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The fixation of pulmonary surfactant for electron microscopy
© 1970 by Academic Press, Inc. J. U L T R A S T R U C T U R E R E S E A R C H
31, 229-246 (1970)
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The Fixation of Pulmonary Surfactant for Electron Microscopy 1,2 II. Transport of Surfactant through the Air-Blood Barrier GERALD B. DERMER
Department of Pathology, Hospital of the Good Samaritan Medical Center, Los Angeles, California 90017 Received July 22, 1969, and in revised form October 6, 1969 The permeability of the alveolar-capillary barrier to pulmonary surfactant used as an electron-opaque tracer has been investigated. Surfactant visualized by tricomplex flocculation was not only localized to the alveolar surface but also to various forms of vesicles and plasmalemmal invaginations of type I alveolar cells and to the basement membrane (interstitial space) that lies between epithelium and endothelium. To a lesser degree, surfactant was also present within endothelial cell vesicles, endothelial intercellular clefts, capillary lumens, and pulmonary macrophages. The observations indicated that surfactant was transported by quanta from the alveolar surface through type I alveolar cells to the basement membrane by plasmalemmal vesicles and through open membrane-lined channels. The channels appeared to be formed by the fusion of the apical plasmalemma at the leading edge of an invagination with the basal plasmalemma with the subsequent elimination of the fused membranes. Pulmonary surfactant did not pass through epithelial intercellular clefts indicating they were tight (zonulae occludentes), but some surfactant seemed to pass between endothelial cells, supporting the concept that these clefts contained maculae occludentes. To some degree, surfactant appeared to be transported from the basement membrane across endothelium by vesicles to the blood and also to be taken up by pulmonary macrophages which were free within alveoli. It is concluded that the images seen represented stages in the normal turnover and removal of surfactant from the lung and that the majority of surfactant left by passage through interstitial spaces to the lymphatic system. Electron m i c r o s c o p i c studies on the passage of m a t e r i a l across cellular barriers have been carried out with the aid of e l e c t r o n - o p a q u e tracer molecules injected intravenously or b y a d m i n i s t r a t i o n of the tracer in vitro. The passage of ferritin (5, 19, 20), c o l l o i d a l particles of t h o r i u m dioxide (20), saccharated i r o n oxide (19, 20), z A preliminary report of this investigation was presented at the Ninth Meeting of the American Society for Cell Biology in Detroit, November, 1969. Supported in part by research award No. 423 from the Los Angeles County Heart Asscciation and funds from the Tuberculosis and Health Association of California.
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colloidal carbon (30) and horseradish peroxidase (21, 40) across the walls of continuous (nonfenestrated) capillary endothelia have been studied in vivo. These investigations and others (23, 24) have suggested that micropinocytotic vesicles are a mode of transport for macromolecules across endothelium. In the lung the transport of protein across the capillary endothelium by pinocytotic vesicles was suggested (40), but its role in the net transport of protein could not be established. In the lung (40) as well as cornea (24), the vesicular transport of tracer molecules across epithelium could not be detected. With the exception of ferritin and blood-borne chylomicra in newborn rats (43), the tracers generally employed have been substances foreign to the body and not physiological. During the course of an electron microscopic examination of lung tissue (8, 9) that had been treated by "tricomplex flocculation" to allow the visualization of the saturated phospholipid dipalmitoyl lecithin--both the major and surface-active component of surfactant--it became obvious that surfactant phospholipid was found at several sites within the air-blood barrier (interalveolar septum). The airblood barrier is composed of a continuous layer of squamous alveolar cells (type I) interspersed by great alveolar cells (type II) and separated from the underlying continuous, nonfenestrated endothelium by a basement membrane. As expected, electron dense deposits corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in tricomplex fixation were found within the alveolar lumen at the surfaces of epithelial cells, often appearing within invaginations of the cell membrane. Similar deposits of material were localized to membrane-bound vesicles within the cytoplasm of type I alveolar epithelial cells and within the basement membrane (interstitial space) that separates epithelium from endothelium. To a lesser degree similar appearing deposits were present within membrane bound vesicles of the capillary endothelium, endothelial intercellular clefts, and within the lumen of capillaries. The data presented here indicated that these observations represented stages in the transport of surfactant away from the alveolar surface related to the normal turnover and removal of surfactant from the lung, a process that has been poorly understood (39). Evidence will be presented which supports the concept that vesicles derived by pinocytosis at one cell front can be active in the transport by quanta of physiologically important molecules across epithelium and endothelium (33, 34). The evidence also indicated that surfactant phospholipid could pass through the intercellular junctions between adjacent capillary endothelial cells confirming the evidence that these junctions were not tight (zonulae occludentes), but were rather maculae occludentes with gaps between the apposed membranes (21, 40). Also in agreement with others (14, 15, 40) the lateral surfaces of pulmonary epithelial cells appeared to be separated by zonulae occludentes (tight junctions) which prevented
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the passage of surfactant from the alveolar lumen into the underlying interstitial space. Data will also be presented indicating the presence within pulmonary epithelium of continuous channels between the alveolar lumen and basement membrane formed by invaginations of the alveolar plasmalemma. Other observations indicated that alveolar macrophages may also function in the degradation and removal of surfactant from the lung. MATERIALS AND METHODS Mature breathing guinea pigs were sacrificed by the injection of 10 ml of a 2 % xylocaine solution into the cisternal cavity. The fixation and processing of the lung tissue including the scheme of surfactant phospholipid fixation by means of tricomplex flocculation have been previously discussed (9). The sections used in this investigation were usually not stained in order to eliminate the heavy metal precipitates that can occur with section staining. In order to increase the contrast of membranes, however, some grids were double stained with uranyl acetate and lead citrate (38). The sections were examined in a Hitachi 8S electron microscope at an electron optical magnification of 5500-36,000 times. The micrographs were enlarged photographically as needed. RESULTS
Pulmonary epithelium Electron dense material in the form of amorphous, lamellar, and particulate deposits corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in the tricomplex fixation scheme was found within the alveolar lumen at the surface of squamous epithelial cells. Many vesicles opening onto the alveolar surface also contained reaction product (Figs. 1 and 2). The membrane bounding these vesicles was continuous with the apical plasmalemma (Figs. 3 and 4). Many of these forms had flask shapes and somewhat elongated necks. It was not uncommon to find rows of these vesicles with similar profiles and containing reaction product opening onto the alveolar surface (Fig. 1). The vesicles intruded varying distances into the cytoplasm, and in some instances they extended through the cell to the plasmalemma bounding the basement membrane (Figs. 1 and 3-5). The impression gained from the micrographs was that the apical plasmalemma at the invaginating end of the intrusions fused with the basal plasmalemma with the eventual elimination of the layers of the two fused membranes, forming a continuous membrane-lined channel between alveolar lumen and basement membrane. In Fig. 3, a flask-shaped vesicle extends through the epithelial cytoplasm and appears fused with the basal plasmalemma. Although one of the dense leaflets of the invaginating plasmalemma is obscured by reaction product, the fusion appears to have produced a five-layered diaphragm which buckles convexly toward the basement membrane.
