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An electron microscope study of the basement membrane of proliferated bile ductules
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
11,
17-27
(1969)
An Electron Microscope Study of the Basement Membrane of Proliferated Bile Ductules KINICHIRO Department
KAJIKAWA
of Pathology.
School
AND
of Medicine,
Received
March
SHOICHIRO Kanazawa
KAKIHARA Universi@.
Kanazawa,
Japan
21, 1969
The epithelial cell is separated from the surrounding connective tissue by the basement membrane. In electron microscopy the basement membrane appears as a continuous layer consisting of fine filaments embedded in an amorphous matrix. This layer, usually called lamina densa (Low, 1961), is separated from the epithelial cells by a narrow transparent space, the lamina lucida (Low, 1961), and on the opposite side connects with the interstitium. The origin of basement membrane is a matter of dispute. Because of its histochemical properties and its close relation to the connective tissue, the basement membrane was considered to be of connective tissue origin (Gersh and Catchpole, 1949). McLaughlin (1966), using tissue cultures, observed that the epidermal cells were surrounded by proliferating fibroblasts in association with the accumulation of PAS-positive materials, and concluded that the basement membrane was formed by mesenchymal products under the influence of the epithelial cells. Recent evidence gathered from autoradiographic (Hay, 1964), immunologic (Pierce and Midgley, 1963; Pierce et al., 1964), chemical (Mukerjee et al. 1965) and electron microscopic (Kurtz and Feldman, 1962) studies has indicated that epithelial cells secrete the basement membranes. This evidence, however, does not explain how the epithelial secretion does not diffuse into the connective tissue, but accumulates just beneath the cells as an organized envelope. It is still uncertain whether the fibroblast is not entirely involved in the formation of the basement membrane and what relation the basement membrane has to the underlying collagen or reticular fibers. In the course of a study of rat hepatic cirrhosis induced by 3’-methyl-diaminoazobenzene, we have encountered many examples to suggest that the fibroblast may participate in the organization of the basement membrane of proliferated bile ductules. In this paper we will discuss the possible role of the fibroblast in formation of the epithelial basement membrane to throw some light on the epithelio-mesenchymal relationship. MATERIALS Twenty male were fed a diet The liver was tinely prepared
AND METHODS
Wistai albino rats, with an initial body weight of 150-200 gm containing 0.06 Y 3’-methyl-diaminoazobenzene for 90-150 days. fixed with neutral formalin, and paraffin sections were roufor light microscopy. 17
18
KAJIKAWA
AND
KAKIHARA
The tissue for electron microscopy was fixed with 2% osmium tetroxide, buffered to pH 7.2 with Verona1 acetate, at 4” C for 1 hour. After dehydration in graded aceton solution, the materials were embedded in Epon 812 (Luft, 1961). Thin sections were cut with a Fernandez-Moran ultramicrotome (Leitz) using glass knives. The sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Electron micrographs were taken with a HU-11 PM type (Hitachi Ltd.) or a JEM-7 type (Japan Electron Optics Laboratory Co. Ltd.) electron microscope at magnifications 3000-10,000. RESULTS Light microscopy revealed that hepatic cirrhosis together with proliferation of bile ductules developed in all of the rats, although there was considerable variability in its severity from one animal to another and among portions of the same liver. By electron microscopy general features of cirrhosis were not different from those previously reported (Popper et al., 1960, 1961; Hutterer et al., 1961; Schaffner and Popper, 1961; Carruthers et al., 1962; Rubin et al., 1963). In many areas branches of the proliferated bile ductules were encompassed by the
FIG. 1. Ductular cell reaction in a cirrhotic liver. Histiocytes around the proliferated ductules (De). No basement membrane tween mesenchymal cells and ductular epithelium. X20,M)O.
