darkly staining osmiophilic granular material seen in younger cysts is gone from the lumen of such older cysts while the myelin figures appear irregular i n shape and are less numerous. Tubular myelin configurations are not present in great numbers. x 9,000,
IN VITRO TYPE 2 CELL CYSTS FROM ADULT RABBIT LUNG R . M . Rosenbaum, P . Picciano, Y. Kress and M . Wittner
PLATE '7
261