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A similar image is seen in Fig. 4. A n o t h e r similar a p p e a r a n c e occurs in Fig. 5 in u n s t a i n e d sections. A l t h o u g h m a n y of the vesicles seen were a t t a c h e d to the p l a s m a l e m m a , a considerable n u m b e r were f o u n d free in the c y t o p l a s m of the p u l m o n a r y type I alveolar cells (Figs. 6 a n d 7). M a n y of these vesicles were filled with reaction product. Generally the vesicles were r o u n d a n d 50-100 m # in diameter. Occasionally larger vesicles were seen, a n d there were indications t h a t this was a result of vesicle coalescence. There were images where a d i a p h r a g m was seen between two fused vesicles (Fig. 8) a n d others where there a p p e a r e d to be a c o n t i n u i t y of vesicular contents (Fig. 9). A l t h o u g h less frequently seen t h a n at the apical cell surface, the basal surface of type I alveolar cells exhibited vesicles which were o p e n e d to the b a s e m e n t m e m b r a n e a n d c o n t a i n e d reaction p r o d u c t (Figs. 2 a n d 6). They generally were n o t f l a s k - s h a p e d with elongated necks, b u t h a d forms whose limiting m e m b r a n e was fused in p a r t with the b a s a l p l a s m a l e m m a . A t the p o i n t of fusion the basal p l a s m a l e m m a generally f o r m e d a shallow infundibulum. Vesicles were seen separated f r o m the interstitial space only by m e m b r a n e m a t e r i a l (Fig. 6) a n d other images showed e l i m i n a t i o n of this m a t e r i a l with a p p a r e n t continuity between vesicular contents a n d interstitial space. F i g u r e 2 shows apical a n d b a s a l p l a s m a l e m m a vesicles o p e n e d onto the alveolar l u m e n a n d interstitial space, respectively. Epithelial intercellular clefts between type I alveolar cells were free of reaction
Key to abbreviations A S alveolar space B M basement membrane CL capillary lumen
En capillary endothelium Ep alveolar epithelium
FIG. 1. Electron opaque material corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in the tricomplex reaction scheme is seen at the alveolar surface of a type I epithelial cell and within a row of vesicles opened onto the surface. These forms have similar profiles, exhibiting flask shapes with somewhat elongated necks. One of these vesicles (arrow) extends nearly across the epithelium to the basal plasmalemma. Except for one vesicle within the endothelium, the rest of the air-blood barrier is free of reaction product. Not section stained. × 67,090. FIG. 2. Reaction product is seen at the alveolar surface of a type I alveolar cell and within vesicles which appear opened to the alveolar surface or closely apposed to the apical plasmalemma. Several of these forms have flask shapes. At the basal surface, one vesicle containing reaction product appears opened to the basement membrane (arrow) while another seems fused with the basal plasmalemma (arrow). The basement membrane contains some electron-opaque material while the endothelium does not appear to contain any. Not section stained. × 67,000. FIGs. 3 and 4. The alveolar surface and vesicles of type I cells opened to the surface contain reaction product. In each figure, one vesicle extends through the epithelial cytoplasm and appears fused with the basal plasmalemma. In Fig. 3, although one of the dense leaflets of the apical plasmalemma is obscured by reaction product, the fusion appears to have produced a five-layered diaphragm which buckles convexly toward the basement membrane (arrow). A similar form is seen in Fig. 4 (arrow). Other portions of the air-blood barrier are free of electron-opaque material. Section stained with uranyl acetate and lead citrate. × 105,000.
CL
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p r o d u c t a n d the o u t e r leaflets of the b o u n d i n g m e m b r a n e s a p p e a r e d fused over a considerable p o r t i o n of their length (Fig. 10).
Basement membrane There were instances where in a d d i t i o n to reaction p r o d u c t at t h e alveolar surface, there was reaction p r o d u c t within the vesicles at the alveolar surface, within vesicles at all levels within the epithelial cytoplasm, b u t where the b a s e m e n t m e m b r a n e cont a i n e d little reaction p r o d u c t (Fig. 6). There were other instances where the basem e n t m e m b r a n e also c o n t a i n e d reaction p r o d u c t (Figs. 2, 5, 7, a n d 10). A t other places within the a i r - b l o o d b a r r i e r the b a s e m e n t m e m b r a n e c o n t a i n e d a l m o s t all of this m a t e r i a l F o r considerable distances, the b a s e m e n t m e m b r a n e could be t r a c e d by following the electron o p a q u e deposits within it (Fig. 11).
Endothelium The e n d o t h e l i u m c o n t a i n e d reaction p r o d u c t b u t n o t to the extent that was seen within the b a s e m e n t m e m b r a n e a n d epithelium (Figs. 7 a n d 12-14). It was n o t unc o m m o n to find the b a s e m e n t m e m b r a n e filled with electron dense particles while the e n d o t h e l i u m c o n t a i n e d very little of this m a t e r i a l (Fig. 11). Very occasionally vesicles filled with reaction p r o d u c t a p p e a r e d in close a p p r o x i m a t i o n to the b a s a l p l a s m a l e m m a of the e n d o t h e l i u m (Figs. 7 a n d 13). M o r e frequently, reaction p r o d u c t was f o u n d within free vesicles within the e n d o t h e l i u m interspersed a m o n g e m p t y vesicles (Figs. 13 a n d 14). I n the epithelium m a n y vesicles were filled with reaction p r o d u c t while within the e n d o t h e l i u m it was m u c h m o r e c o m m o n to find only a p o r t i o n of some of the vesicles filled with this material. Vesicles containing reaction FIG. 5. Reaction product at the alveolar surface extends into vesicular forms which traverse the cytoplasm of a type I cell. The invaginating edges of these forms appear fused with the basal plasmalemma (arrows). One of these invaginations appears flask-shaped. Other vesicles opened to the alveolar surface and containing reaction product intrude only a small distance into the cytoplasm. In this case, the basement membrane contains a considerable amount of reaction product. Not section stained. × 65,000. FIG. 6. The cytoplasm of a type I alveolar cell is filled with vesicles containing reaction product. These vesicles appear free within the cytoplasm and exhibit a bounding membrane. Electron opaque material is also seen at the alveolar surface while the basement membrane contains little of this material. At the basal surface of this cell, vesicular membranes appear fused with the plasmalemma exhibiting diaphragms which separate vesicular contents from the basement membrane (arrows). At the points of fusion the basal plasmalemma forms a shallow infundibulum. Not section stained. x 69,000. FIG. 7. Electron opaque material is seen at the alveolar surface, within cytoplasmic vesicles of a type I alveolar cell, within vesicles of the endothelinm, and also within the basement membrane. The capillary lumen is free of this material. Not section stained, x 67,000. FIa. 8. Two fused vesicles, one of which contains electron opaque material; are seen close to the basal plasmalemma of the epithelium. A diaphragm is present between the two forms. Much electronopaque material is seen at the alveolar surface, Section stained with uranyl acetate and lead citrate. x 90,000.
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product also appeared fused with the apical plasmalemma at the luminal surface of capillaries (Fig. 12). There were instances where there was reaction product within the basement membrane, within some vesicles in the endothelial cytoplasm but where the capillary lumen contained none of this material (Figs. 7 and 14). There were other images where the capillary lumen also contained reaction product (Fig. 12). Intercellular clefts between endothelial cells contained reaction product (Fig. 14). Near to the capillary lumen, the adjacent plasma membranes appeared closely approximated to form a cell junction (Fig. 15). Reaction product was rarely found within the junctions.
Pulmonary macrophages Within alveoli, electron dense deposits were found lining the surfaces of free macrophages after tricomplex fixation (Figs. 16 and 17). Occasionally vesicles opening onto the surfaces of these cells contained reaction product (Fig. 16) and near the plasmalemma, free vesicles containing this material were also occasionally observed (Fig. 16). Deeper within the cytoplasm, vesicles containing reaction product were not seen although electron-opaque material of this sort was observed with the cytoplasmic matrix of the macrophages. In Fig. 17 similar appearing electron dense material is seen at the surface and in the cytoplasm and in the latter case does not appear within cisternae of cytoplasmic membranes. This can perhaps be better visualized in a higher power illustration (Fig. 18). Inclusion bodies of several sorts were seen within the cytoplasm of the pulmonary macrophages. Some contained granular material while others contained both lamellar and granular forms (Fig. 17). DISCUSSION Tricomplex fixation has allowed the electron microscopic visualization of the major component of pulmonary surfactant, dipalmitoyl lecithin, which as expected was found at the alveolar surface (8, 9). Surfactant was detected by the presence of electron-opaque deposits and lamellar structures corresponding to the reaction proFIG. 9. A continuity of vesicular contents, which consists of electron opaque material, appears to exist between two fused vesicles of a type I alvolar cell (arrow). Reaction product is also seen at the alveolar surface and to a small extent within the basement membrane. Not section stained. × 78,000. FIG. 10. The epithelial intercellular cleft between two type I alveolar cells is free of reaction product and the bounding membranes appear tightly fused (arrows). However, reaction product is found at the alveolar surface, within a flask-shaped vesicle opened to the surface, within a free cytoplasmic vesicle, and within the basement membrane. Not section stained, x 69,000. Fit. 11. The basement membrane between pulmonary epithelium and endothelium is filled with electron-opaque material while the cellular portions of the air-blood barrier and the capillary lumen containing a red blood cell (RBC)has little of this material. Some electron-opaque material is also seen within the alveolus. Section stained with uranyl acetate and lead citrate, x 24,000.