(Hs) and fibroblast (Fb) are seen is seen. Note intimate contact be-
BASEMENT
MEMBRANE
OF BILE
DUCTULES
19
fibrous connective tissue, whereas in some areas the ductular cell reaction (Popper et al., 1957) was evident. Apparently proliferating ductular epithelium consisted of cells with a large nucleus and relatively numerous profiles of rough-surfaced endoplasmic reticulum. Cisternae of the endoplasmic reticulum were slightly to moderately distended (Figs. 1, 2, and 4). Ribosomes were distributed throughout the cytoplasm in small clusters. Mitochondria, round or oval in shape, were relatively numerous. The Golgi complex appeared prominent. Lateral surfaces of the cells were relatively smooth, except for occasional interdigitations. Desmosomes were found in some places. The luminal surface had microvilli. The basal surface appeared slightly irregular having small cytoplasmic projections. Where the ductural cell reaction was evident, no basement membrane was found (Fig. 1). In this area large numbers of inflammatory cells-including histiocytes, leucocytes, and plasma cells-were found around the ductules. Fibroblasts were not common, but in some instances were found scattered among the inflammatory cells. The fibroblasts had elongated cytoplasm with extensive rough-surfaced endoplasmic reticulum. They were distinguished by this characteristic from histiocytes which were rich in smooth-surfaced vesi-
FIG. 2. Ductular cell reaction in a cirrhotic liver. Histiocytes present near the ductules (De). Intercellular space is filled with basement membrane is seen. X16,000.
(Hs) and fibroblasts (Fb) are a dense material. No distinct
20
KAJIKAWA
AND
KAKIHARA
cles and lysosomes (Gieseking, 1963; Kajikawa, 1964). The histiocytes and fibroblasts were close to the ductular epithelium or came directly in contact with it without any intervening basement membrane (Figs. 1 and 3). The intercellular space contained collagen fibrils and amorphous materials. An interesting finding was the accumulation of a dense material in the intercellular spaces near the ductules. This material, at higher magnifications, was similar in appearance to the basement membrane material in that it consisted of ill-defined fine filaments embedded in an amorphous matrix of moderate density (Fig. 5). There was considerable variation in quantity of the dense material, probably depending on the development of cirrhosis. In areas where the proliferation of the fibroblasts and histiocytes was still prominent, the dense material was found diffusely in the intercellular space and the basement
FIG. 3. Portion of a cirrhotic liver. Ductular epithelium (De) is surrounded by the basement membrane-like material (Bm) which, in part, is continuous with a similar material around the fibroblast (Fb) that is in direct contact with the ductule. Fibrocytes (Fbb) extend attenuated cytoplasmic processes around the ductule. Aggregation of histiocytes (Hs) is evident at a distance from the ductule. X7500.
BASEMENT
MEMBRANE
OF
BILE
DUCTULES
21
membrane of the ductules was hardly discerned (Fig. 2). In advanced cirrhosis proliferated ductules were surrounded by a laying down of the dense material. The fibroblasts which were close to the ductules extended ramified cytoplasmic processes as if to enclose the material (Figs. 4 and 5). The dense material in this area assumed an appearance similar to the basement membrane, except that it was much thicker than the normal basement membrane and that the lamina lucida was absent in many portions. The fibroblasts in the periductular regions were in contact over variable portions of the cell surface with the dense material. Sometimes there was a direct continuity between the dense material around the fibroblasts and the ductules (Figs. 3 and 7). Frequently the dense material around the fibroblasts was associated with fine fibrils and, in some instances, appeared to have been replayed by progressive deposition of the fibrils (Fig. 7). The fibrils were 100150 A in diameter and had no distinct periodicity. These have been observed in various connective tissue and were called microfibrils (Low, 1962; Haust, 1965). Where the amount of collagen fibrils was large, both the dense material and microfibrils were small in amount. It was noted that massive accumulation of the dense material appeared to be associated with inhibited formation of collagen fibrils in the periductular
FIG. 4. This portion of cytoplasmic processes ductular fibrous stroma.
of ductular epithelium (De) of fibroblasts (Fb) appears x 16,000.
is surrounded to demarcate
by the dense material. the dense zone and
A layer the peri-
22
KAJIKAWA
AND
KAKIHARA
FIG. 5. The space between the ductular epithelium (De) and attenuated cytoplasmic processes of the fibroblast (Fb) with the dense material. The lamina lucida is not distinct and only suggestive, at best. Small round bodies are scattered within the material as indicated by the arrows. We have not determined whether these bodies represent cytoplasmic projections or secretory products. The interstitium further peripheral to the fibroblasts contains numerous collagen fibrils (Cf) and sparse microfibrils (Mf). X40,000.