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Fio. 12. Reaction product is found within the basement membrane, within a capillary, some of which appears at the surface of red blood cell, and within several vesicles of the endothelium. These vesicles appear fused with or closely apposed to the apical plasmalemma of the endothelial cell (arrows). Not section stained, x 67,000.
duct formed between surfactant phospholipid a n d the heavy metal salts used i n the tricomplex reaction scheme. This material was n o t observed when lung was fixed only in glutaraldehyde a n d osmium. The observations reported here i n d i c a t e d that surfactant was f o u n d at other sites within the air-blood barrier suggestive of pathways by which surfactant was degraded a n d removed from the lung. These processes have been poorly u n d e r s t o o d (39) b u t the alveolar lining of surfactant did appear to have a short lifetime as c o m p a r e d to the cells of the lung (6). P u l m o n a r y surfactant after tricomplex fixation acted as a n electron-opaque tracer which has been used here to investigate the permeability of the alveolar-capillary barrier to this physiologically i m p o r t a n t material. Palade a n d Bruns (35) have described structural m o d u l a t i o n s of p l a s m a l e m m a l vesicles of vascular endothelia. They proposed a rationalization of these structural FIG. 13. This low power micrograph illustrates vesicles filled with reaction product within pulmonary endothelium. The vesicles appear in a row fused with or closely apposed to the basal plasmalemma (arrows). Elsewhere they are found as free vesicles at different levels within the cytoplasm of the endothelium. The alveolar surface contains much reaction product while the basement membrane and capillary lumen contain none of this material. Not section stained, x 28,000. FIO. 14. The intercellular cleft between two endothelial cells contains reaction product (arrows), as does the basement membrane and free vesicles within the endothelial cytoplasm. The capillary lumen contains none of this material. Not section stained, x 52,000. Flo. 15. Reaction product is found within a cell junction between two endothelial cells (arrows). Much reaction product is seen at the alveolar surface and to some extent within the basement membrane, but the capillary lumen is free of this material. Not section stained, x 67,000. FIG. 16. Reaction product is found at the surface of a pulmonary macrophage and within a vesicle opened onto its surface (arrow). Similar material is found within a free vesicle near the surface (arrow), but other vesiclesand inclusionsdeeper within the cytoplasm do not appear to contain material of similar electron opacity. Section stained with uranyl acetate and lead citrate x 30,000.
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modulations of vesicles in terms of their involvement in transport in quanta across endothelium. Different forms of vesicles as described by Palade and Bruns have been observed in this investigation to contain surfactant, supporting the concept that these forms are involved in the transport of molecules across cells. The present observations also extend their views by observations of structural modulations of plasmalemmal vesicles within pulmonary epithelium and endothelium and the observation of a new vesicular form. Vesicles containing reaction product and opened onto the alveolar surface have been frequently observed. Most of these forms have flask shapes and somewhat elongated necks. It has been proposed (35) that forms of this type represent stages in the formation and loading of membrane invaginations, followed by their being pinched off to form isolated vesicles. The pinching off process has been thought to be slow in endothelia (16, 35), and this seems to hold true for pulmonary epithelium also because rows of these invaginations in approximately the same stage of pinching off were often seen at the alveolar surface. Most of the invaginations at the alveolar plasmalemma appeared to pinch off forming vesicles many of which were filled with reaction product. These free vesicles were seen at all levels within the cytoplasm of the squamous epithelium. It was possible, however, that some of these forms represented invaginations whose necks were opened to the alveolar surface at some other level of sectioning. The basal surface of the pulmonary epithelium exhibited vesicles containing reaction product which were either fused with the basal plasmalemma or were opened to the basement membrane by the elimination of the fused membranes. These forms have been assumed to represent stages in the discharge process of vesicular contents (35). Within the epithelial cytoplasm, membrane diaphragms were seen between fused vesicles. Other images showed forms where surfactant appeared to be continuous from one vesicle to another after elimination of the fusion layer. These forms apparently gave rise to the large vesicles which were seen within alveolar type I cells. It has been suggested that chains of vesicles might be formed which open simultaneously on both cell fronts but, as in muscle capillaries (19), this observation has not yet been made. However, separate vesicles containing reaction product which opened simultaneously on two cell fronts were a frequent observation. The endothelium of visceral capillaries is interrupted by fenestrae generally closed by apertures or diaphragms (12), as is the endothelium of tongue capillaries (4). Fusion of the plasmalemma on the blood front with the plasmalemma on the tissue front of the cell followed by a progressive elimination of membrane layers appeared to result in the formation of these fenestrae (35). This process has been thought to be confined to vascular endothelium but the observations presented here indicated a similar mechanism might be working within pulmonary epithelium. Flask-shaped
FIG. 17. Reaction product is found at the surface of a pulmonary macrophage and similar appearing electron-opaque material is also seen with the cytoplasmic matrix (arrows). This cytoplasmic material is not found within cisternae of intracellular membranes. Some inclusion bodies within the cytoplasm contain granular material while others show lamellar and granular forms (arrow). Section stained with uranyl acetate and lead citrate, x 25,000. FIG. 18. Electron-opaque material is seen within the cytoplasm of a pulmonary macrophage while the cisternae of membrane-bound vesicles do not contain any of this material. At several locations the cytoplasmic material completely surrounds these vesicles and the vesicular membranes can often be detected at the boundaries of the electron opaque material (arrows). Section stained with uranyi acetate and lead citrate, x 54,000.
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vesicles filled with reaction product and opened onto the alveolar surface extended through the epithelial cytoplasm, and the plasmalemma on the alveolar front became fused with the plasmalemma on the basement membrane front of the cell. The fusion generally produced a five-layered diaphragm similar to what was seen in tongue capillaries (35). This type of diaphragm had been considered to be an intermediate stage of membrane fusion which led to an eventual removal of the fusion layer (35). Forms suggestive of more advanced stages of diaphragm elimination have been seen. These forms would give rise to continuous membrane-lined pores between the alveolar lumen and basement membrane, which were apparently very short lived or occurred infrequently because few examples have been found. Alveolar surfactant did not penetrate the intercellular spaces between adjacent type I pulmonary epithelial cells, supporting the data which indicated that junctions between epithelial cells were tight (zonulae occludentes) (i4, 15, 25, 40). If the rationalization of different structural forms of plasmalemmal vesicles as proposed by Palade and Bruns (35) in terms of their involvement in transport by quanta across cells was correct, then surfactant was loaded into membrane invaginations at the alveolar surface. Most of these invaginations then pinched off to form free vesicles which moved to and fused with the basal plasmalemma, with the elimination of the fused membranes and the discharge of surfactant into the interstitial space. The vesicular forms seen at the alveolar and interstitial fronts of the cell supported this interpretation. There also appeared to be an alternative route by which surfactant could be transported from alveolar lumen to the basement membrane. This would take place by the fusion of the apical plasmalemma at the leading edge of an invagination with the basal plasmalemma with the elimination of the fused membranes forming an open membrane-lined channel between the alveolar lumen and basement membrane. This has not been a time sequence study of the transport of an electron, opaque tracer since injections of the tracer molecule were not performed. Rather, this has been a study of the distribution of a naturally occurring material present in normal, mature lungs at all times. Therefore, data obtained at early and late stages after tracer injection indicating the direction of transport were impossible to obtain. The data, however, suggested that surfactant transport takes place through the air-blood barrier and it appeared that different regions of one lung illustrated various stages of the process. The vesicular forms seen at the alveolar and interstitial fronts of the type I epithelial cell indicated that the transport of surfactant by quanta was inward from the alveolar surface to the basement membrane, not in the opposite direction. Other considerations also supported this conclusion. There were instances when there was much reaction product at the alveolar surface, within vesicles at the alveolar surface, within free vesicles of the epithelial cytoplasm, within vesicles at the
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basal surface of the cell interposed among empty vesicles and where the basement membrane itself did not contain any reaction product. This sort of evidence (21) would suggest that the vesicles had moved across the epithelium from the alveolar surface. Furthermore, there is considerable circumstantial evidence (2, 18, 26, 29, 42) which indicates that surfactant is synthesized within type II alveolar cells and that it is secreted from these cells into the alveolus forming the surface lining layer. It would be expected therefore that, in the degradation and turnover of surfactant, material would leave the alveolar surface and not be transported to it. The transport of surfactant through pulmonary epithelium took place only at alveolar type I cells, for surfactant transport through type II alveolar cells has not been observed. Physiological studies (1, 10, 11, 32, 41) have shown that albumin solutions introduced into pulmonary alveoli are absorbed by the lung in an intact form (1, 41). The delayed removal of protein from alveoli appeared to occur in the removal of protein from the interstitial space which had accumulated a large amount of albumin, rather than in passage through the alveolar epithelium (32). The interstitial space within the intra-alveolar septum is continuous with spaces surrounding pulmonary vessels and bronchi where pulmonary lymphatics exist (32, 44) and the data indicated that the lymphatic system removed some of the albumin which had been absorbed from alveoli (11, 32). Although for albumin, most of the absorbed protein was found in the pulmonary blood (32). These data supported the suggestion that pulmonary lymphatics may function to remove foreign material or fluid from alveoli (44). Thus it appears that the epithelial surface of the lung is active in the absorption of protein as well as surfactant and the physiological and electron microscopic data indicate that both may be transported by similar mechanisms. Serum albumin has a molecular weight of 69,000 with a diameter of 3.8 nm and molecules of this size appear physiologically to be transported across cells by vesicles (37) or through pores larger than about 4.0 nm in diameter (17, 31, 36, 37). These conclusions are consistent with the present electron microsciopic study indicating that surfactant is also transported across the pulmonary epithelium by vesicles and possibly through membrane-lined pores. Therefore pulmonary surfactant appears to behave as a protein-like macromolecule even though the molecular weight of dipalmitoyl lecithin is only 754. However, the alveolar surface lining layer of surfactant after tricomplex fixation appeared to be made up of small rounded particles 5.0-10.0 nm in diameter (9) in which the phospholipid molecules seemed to be associated as molecular aggregates called micelles. Also, it is known that a micellar solution of phospholipid does occur in the phase diagram of lecithin-water system (28) and negatively stained preparations of lecithin have exhibited particles about 4.0 nm in diameter (27). The size of these micelles would be in the range of a molecule with dimensions like serum albumin. It also seems interesting that albumin
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absorbed from the alveolus had accumulated within the basement membrane (32), for the same holds true when considering surfactant transport through the alveolarcapillary barrier. The basement membrane contained much reaction product due to its accumulation there after transport through the pulmonary epithelium. For long distances the basement membrane could be traced by following the electron dense deposits within it and since this interstitial space is continuous with pulmonary lymphatics, it appeared that the majority of surfactant was removed from the lung by the lymphatic system. This does not seem surprising when one considers that long-chain triglycerides are removed from the intestine by the lymph after absorption (3). Dipalmitoyl lecithin, the major component of pulmonary surfactant, is a phospholipid containing two long-chain fatty acids. Only a small amount of surfactant within the basement membrane appeared to be transported across the endothelium into the blood in contrast to protein absorption where the capillaries seemed to remove the majority of the albumin absorbed from the alveolus (32). Again the similarity to intestinal absorption seems noteworthy, for digested protein is also removed from the intestine by the blood. Transport of surfactant across the endothelium appeared to be vesicular and between cells through intercellular clefts. Most images showed reaction product within the basement membrane, within some endothelial vesicles, within intercellular clefts, but where the capillary lumens contained little or none of this material. This was suggestive of transport from the basement membrane to the blood, but images representing vesicular loading at the basal surface of the endothelium have been difficult to obtain. Furthermore, it was not known what proportion of this endothelial transport was vesicular or through the intercellular space. The vesicular transport of surfactant through pulmonary endothelium would represent an inversion of the process of pinocytosis since material loaded into vesicles at the basal surface was carried through the cytoplasm and discharged at the apical surface into a capillary. A similar observation has been made on corneal epithelium (23). Although reaction product was found within endothelial intercellular clefts, it was quite rare to find any within cell junctions where the adjacent cell membranes appeared closely apposed. It has been shown, however, that endothelial cell junctions have a gap between the adjacent plasma membranes of about 4.0 m# which are permeable to peroxidase used as an electron opaque tracer (20, 21). Therefore, these junctions are probably also permeable to pulmonary surfactant. The detection of reaction product within pulmonary epithelium and endothelium and also within endothelial intercellular spaces and the basement membrane, indicated that surfactant was not degraded at these sites. The tricomplex reaction is specific for phospholipids (13), and if the choline residue had been split off, reaction product would not have been formed.
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Pulmonary macrophages also seemed to be involved in the removal of surfactant from the lung. Reaction product at the surface of macrophages was occasionally found within invaginations of the plasmalemma and within free vesicles near the surface. However, reaction product was not found in vesicles deeper within the cytoplasm and the free vesicles near the surface might have represented invaginations opened to the surface at some other level of sectioning. The alveolar macrophage has been shown to be phagocytic (22) however and the uptake of surfactant by this process might be expected. The inclusion bodies within these cells showed acid phosphatase activity (7) and were thought to be concerned in the digestion of exogenous material (7), and this material might in part be surfactant. The lamellar forms seen within these inclusions exhibited a repeat of about 5.0 nm (22) and were very similar to lamellar forms seen within the alveolar surface lining material (8, 9). The laminated and granular forms found within these inclusion bodies might in part represent the end products of the intracellular digestion of pulmonary surfactant by macrophages. It appeared that surfactant could be internalized by macrophages by another mechanism besides pinocytosis, for electron dense material similar to reaction product seen at the surfaces of pulmonary cells was found within the cytoplasmic ground substance of macrophages. Reaction product appeared to be excluded from cisternae of cytoplasmic membranes, and it seemed that surfactant was transported through the plasmalemma of the macrophages, solubilized within its cytoplasm, and possibly stored in a digested form within the lysosome-like inclusions. The author is grateful to Dr. Harry B. Neustein for his critical evaluation of the manuscript. REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9.
10. 11. 12. 13.
BENSCH,K., DOMINGUEZ,E. and LIEBOW,A. A., Science 157, 1204 (1967). BENSCH, K., SCHAEFER,K. and AVERY, M. E., Science 145, 1318 (1964). BORGSTROM,B., Biochem. J. 58, 600 (1954). BRUNS, R. R. and PALADE, G. E., J. Cell Biol. 37, 244 (1968). -ibid. 37, 277 (1968). CLEMENTS, J. A., Ciba Found. Syrup. Develop. Lung, p. 202. Little, Brown, Boston, Massachusetts, 1967. CORRIN, B., CLARK, A. E. and SPENCER, H., J. Anat. 104, 65 (1969). DERMER, G. B., J. Cell Biol. 39, 33a (1968). -J. Ultrastruct. Res. 27, 88 (1969). DOMINGUEZ,E. A. M., LIEBOW,A. A. and BENSCH,K. G., Lab. Invest. 16, 905 (1967). DRINKER,C. K. and HARDENBER~H,E., J. Exptl. Med. 86, 7 (1947). EKHOLM,R. and SJ6STRAND,F. S., J. Ultrastruct. Res. 1, 178 (1957). ELBFRS,P. F., VERVER6AERT,P. H. J. T. and DEMEL,R., J. Cell Biol. 24, 23 (1965).