regions. The space between the ductular epithelium and subjacent fibroblasts was occupied by the dense material, whereas the stroma on the opposite side of the fibroblasts was filled with collagen fibrils arranged in bundles (Figs. 4 and 5). In some places, the dense material was alienated from the fibroblasts by small amount of fine fibrils (Fig. 4). In this area the lamina lucida was apparent. Tn Fig. 6, the dense material in the periductular space was largely absent and presumably had been replaced by collagen which seemed to be deposited progressively from the vicinity of the fibroblasts towards the epithelia. These collagen fibrils were smaller in diameter (about 300 A wide) than those elsewhere. They appeared loosely arranged, being intermingled with the microfibrils and occasional fragments of the dense material. These fibrils seemed to correspond to the reticular fibers in light microscopy. The periductular collagen deposition did not reach the basal surface of the ductular epithelium so that the dense material remained just beneath the epithelium as a continuous thin layer (lamina densa) (Fig. 7). Thus, presumably mature basement membrane appeared to fuse with the stromal microfibrils and collagen fibrils without distinct demarcation.
BASEMENT
MEMBRANE
OF
BILE
DUCTULES
23
With collagen deposition, in the periductular regions the stromal cells, presumably fibrocytes, were scant and appeared in a given plane of section mostly as thin strands of cytoplasm scattered among the collagen fibrils that were well oriented in small bundles. At a distance from the ductules and further distal from the area where collagen fibrils were in bundles, however, the interstitium had numerous fibroblasts and chronic inflammatory cells (Figs. 3, 6, and 7). What appeared to be fully developed basement membrane was associated with the ductular epithelium that had cytoplasmic structures similar to those in the normal liver (Schaffner and Popper, 1961; Steiner and Carruthers, 1961). The cytoplasm contained small mitochondria and narrow strands of rough-surfaced endoplasmic reticulum. Free ribosomes appeared as a few scattered clusters. Occasionally lysosomes and lipid droplets were observed. The basal surface resting on the basement membrane was smooth. DISCUSSION It was a noticeable finding in this study that the basement membrane did not surround the ductules where inflammatory cells were near them, or where fibroblasts and histiocytes were in direct contact with them. In this region the ductular epithelium was relatively rich in free ribosomes in the cytoplasm
FIG. density, collagen
6. Concomitant with the reduction of areas occupied by the diffuse and amorphous which results in a more defined dense zone like basement membrane, a narrow layer of fibrils (Cf) appears on the luminal side of the periductular fibroblasts (Fb). ~20,000.
24
KAJIKAWA
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,KAKIHARA
FIG. 7. Well-organized basement membrane (Bm) is separated from the ductular epithelium (De) by the lamina lucida. The amorphous density (Gs) around the process of fibroblast (Fb) appears partly continuous with the basement membrane. In the right corner a portion of fibroblast with wide cytoplasm is seen. The microfibrils (Mf) are often seen close to the fibroblasts. X~O,OOO.
and showed occasionally cytoplasmic projections at the basal surface. These features may reflect immaturity of the ductular epithelium. In cirrhotic areas, however, the ductules which had apparently similar cytoplasmic structure, were surrounded by the basement membrane-like material. Thus the absence of the basement membrane in the region of ductular cell reaction does not appear to be owing to immaturity of the epithelial cells, but rather to environmental conditions which seem to be controlled by the underlying connective tissue,; we are inclined to think that the inflammatory cells could not provide a suitable environment for the formation of the basement membrane. Our observation showed that a dense material was deposited in the space between the ductular epithelium and subjacent fibroblasts. The origin of the material is not clear. One is tempted to speculate that the fibroblasts may produce some components of the material, since it appears structurally very similar to the material (presumably the stromal ground substance) around the fibroblasts which are located freely in the stroma, and since these two materials are continuous in places.