246 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
DERMER FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). ibid. 26, 263 (1965). -FAWCETT,D. W., J. Histochem. Cytochem. 13, 75 (1965). GROTTE, G., Acta Chic. Scand., Suppl. 211, 84 (1956). HATASA,H. and NAKAMURA,T., Z. Zellforsch. Mikroskop. Anat. 68, 266 (1965). JENNINGS,M. A. and FLOREY, I-I., Proc. Roy Soc. B167, 39 (1967). JENNINGS,M. A., MARCHESI,V. T. and FLOREY, H., Proc. Roy. Soc. B156, 14 (1962). KARNOVSKY,M. J., J. Cell Biol. 35, 213 (1967). KARRER, H. E., J. Biochem. Biophys. Cytol. 7, 357 (1960). KAYE, G. I. and PAPPAS, G. E., J. Cell Biol. 12, 457 (1962). KAYE, G. I., PAPPAS, G. E., DONN, A. and MALLETT,N., J. Cell Biol. 12, 481 (1962). KISTLER,G. S., CALDWELL,P. R. B. and WEmEL, E. R., J. Cell Biol. 32, 605 (1967). KLAUS, M., REISS, O. K., TOOLEY, W. H., PIEL, C. and CLEMENTS,J. A., Science 137, 750 (1962). LucY, J. A. and GLAUERT,A. M., or. )Viol. Biol. 8, 727, (1964). LUZZATI,V., REISS-HuSSON,F., RIVAS, E. and GULm-KRZYWlCKI,T., Ann. N. Y. Aead. Sci, 137, 409 (1966). MACKLIN,C. C., Lancet I, 1099 (1954). MARCnESl, V. T., Proc. Roy. Soc. B 156, 550 (1962). MAYERSON,A. S., WOLFRAM,C. G., SHIRLEY,H. H. and WASSERMAN,K., Am. J. Physiol. 198, 155 (1960). MEYER, E. C., DOMINGUEZ,E. A. M. and BENSCH, K. G., Lab. Invest. 20, 1 (1969). PALADE,G. E., J. Appl. Phys. 24, 1424 (1953). Anat. Record 136, 254 (1960). -PALADE, G. E. and BRUNS, R. R., J. Cell BioL 37, 633 (1968). PAPt'ENHEIMER,J. R., RENKIN, E. M. and BORRERO, L. M., Am. J. Physiol. 167, 13 (1951). RENKIN,E. M., Physiologist 7, 13 (1964). REYNOLDS,E. S., ar. Cell Biol. 17, 208 (1963). SCARPELLI,E. M., The Surfactant System of the Lung. Lea & Febiger, Philadelphia, Pennsylvania, 1968. SCHNEEBERGER-KEELEY,E. E. and KARNOVSKY,M. J., J. Cell Biol. 37, 781 (1968). SCHULTZ,A. A., GRISNER,J. T., WADA, S. and GRANDE, F., Am. J. Physiol. 207, 1300 (1964). SOROKIN,S. P., J. Histochem. Cytochem. 14, 884 (1967). SUTER, E. R. and MAJNO, G., J. Cell Biol. 27, 163 (1965). TOmN, C. E., Anat. Record 120, 625 (1954).
31, 229-246 (1970)
229
The Fixation of Pulmonary Surfactant for Electron Microscopy 1,2 II. Transport of Surfactant through the Air-Blood Barrier GERALD B. DERMER
Department of Pathology, Hospital of the Good Samaritan Medical Center, Los Angeles, California 90017 Received July 22, 1969, and in revised form October 6, 1969 The permeability of the alveolar-capillary barrier to pulmonary surfactant used as an electron-opaque tracer has been investigated. Surfactant visualized by tricomplex flocculation was not only localized to the alveolar surface but also to various forms of vesicles and plasmalemmal invaginations of type I alveolar cells and to the basement membrane (interstitial space) that lies between epithelium and endothelium. To a lesser degree, surfactant was also present within endothelial cell vesicles, endothelial intercellular clefts, capillary lumens, and pulmonary macrophages. The observations indicated that surfactant was transported by quanta from the alveolar surface through type I alveolar cells to the basement membrane by plasmalemmal vesicles and through open membrane-lined channels. The channels appeared to be formed by the fusion of the apical plasmalemma at the leading edge of an invagination with the basal plasmalemma with the subsequent elimination of the fused membranes. Pulmonary surfactant did not pass through epithelial intercellular clefts indicating they were tight (zonulae occludentes), but some surfactant seemed to pass between endothelial cells, supporting the concept that these clefts contained maculae occludentes. To some degree, surfactant appeared to be transported from the basement membrane across endothelium by vesicles to the blood and also to be taken up by pulmonary macrophages which were free within alveoli. It is concluded that the images seen represented stages in the normal turnover and removal of surfactant from the lung and that the majority of surfactant left by passage through interstitial spaces to the lymphatic system. Electron m i c r o s c o p i c studies on the passage of m a t e r i a l across cellular barriers have been carried out with the aid of e l e c t r o n - o p a q u e tracer molecules injected intravenously or b y a d m i n i s t r a t i o n of the tracer in vitro. The passage of ferritin (5, 19, 20), c o l l o i d a l particles of t h o r i u m dioxide (20), saccharated i r o n oxide (19, 20), z A preliminary report of this investigation was presented at the Ninth Meeting of the American Society for Cell Biology in Detroit, November, 1969. Supported in part by research award No. 423 from the Los Angeles County Heart Asscciation and funds from the Tuberculosis and Health Association of California.
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colloidal carbon (30) and horseradish peroxidase (21, 40) across the walls of continuous (nonfenestrated) capillary endothelia have been studied in vivo. These investigations and others (23, 24) have suggested that micropinocytotic vesicles are a mode of transport for macromolecules across endothelium. In the lung the transport of protein across the capillary endothelium by pinocytotic vesicles was suggested (40), but its role in the net transport of protein could not be established. In the lung (40) as well as cornea (24), the vesicular transport of tracer molecules across epithelium could not be detected. With the exception of ferritin and blood-borne chylomicra in newborn rats (43), the tracers generally employed have been substances foreign to the body and not physiological. During the course of an electron microscopic examination of lung tissue (8, 9) that had been treated by "tricomplex flocculation" to allow the visualization of the saturated phospholipid dipalmitoyl lecithin--both the major and surface-active component of surfactant--it became obvious that surfactant phospholipid was found at several sites within the air-blood barrier (interalveolar septum). The airblood barrier is composed of a continuous layer of squamous alveolar cells (type I) interspersed by great alveolar cells (type II) and separated from the underlying continuous, nonfenestrated endothelium by a basement membrane. As expected, electron dense deposits corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in tricomplex fixation were found within the alveolar lumen at the surfaces of epithelial cells, often appearing within invaginations of the cell membrane. Similar deposits of material were localized to membrane-bound vesicles within the cytoplasm of type I alveolar epithelial cells and within the basement membrane (interstitial space) that separates epithelium from endothelium. To a lesser degree similar appearing deposits were present within membrane bound vesicles of the capillary endothelium, endothelial intercellular clefts, and within the lumen of capillaries. The data presented here indicated that these observations represented stages in the transport of surfactant away from the alveolar surface related to the normal turnover and removal of surfactant from the lung, a process that has been poorly understood (39). Evidence will be presented which supports the concept that vesicles derived by pinocytosis at one cell front can be active in the transport by quanta of physiologically important molecules across epithelium and endothelium (33, 34). The evidence also indicated that surfactant phospholipid could pass through the intercellular junctions between adjacent capillary endothelial cells confirming the evidence that these junctions were not tight (zonulae occludentes), but were rather maculae occludentes with gaps between the apposed membranes (21, 40). Also in agreement with others (14, 15, 40) the lateral surfaces of pulmonary epithelial cells appeared to be separated by zonulae occludentes (tight junctions) which prevented
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the passage of surfactant from the alveolar lumen into the underlying interstitial space. Data will also be presented indicating the presence within pulmonary epithelium of continuous channels between the alveolar lumen and basement membrane formed by invaginations of the alveolar plasmalemma. Other observations indicated that alveolar macrophages may also function in the degradation and removal of surfactant from the lung. MATERIALS AND METHODS Mature breathing guinea pigs were sacrificed by the injection of 10 ml of a 2 % xylocaine solution into the cisternal cavity. The fixation and processing of the lung tissue including the scheme of surfactant phospholipid fixation by means of tricomplex flocculation have been previously discussed (9). The sections used in this investigation were usually not stained in order to eliminate the heavy metal precipitates that can occur with section staining. In order to increase the contrast of membranes, however, some grids were double stained with uranyl acetate and lead citrate (38). The sections were examined in a Hitachi 8S electron microscope at an electron optical magnification of 5500-36,000 times. The micrographs were enlarged photographically as needed. RESULTS
Pulmonary epithelium Electron dense material in the form of amorphous, lamellar, and particulate deposits corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in the tricomplex fixation scheme was found within the alveolar lumen at the surface of squamous epithelial cells. Many vesicles opening onto the alveolar surface also contained reaction product (Figs. 1 and 2). The membrane bounding these vesicles was continuous with the apical plasmalemma (Figs. 3 and 4). Many of these forms had flask shapes and somewhat elongated necks. It was not uncommon to find rows of these vesicles with similar profiles and containing reaction product opening onto the alveolar surface (Fig. 1). The vesicles intruded varying distances into the cytoplasm, and in some instances they extended through the cell to the plasmalemma bounding the basement membrane (Figs. 1 and 3-5). The impression gained from the micrographs was that the apical plasmalemma at the invaginating end of the intrusions fused with the basal plasmalemma with the eventual elimination of the layers of the two fused membranes, forming a continuous membrane-lined channel between alveolar lumen and basement membrane. In Fig. 3, a flask-shaped vesicle extends through the epithelial cytoplasm and appears fused with the basal plasmalemma. Although one of the dense leaflets of the invaginating plasmalemma is obscured by reaction product, the fusion appears to have produced a five-layered diaphragm which buckles convexly toward the basement membrane.
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A similar image is seen in Fig. 4. A n o t h e r similar a p p e a r a n c e occurs in Fig. 5 in u n s t a i n e d sections. A l t h o u g h m a n y of the vesicles seen were a t t a c h e d to the p l a s m a l e m m a , a considerable n u m b e r were f o u n d free in the c y t o p l a s m of the p u l m o n a r y type I alveolar cells (Figs. 6 a n d 7). M a n y of these vesicles were filled with reaction product. Generally the vesicles were r o u n d a n d 50-100 m # in diameter. Occasionally larger vesicles were seen, a n d there were indications t h a t this was a result of vesicle coalescence. There were images where a d i a p h r a g m was seen between two fused vesicles (Fig. 8) a n d others where there a p p e a r e d to be a c o n t i n u i t y of vesicular contents (Fig. 9). A l t h o u g h less frequently seen t h a n at the apical cell surface, the basal surface of type I alveolar cells exhibited vesicles which were o p e n e d to the b a s e m e n t m e m b r a n e a n d c o n t a i n e d reaction p r o d u c t (Figs. 2 a n d 6). They generally were n o t f l a s k - s h a p e d with elongated necks, b u t h a d forms whose limiting m e m b r a n e was fused in p a r t with the b a s a l p l a s m a l e m m a . A t the p o i n t of fusion the basal p l a s m a l e m m a generally f o r m e d a shallow infundibulum. Vesicles were seen separated f r o m the interstitial space only by m e m b r a n e m a t e r i a l (Fig. 6) a n d other images showed e l i m i n a t i o n of this m a t e r i a l with a p p a r e n t continuity between vesicular contents a n d interstitial space. F i g u r e 2 shows apical a n d b a s a l p l a s m a l e m m a vesicles o p e n e d onto the alveolar l u m e n a n d interstitial space, respectively. Epithelial intercellular clefts between type I alveolar cells were free of reaction
Key to abbreviations A S alveolar space B M basement membrane CL capillary lumen
En capillary endothelium Ep alveolar epithelium
FIG. 1. Electron opaque material corresponding to the reaction product between surfactant phospholipid and the heavy metal salts used in the tricomplex reaction scheme is seen at the alveolar surface of a type I epithelial cell and within a row of vesicles opened onto the surface. These forms have similar profiles, exhibiting flask shapes with somewhat elongated necks. One of these vesicles (arrow) extends nearly across the epithelium to the basal plasmalemma. Except for one vesicle within the endothelium, the rest of the air-blood barrier is free of reaction product. Not section stained. × 67,090. FIG. 2. Reaction product is seen at the alveolar surface of a type I alveolar cell and within vesicles which appear opened to the alveolar surface or closely apposed to the apical plasmalemma. Several of these forms have flask shapes. At the basal surface, one vesicle containing reaction product appears opened to the basement membrane (arrow) while another seems fused with the basal plasmalemma (arrow). The basement membrane contains some electron-opaque material while the endothelium does not appear to contain any. Not section stained. × 67,000. FIGs. 3 and 4. The alveolar surface and vesicles of type I cells opened to the surface contain reaction product. In each figure, one vesicle extends through the epithelial cytoplasm and appears fused with the basal plasmalemma. In Fig. 3, although one of the dense leaflets of the apical plasmalemma is obscured by reaction product, the fusion appears to have produced a five-layered diaphragm which buckles convexly toward the basement membrane (arrow). A similar form is seen in Fig. 4 (arrow). Other portions of the air-blood barrier are free of electron-opaque material. Section stained with uranyl acetate and lead citrate. × 105,000.
CL
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p r o d u c t a n d the o u t e r leaflets of the b o u n d i n g m e m b r a n e s a p p e a r e d fused over a considerable p o r t i o n of their length (Fig. 10).
Basement membrane There were instances where in a d d i t i o n to reaction p r o d u c t at t h e alveolar surface, there was reaction p r o d u c t within the vesicles at the alveolar surface, within vesicles at all levels within the epithelial cytoplasm, b u t where the b a s e m e n t m e m b r a n e cont a i n e d little reaction p r o d u c t (Fig. 6). There were other instances where the basem e n t m e m b r a n e also c o n t a i n e d reaction p r o d u c t (Figs. 2, 5, 7, a n d 10). A t other places within the a i r - b l o o d b a r r i e r the b a s e m e n t m e m b r a n e c o n t a i n e d a l m o s t all of this m a t e r i a l F o r considerable distances, the b a s e m e n t m e m b r a n e could be t r a c e d by following the electron o p a q u e deposits within it (Fig. 11).
Endothelium The e n d o t h e l i u m c o n t a i n e d reaction p r o d u c t b u t n o t to the extent that was seen within the b a s e m e n t m e m b r a n e a n d epithelium (Figs. 7 a n d 12-14). It was n o t unc o m m o n to find the b a s e m e n t m e m b r a n e filled with electron dense particles while the e n d o t h e l i u m c o n t a i n e d very little of this m a t e r i a l (Fig. 11). Very occasionally vesicles filled with reaction p r o d u c t a p p e a r e d in close a p p r o x i m a t i o n to the b a s a l p l a s m a l e m m a of the e n d o t h e l i u m (Figs. 7 a n d 13). M o r e frequently, reaction p r o d u c t was f o u n d within free vesicles within the e n d o t h e l i u m interspersed a m o n g e m p t y vesicles (Figs. 13 a n d 14). I n the epithelium m a n y vesicles were filled with reaction p r o d u c t while within the e n d o t h e l i u m it was m u c h m o r e c o m m o n to find only a p o r t i o n of some of the vesicles filled with this material. Vesicles containing reaction FIG. 5. Reaction product at the alveolar surface extends into vesicular forms which traverse the cytoplasm of a type I cell. The invaginating edges of these forms appear fused with the basal plasmalemma (arrows). One of these invaginations appears flask-shaped. Other vesicles opened to the alveolar surface and containing reaction product intrude only a small distance into the cytoplasm. In this case, the basement membrane contains a considerable amount of reaction product. Not section stained. × 65,000. FIG. 6. The cytoplasm of a type I alveolar cell is filled with vesicles containing reaction product. These vesicles appear free within the cytoplasm and exhibit a bounding membrane. Electron opaque material is also seen at the alveolar surface while the basement membrane contains little of this material. At the basal surface of this cell, vesicular membranes appear fused with the plasmalemma exhibiting diaphragms which separate vesicular contents from the basement membrane (arrows). At the points of fusion the basal plasmalemma forms a shallow infundibulum. Not section stained. x 69,000. FIG. 7. Electron opaque material is seen at the alveolar surface, within cytoplasmic vesicles of a type I alveolar cell, within vesicles of the endothelinm, and also within the basement membrane. The capillary lumen is free of this material. Not section stained, x 67,000. FIa. 8. Two fused vesicles, one of which contains electron opaque material; are seen close to the basal plasmalemma of the epithelium. A diaphragm is present between the two forms. Much electronopaque material is seen at the alveolar surface, Section stained with uranyl acetate and lead citrate. x 90,000.
&S
16 -- '701825
J . Ultrastructure J~esearch
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product also appeared fused with the apical plasmalemma at the luminal surface of capillaries (Fig. 12). There were instances where there was reaction product within the basement membrane, within some vesicles in the endothelial cytoplasm but where the capillary lumen contained none of this material (Figs. 7 and 14). There were other images where the capillary lumen also contained reaction product (Fig. 12). Intercellular clefts between endothelial cells contained reaction product (Fig. 14). Near to the capillary lumen, the adjacent plasma membranes appeared closely approximated to form a cell junction (Fig. 15). Reaction product was rarely found within the junctions.