BASEMENT
MEMBRANE
OF
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DUCTULES
25
On the other hand, autoradiographic study by Hay (1964) has indicated that the epithelial cells secrete proline-rich protein which apparently contributes to the formation of their own basement membrane. Similarly, on the basis of chemical and immunological studies, Pierce et al. (1963, 1964), and Mukerjee et al. (1965) concluded that the basement membrane was an epithelial secretion and differed from the connective tissue elements. Since these studies seem to be indicate quite convincingly that the epithelial cells do secrete the basement membrane material, one could not rule out the possibility that the dense material beneath the ductular epithelium, at least in part, may be of epithelial origin. Chemical studies (Bruckhausen and Merker, 1965; Mukerjee et al., 1965) have indicated that the basement membrane contains polysaccharides and collagen as do the products of fibroblasts. The fibroblasts and epithelial cells may produce common materials which are concerned with the formation of the basement membrane. For the moment, it seems reasonable to assume that the deposits in the space between the epithelium and subjacent fibroblasts are occupied by a mixture of materials which are produced by both cells. The dense material beneath the epithelium appeared to be converted into the basement membrane, concurrent with collagen deposition which was most likely induced by the underlying fibroblast (Figs, 4-7). Grobstein and gallman (1965) proposed the view that the epithelial surface provides a favorable site for collagen polymerization. Our observation, however, indicated that the collagen fibrils in the periductular regions were smaller in number and diameter than those in the stroma on the opposite side of the underlying fibroblasts. This suggests that the fiber formation may be inhibited in the periductular regions. The reason for this is unknown, but it is assumed that the epithelial cells exert influence to the surrounding connective tissue, by which collagen polymerization or fibroblastic activity may be affected. Wood (1960) demonstrated that mucopolysaccharides could alter the rate of collagen polymerization in vitro. The basement membrane material is known to be relatively rich in mucopolysaccharides and these would seem to inhibit further polymerization of collagen produced by the fibroblasts. In addition it should be considered that collagen synthesis by fibroblasts may be reduced in the periductular region, since the fibroblasts in this region appeared to be rapidly transformed into fibrocytes. We do not feel that the situation is as simple as to categorically state that the proliferating ductular epithelium stimulate fibroblastic proliferation and collagen deposition (Popper et al., 1960, 1961; Carruthers et al., 1961; Grishman and Hartroft, 1961; Hutterer et al., 1961), and that the newly formed basement membrane merely provides an environment conducive to collagen deposition (Thomas, 1964). Further investigation seems required to understand the precise nature of interaction between the epithelial cells and fibroblasts. From the findings presented in this paper, however, we concluded that the epithelial basement membrane is probably elaborated by a combined effort of the epithelial cells and the underlying fibroblasts.
26
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SUMMARY
An electron microscopic study was made on hepatic cirrhosis of rats fed a diet containing 3’methyl-diaminoazobenzene. Special attention was focused on the possible role of the fibroblast in the organization of basement membrane of proliferated bile ductules. It was noted that no basement membrane was seen where the ductular cell reaction was evident, and that the dense material deposited beneath the ductules appeared to be converted into the basement membrane concurrent to collagen deposition which was most likely induced by the underlying fibroblasts. Our results suggest that the epithelial cells exert some influence over polymerization of collagen and differentiation of fibroblasts in the juxtaepithelial regions. Data obtained in this study strongly suggest that the epithelial basement membrane is elaborated by cooperation of the epithelial cells and fibroblasts. REFERENCES
BRUCHHAUSEN,F. V. and MERKER, H. L. (1965). Gewinnung und morphologische Charakterisienmg einer Basalmembranfraktion aus der Nierenrinde der Ratte. Arch. Exptl. Pathol. Phurmakol.
251, l-12.
CARRUTHERS,J. S., KALIFAT, S. R. and STEINER, J. W. (1962). The ductular cell reaction of rat liver in extrahepatic cholestasis. II. The proliferation of connective tissue. Exptl. Mol. Pathol. 1, 377-396.