Pulmonary macrophages Within alveoli, electron dense deposits were found lining the surfaces of free macrophages after tricomplex fixation (Figs. 16 and 17). Occasionally vesicles opening onto the surfaces of these cells contained reaction product (Fig. 16) and near the plasmalemma, free vesicles containing this material were also occasionally observed (Fig. 16). Deeper within the cytoplasm, vesicles containing reaction product were not seen although electron-opaque material of this sort was observed with the cytoplasmic matrix of the macrophages. In Fig. 17 similar appearing electron dense material is seen at the surface and in the cytoplasm and in the latter case does not appear within cisternae of cytoplasmic membranes. This can perhaps be better visualized in a higher power illustration (Fig. 18). Inclusion bodies of several sorts were seen within the cytoplasm of the pulmonary macrophages. Some contained granular material while others contained both lamellar and granular forms (Fig. 17). DISCUSSION Tricomplex fixation has allowed the electron microscopic visualization of the major component of pulmonary surfactant, dipalmitoyl lecithin, which as expected was found at the alveolar surface (8, 9). Surfactant was detected by the presence of electron-opaque deposits and lamellar structures corresponding to the reaction proFIG. 9. A continuity of vesicular contents, which consists of electron opaque material, appears to exist between two fused vesicles of a type I alvolar cell (arrow). Reaction product is also seen at the alveolar surface and to a small extent within the basement membrane. Not section stained. × 78,000. FIG. 10. The epithelial intercellular cleft between two type I alveolar cells is free of reaction product and the bounding membranes appear tightly fused (arrows). However, reaction product is found at the alveolar surface, within a flask-shaped vesicle opened to the surface, within a free cytoplasmic vesicle, and within the basement membrane. Not section stained, x 69,000. Fit. 11. The basement membrane between pulmonary epithelium and endothelium is filled with electron-opaque material while the cellular portions of the air-blood barrier and the capillary lumen containing a red blood cell (RBC)has little of this material. Some electron-opaque material is also seen within the alveolus. Section stained with uranyl acetate and lead citrate, x 24,000.
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Fio. 12. Reaction product is found within the basement membrane, within a capillary, some of which appears at the surface of red blood cell, and within several vesicles of the endothelium. These vesicles appear fused with or closely apposed to the apical plasmalemma of the endothelial cell (arrows). Not section stained, x 67,000.
duct formed between surfactant phospholipid a n d the heavy metal salts used i n the tricomplex reaction scheme. This material was n o t observed when lung was fixed only in glutaraldehyde a n d osmium. The observations reported here i n d i c a t e d that surfactant was f o u n d at other sites within the air-blood barrier suggestive of pathways by which surfactant was degraded a n d removed from the lung. These processes have been poorly u n d e r s t o o d (39) b u t the alveolar lining of surfactant did appear to have a short lifetime as c o m p a r e d to the cells of the lung (6). P u l m o n a r y surfactant after tricomplex fixation acted as a n electron-opaque tracer which has been used here to investigate the permeability of the alveolar-capillary barrier to this physiologically i m p o r t a n t material. Palade a n d Bruns (35) have described structural m o d u l a t i o n s of p l a s m a l e m m a l vesicles of vascular endothelia. They proposed a rationalization of these structural FIG. 13. This low power micrograph illustrates vesicles filled with reaction product within pulmonary endothelium. The vesicles appear in a row fused with or closely apposed to the basal plasmalemma (arrows). Elsewhere they are found as free vesicles at different levels within the cytoplasm of the endothelium. The alveolar surface contains much reaction product while the basement membrane and capillary lumen contain none of this material. Not section stained, x 28,000. FIO. 14. The intercellular cleft between two endothelial cells contains reaction product (arrows), as does the basement membrane and free vesicles within the endothelial cytoplasm. The capillary lumen contains none of this material. Not section stained, x 52,000. Flo. 15. Reaction product is found within a cell junction between two endothelial cells (arrows). Much reaction product is seen at the alveolar surface and to some extent within the basement membrane, but the capillary lumen is free of this material. Not section stained, x 67,000. FIG. 16. Reaction product is found at the surface of a pulmonary macrophage and within a vesicle opened onto its surface (arrow). Similar material is found within a free vesicle near the surface (arrow), but other vesiclesand inclusionsdeeper within the cytoplasm do not appear to contain material of similar electron opacity. Section stained with uranyl acetate and lead citrate x 30,000.
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modulations of vesicles in terms of their involvement in transport in quanta across endothelium. Different forms of vesicles as described by Palade and Bruns have been observed in this investigation to contain surfactant, supporting the concept that these forms are involved in the transport of molecules across cells. The present observations also extend their views by observations of structural modulations of plasmalemmal vesicles within pulmonary epithelium and endothelium and the observation of a new vesicular form. Vesicles containing reaction product and opened onto the alveolar surface have been frequently observed. Most of these forms have flask shapes and somewhat elongated necks. It has been proposed (35) that forms of this type represent stages in the formation and loading of membrane invaginations, followed by their being pinched off to form isolated vesicles. The pinching off process has been thought to be slow in endothelia (16, 35), and this seems to hold true for pulmonary epithelium also because rows of these invaginations in approximately the same stage of pinching off were often seen at the alveolar surface. Most of the invaginations at the alveolar plasmalemma appeared to pinch off forming vesicles many of which were filled with reaction product. These free vesicles were seen at all levels within the cytoplasm of the squamous epithelium. It was possible, however, that some of these forms represented invaginations whose necks were opened to the alveolar surface at some other level of sectioning. The basal surface of the pulmonary epithelium exhibited vesicles containing reaction product which were either fused with the basal plasmalemma or were opened to the basement membrane by the elimination of the fused membranes. These forms have been assumed to represent stages in the discharge process of vesicular contents (35). Within the epithelial cytoplasm, membrane diaphragms were seen between fused vesicles. Other images showed forms where surfactant appeared to be continuous from one vesicle to another after elimination of the fusion layer. These forms apparently gave rise to the large vesicles which were seen within alveolar type I cells. It has been suggested that chains of vesicles might be formed which open simultaneously on both cell fronts but, as in muscle capillaries (19), this observation has not yet been made. However, separate vesicles containing reaction product which opened simultaneously on two cell fronts were a frequent observation. The endothelium of visceral capillaries is interrupted by fenestrae generally closed by apertures or diaphragms (12), as is the endothelium of tongue capillaries (4). Fusion of the plasmalemma on the blood front with the plasmalemma on the tissue front of the cell followed by a progressive elimination of membrane layers appeared to result in the formation of these fenestrae (35). This process has been thought to be confined to vascular endothelium but the observations presented here indicated a similar mechanism might be working within pulmonary epithelium. Flask-shaped
FIG. 17. Reaction product is found at the surface of a pulmonary macrophage and similar appearing electron-opaque material is also seen with the cytoplasmic matrix (arrows). This cytoplasmic material is not found within cisternae of intracellular membranes. Some inclusion bodies within the cytoplasm contain granular material while others show lamellar and granular forms (arrow). Section stained with uranyl acetate and lead citrate, x 25,000. FIG. 18. Electron-opaque material is seen within the cytoplasm of a pulmonary macrophage while the cisternae of membrane-bound vesicles do not contain any of this material. At several locations the cytoplasmic material completely surrounds these vesicles and the vesicular membranes can often be detected at the boundaries of the electron opaque material (arrows). Section stained with uranyi acetate and lead citrate, x 54,000.