GIESKING, R. (1963). Submikroskopische Strukturunterschiede zwischen Histiozyten und Fibroblasten. Beitr. Pathol. Anut. Allgem. Pathol. 128, 259-282. GRISHAM, J. W. and HARTROFT, W. S. (1961). Morphologic identification by electron microscopy of “oval cells” in experimental hepatic degeneration. Lab. Znuest. 10, 317-332. HAUST, M. D. (1965). Fine fibrils of extracellular space (microfibrils). Their structure and role in connective tissue organization. Am. J. Pathol. 47, 1113-1137. HAY, E. D. (1964). Secretion of a connective tissue protein by developing epidermis. In “The Epidermis” (W. Montagna and W. C. Lobitz, Jr., eds.), pp. 97-116. Academic Press, New York. HUTTERER, F., RUBIN, E., SINGER, E. J., and POPPER,H. (1961). Quantitative relation of cell proliferation and fibrogenesis in the liver. Cancer Res. 21, 206-215. KAJIKAWA, K. (1964). Electron microscopic studies on histiocytes. Tohoku J. Exptl. Med. 81, 350-365.
KURTZ, S. M. and FELDMAN, J. D. (1962). Experimental studies on the formation of the glomerular basement membrane. J. Ultrastruct. Res. 6, 19-27. Low, F. N. (1961). The extracellular portion of the human blood-air barrier and its relation to tissue space. Anut. Record 139, 105-123. Low, F. N. (1962). Microfibrils: Fine filamentous components of the tissue space. Anat. Record 142, 131-137. Lua, J. H. (1961). 9, 409-414. MC LOUGHLIN, B.
Improvements in epoxy resin embedding methods. J. Biophys.
Biochem.
Cytol.
(1960). The connective tissue substrate. In “Biological Approaches to Cancer Chemotherapy” (R. J. C. Harris, ed.), pp. 371-385, Academic Press, New York. MUKERJEE, H., RAM, J. S. and PIERCE, G. B. (1965). Basement membranes. V. Chemical composition of neoplastic basement membrane mucoprotein. Am. J. Pathol. 46, 49-57. PIERCE, G. B. and MIDGLEY, A. R. (1963). The histogenesis of basement membranes. J. Exptl. Med. 117, 339-348. PIERCE, G. B., BEALS, T. F., RAM, J. S. and MIDGLEY, A. R. (1964). Basement membranes. IV. Epithelial origin and immunologic cross reactions. Am. J. Pathol. 45, 929-961. POPPER, H., KENT, G. and STEIN, R. (1957). Ductular cell reaction in the liver in hepatic injury. J. Mt. POPPER,
Sinai
Hosp.,
N.Y.
24, 551-556.
H., SCHAFFNER, F., HUTTERER, F. PAFCONE?TO. G. and BARKA, T. (1960). Parenchymal fibrogenesis: The liver. Ann. N.Y. Acad. Sci. 86, 1075-1088. POPPER, H., PARONETTO, F., SCHAFFNER, F. and PEREZ, V. (1961). Studies on hepatic fibrosis. Lab.
Znuest.
10, 265-290.
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MEMBRANE
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REYNOLDS,E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RUBIN, E., HLJTTERER,F. and POPPER, H. (1963). Cell proliferation and fiber formation in chronic carbon tetrachloride intoxication. A morphologic and chemical study. Am. J. Puthol. 42, 715728.
SCHAFFNER,F. and POPPER, H. (1961). Electron microscopic studies of normal and proliferating bile ductules. Am. J. Puthol. 38, 393-410. STEINER, J. W. and CARRUTHERS,J. S. (1961). Studies on the fine structure of the terminal branches of the biliary tree. 1. The morphology of normal bile canaliculi, bile pre-ductules (ductus of Hering) and bile ductules. Am. J. Pathol. 38, 639-661. THOMAS, P. K. (1964). The deposition of collagen in relation to Schwann cell basement membrane during peripheral nerve regeneration. J. Cell Biol. 23, 375-382. WOOD, G. C. (1960). The formation of fibrils from collagen solution. 3. Effect of chondroitin sulfate and some other naturally occurring polyanions on the rate of formation. Biochem. J. 75, 605-612.
AND
MOLECULAR
PATHOLOGY
11,
17-27
(1969)
An Electron Microscope Study of the Basement Membrane of Proliferated Bile Ductules KINICHIRO Department
KAJIKAWA
of Pathology.
School
AND
of Medicine,
Received
March
SHOICHIRO Kanazawa
KAKIHARA Universi@.
Kanazawa,
Japan
21, 1969
The epithelial cell is separated from the surrounding connective tissue by the basement membrane. In electron microscopy the basement membrane appears as a continuous layer consisting of fine filaments embedded in an amorphous matrix. This layer, usually called lamina densa (Low, 1961), is separated from the epithelial cells by a narrow transparent space, the lamina lucida (Low, 1961), and on the opposite side connects with the interstitium. The origin of basement membrane is a matter of dispute. Because of its histochemical properties and its close relation to the connective tissue, the basement membrane was considered to be of connective tissue origin (Gersh and Catchpole, 1949). McLaughlin (1966), using tissue cultures, observed that the epidermal cells were surrounded by proliferating fibroblasts in association with the accumulation of PAS-positive materials, and concluded that the basement membrane was formed by mesenchymal products under the influence of the epithelial cells. Recent evidence gathered from autoradiographic (Hay, 1964), immunologic (Pierce and Midgley, 1963; Pierce et al., 1964), chemical (Mukerjee et al. 1965) and electron microscopic (Kurtz and Feldman, 1962) studies has indicated that epithelial cells secrete the basement membranes. This evidence, however, does not explain how the epithelial secretion does not diffuse into the connective tissue, but accumulates just beneath the cells as an organized envelope. It is still uncertain whether the fibroblast is not entirely involved in the formation of the basement membrane and what relation the basement membrane has to the underlying collagen or reticular fibers. In the course of a study of rat hepatic cirrhosis induced by 3’-methyl-diaminoazobenzene, we have encountered many examples to suggest that the fibroblast may participate in the organization of the basement membrane of proliferated bile ductules. In this paper we will discuss the possible role of the fibroblast in formation of the epithelial basement membrane to throw some light on the epithelio-mesenchymal relationship. MATERIALS Twenty male were fed a diet The liver was tinely prepared
AND METHODS
Wistai albino rats, with an initial body weight of 150-200 gm containing 0.06 Y 3’-methyl-diaminoazobenzene for 90-150 days. fixed with neutral formalin, and paraffin sections were roufor light microscopy. 17
18
KAJIKAWA
AND
KAKIHARA
The tissue for electron microscopy was fixed with 2% osmium tetroxide, buffered to pH 7.2 with Verona1 acetate, at 4” C for 1 hour. After dehydration in graded aceton solution, the materials were embedded in Epon 812 (Luft, 1961). Thin sections were cut with a Fernandez-Moran ultramicrotome (Leitz) using glass knives. The sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Electron micrographs were taken with a HU-11 PM type (Hitachi Ltd.) or a JEM-7 type (Japan Electron Optics Laboratory Co. Ltd.) electron microscope at magnifications 3000-10,000. RESULTS Light microscopy revealed that hepatic cirrhosis together with proliferation of bile ductules developed in all of the rats, although there was considerable variability in its severity from one animal to another and among portions of the same liver. By electron microscopy general features of cirrhosis were not different from those previously reported (Popper et al., 1960, 1961; Hutterer et al., 1961; Schaffner and Popper, 1961; Carruthers et al., 1962; Rubin et al., 1963). In many areas branches of the proliferated bile ductules were encompassed by the
FIG. 1. Ductular cell reaction in a cirrhotic liver. Histiocytes around the proliferated ductules (De). No basement membrane tween mesenchymal cells and ductular epithelium. X20,M)O.
(Hs) and fibroblast (Fb) are seen is seen. Note intimate contact be-
BASEMENT
MEMBRANE
OF BILE
DUCTULES
19
fibrous connective tissue, whereas in some areas the ductular cell reaction (Popper et al., 1957) was evident. Apparently proliferating ductular epithelium consisted of cells with a large nucleus and relatively numerous profiles of rough-surfaced endoplasmic reticulum. Cisternae of the endoplasmic reticulum were slightly to moderately distended (Figs. 1, 2, and 4). Ribosomes were distributed throughout the cytoplasm in small clusters. Mitochondria, round or oval in shape, were relatively numerous. The Golgi complex appeared prominent. Lateral surfaces of the cells were relatively smooth, except for occasional interdigitations. Desmosomes were found in some places. The luminal surface had microvilli. The basal surface appeared slightly irregular having small cytoplasmic projections. Where the ductural cell reaction was evident, no basement membrane was found (Fig. 1). In this area large numbers of inflammatory cells-including histiocytes, leucocytes, and plasma cells-were found around the ductules. Fibroblasts were not common, but in some instances were found scattered among the inflammatory cells. The fibroblasts had elongated cytoplasm with extensive rough-surfaced endoplasmic reticulum. They were distinguished by this characteristic from histiocytes which were rich in smooth-surfaced vesi-
FIG. 2. Ductular cell reaction in a cirrhotic liver. Histiocytes present near the ductules (De). Intercellular space is filled with basement membrane is seen. X16,000.