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vesicles filled with reaction product and opened onto the alveolar surface extended through the epithelial cytoplasm, and the plasmalemma on the alveolar front became fused with the plasmalemma on the basement membrane front of the cell. The fusion generally produced a five-layered diaphragm similar to what was seen in tongue capillaries (35). This type of diaphragm had been considered to be an intermediate stage of membrane fusion which led to an eventual removal of the fusion layer (35). Forms suggestive of more advanced stages of diaphragm elimination have been seen. These forms would give rise to continuous membrane-lined pores between the alveolar lumen and basement membrane, which were apparently very short lived or occurred infrequently because few examples have been found. Alveolar surfactant did not penetrate the intercellular spaces between adjacent type I pulmonary epithelial cells, supporting the data which indicated that junctions between epithelial cells were tight (zonulae occludentes) (i4, 15, 25, 40). If the rationalization of different structural forms of plasmalemmal vesicles as proposed by Palade and Bruns (35) in terms of their involvement in transport by quanta across cells was correct, then surfactant was loaded into membrane invaginations at the alveolar surface. Most of these invaginations then pinched off to form free vesicles which moved to and fused with the basal plasmalemma, with the elimination of the fused membranes and the discharge of surfactant into the interstitial space. The vesicular forms seen at the alveolar and interstitial fronts of the cell supported this interpretation. There also appeared to be an alternative route by which surfactant could be transported from alveolar lumen to the basement membrane. This would take place by the fusion of the apical plasmalemma at the leading edge of an invagination with the basal plasmalemma with the elimination of the fused membranes forming an open membrane-lined channel between the alveolar lumen and basement membrane. This has not been a time sequence study of the transport of an electron, opaque tracer since injections of the tracer molecule were not performed. Rather, this has been a study of the distribution of a naturally occurring material present in normal, mature lungs at all times. Therefore, data obtained at early and late stages after tracer injection indicating the direction of transport were impossible to obtain. The data, however, suggested that surfactant transport takes place through the air-blood barrier and it appeared that different regions of one lung illustrated various stages of the process. The vesicular forms seen at the alveolar and interstitial fronts of the type I epithelial cell indicated that the transport of surfactant by quanta was inward from the alveolar surface to the basement membrane, not in the opposite direction. Other considerations also supported this conclusion. There were instances when there was much reaction product at the alveolar surface, within vesicles at the alveolar surface, within free vesicles of the epithelial cytoplasm, within vesicles at the
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basal surface of the cell interposed among empty vesicles and where the basement membrane itself did not contain any reaction product. This sort of evidence (21) would suggest that the vesicles had moved across the epithelium from the alveolar surface. Furthermore, there is considerable circumstantial evidence (2, 18, 26, 29, 42) which indicates that surfactant is synthesized within type II alveolar cells and that it is secreted from these cells into the alveolus forming the surface lining layer. It would be expected therefore that, in the degradation and turnover of surfactant, material would leave the alveolar surface and not be transported to it. The transport of surfactant through pulmonary epithelium took place only at alveolar type I cells, for surfactant transport through type II alveolar cells has not been observed. Physiological studies (1, 10, 11, 32, 41) have shown that albumin solutions introduced into pulmonary alveoli are absorbed by the lung in an intact form (1, 41). The delayed removal of protein from alveoli appeared to occur in the removal of protein from the interstitial space which had accumulated a large amount of albumin, rather than in passage through the alveolar epithelium (32). The interstitial space within the intra-alveolar septum is continuous with spaces surrounding pulmonary vessels and bronchi where pulmonary lymphatics exist (32, 44) and the data indicated that the lymphatic system removed some of the albumin which had been absorbed from alveoli (11, 32). Although for albumin, most of the absorbed protein was found in the pulmonary blood (32). These data supported the suggestion that pulmonary lymphatics may function to remove foreign material or fluid from alveoli (44). Thus it appears that the epithelial surface of the lung is active in the absorption of protein as well as surfactant and the physiological and electron microscopic data indicate that both may be transported by similar mechanisms. Serum albumin has a molecular weight of 69,000 with a diameter of 3.8 nm and molecules of this size appear physiologically to be transported across cells by vesicles (37) or through pores larger than about 4.0 nm in diameter (17, 31, 36, 37). These conclusions are consistent with the present electron microsciopic study indicating that surfactant is also transported across the pulmonary epithelium by vesicles and possibly through membrane-lined pores. Therefore pulmonary surfactant appears to behave as a protein-like macromolecule even though the molecular weight of dipalmitoyl lecithin is only 754. However, the alveolar surface lining layer of surfactant after tricomplex fixation appeared to be made up of small rounded particles 5.0-10.0 nm in diameter (9) in which the phospholipid molecules seemed to be associated as molecular aggregates called micelles. Also, it is known that a micellar solution of phospholipid does occur in the phase diagram of lecithin-water system (28) and negatively stained preparations of lecithin have exhibited particles about 4.0 nm in diameter (27). The size of these micelles would be in the range of a molecule with dimensions like serum albumin. It also seems interesting that albumin
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absorbed from the alveolus had accumulated within the basement membrane (32), for the same holds true when considering surfactant transport through the alveolarcapillary barrier. The basement membrane contained much reaction product due to its accumulation there after transport through the pulmonary epithelium. For long distances the basement membrane could be traced by following the electron dense deposits within it and since this interstitial space is continuous with pulmonary lymphatics, it appeared that the majority of surfactant was removed from the lung by the lymphatic system. This does not seem surprising when one considers that long-chain triglycerides are removed from the intestine by the lymph after absorption (3). Dipalmitoyl lecithin, the major component of pulmonary surfactant, is a phospholipid containing two long-chain fatty acids. Only a small amount of surfactant within the basement membrane appeared to be transported across the endothelium into the blood in contrast to protein absorption where the capillaries seemed to remove the majority of the albumin absorbed from the alveolus (32). Again the similarity to intestinal absorption seems noteworthy, for digested protein is also removed from the intestine by the blood. Transport of surfactant across the endothelium appeared to be vesicular and between cells through intercellular clefts. Most images showed reaction product within the basement membrane, within some endothelial vesicles, within intercellular clefts, but where the capillary lumens contained little or none of this material. This was suggestive of transport from the basement membrane to the blood, but images representing vesicular loading at the basal surface of the endothelium have been difficult to obtain. Furthermore, it was not known what proportion of this endothelial transport was vesicular or through the intercellular space. The vesicular transport of surfactant through pulmonary endothelium would represent an inversion of the process of pinocytosis since material loaded into vesicles at the basal surface was carried through the cytoplasm and discharged at the apical surface into a capillary. A similar observation has been made on corneal epithelium (23). Although reaction product was found within endothelial intercellular clefts, it was quite rare to find any within cell junctions where the adjacent cell membranes appeared closely apposed. It has been shown, however, that endothelial cell junctions have a gap between the adjacent plasma membranes of about 4.0 m# which are permeable to peroxidase used as an electron opaque tracer (20, 21). Therefore, these junctions are probably also permeable to pulmonary surfactant. The detection of reaction product within pulmonary epithelium and endothelium and also within endothelial intercellular spaces and the basement membrane, indicated that surfactant was not degraded at these sites. The tricomplex reaction is specific for phospholipids (13), and if the choline residue had been split off, reaction product would not have been formed.
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Pulmonary macrophages also seemed to be involved in the removal of surfactant from the lung. Reaction product at the surface of macrophages was occasionally found within invaginations of the plasmalemma and within free vesicles near the surface. However, reaction product was not found in vesicles deeper within the cytoplasm and the free vesicles near the surface might have represented invaginations opened to the surface at some other level of sectioning. The alveolar macrophage has been shown to be phagocytic (22) however and the uptake of surfactant by this process might be expected. The inclusion bodies within these cells showed acid phosphatase activity (7) and were thought to be concerned in the digestion of exogenous material (7), and this material might in part be surfactant. The lamellar forms seen within these inclusions exhibited a repeat of about 5.0 nm (22) and were very similar to lamellar forms seen within the alveolar surface lining material (8, 9). The laminated and granular forms found within these inclusion bodies might in part represent the end products of the intracellular digestion of pulmonary surfactant by macrophages. It appeared that surfactant could be internalized by macrophages by another mechanism besides pinocytosis, for electron dense material similar to reaction product seen at the surfaces of pulmonary cells was found within the cytoplasmic ground substance of macrophages. Reaction product appeared to be excluded from cisternae of cytoplasmic membranes, and it seemed that surfactant was transported through the plasmalemma of the macrophages, solubilized within its cytoplasm, and possibly stored in a digested form within the lysosome-like inclusions. The author is grateful to Dr. Harry B. Neustein for his critical evaluation of the manuscript. REFERENCES 1. 2. 3. 4. 5.
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