(Hs) and fibroblasts (Fb) are a dense material. No distinct
20
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cles and lysosomes (Gieseking, 1963; Kajikawa, 1964). The histiocytes and fibroblasts were close to the ductular epithelium or came directly in contact with it without any intervening basement membrane (Figs. 1 and 3). The intercellular space contained collagen fibrils and amorphous materials. An interesting finding was the accumulation of a dense material in the intercellular spaces near the ductules. This material, at higher magnifications, was similar in appearance to the basement membrane material in that it consisted of ill-defined fine filaments embedded in an amorphous matrix of moderate density (Fig. 5). There was considerable variation in quantity of the dense material, probably depending on the development of cirrhosis. In areas where the proliferation of the fibroblasts and histiocytes was still prominent, the dense material was found diffusely in the intercellular space and the basement
FIG. 3. Portion of a cirrhotic liver. Ductular epithelium (De) is surrounded by the basement membrane-like material (Bm) which, in part, is continuous with a similar material around the fibroblast (Fb) that is in direct contact with the ductule. Fibrocytes (Fbb) extend attenuated cytoplasmic processes around the ductule. Aggregation of histiocytes (Hs) is evident at a distance from the ductule. X7500.
BASEMENT
MEMBRANE
OF
BILE
DUCTULES
21
membrane of the ductules was hardly discerned (Fig. 2). In advanced cirrhosis proliferated ductules were surrounded by a laying down of the dense material. The fibroblasts which were close to the ductules extended ramified cytoplasmic processes as if to enclose the material (Figs. 4 and 5). The dense material in this area assumed an appearance similar to the basement membrane, except that it was much thicker than the normal basement membrane and that the lamina lucida was absent in many portions. The fibroblasts in the periductular regions were in contact over variable portions of the cell surface with the dense material. Sometimes there was a direct continuity between the dense material around the fibroblasts and the ductules (Figs. 3 and 7). Frequently the dense material around the fibroblasts was associated with fine fibrils and, in some instances, appeared to have been replayed by progressive deposition of the fibrils (Fig. 7). The fibrils were 100150 A in diameter and had no distinct periodicity. These have been observed in various connective tissue and were called microfibrils (Low, 1962; Haust, 1965). Where the amount of collagen fibrils was large, both the dense material and microfibrils were small in amount. It was noted that massive accumulation of the dense material appeared to be associated with inhibited formation of collagen fibrils in the periductular
FIG. 4. This portion of cytoplasmic processes ductular fibrous stroma.
of ductular epithelium (De) of fibroblasts (Fb) appears x 16,000.
is surrounded to demarcate
by the dense material. the dense zone and
A layer the peri-
22
KAJIKAWA
AND
KAKIHARA
FIG. 5. The space between the ductular epithelium (De) and attenuated cytoplasmic processes of the fibroblast (Fb) with the dense material. The lamina lucida is not distinct and only suggestive, at best. Small round bodies are scattered within the material as indicated by the arrows. We have not determined whether these bodies represent cytoplasmic projections or secretory products. The interstitium further peripheral to the fibroblasts contains numerous collagen fibrils (Cf) and sparse microfibrils (Mf). X40,000.
regions. The space between the ductular epithelium and subjacent fibroblasts was occupied by the dense material, whereas the stroma on the opposite side of the fibroblasts was filled with collagen fibrils arranged in bundles (Figs. 4 and 5). In some places, the dense material was alienated from the fibroblasts by small amount of fine fibrils (Fig. 4). In this area the lamina lucida was apparent. Tn Fig. 6, the dense material in the periductular space was largely absent and presumably had been replaced by collagen which seemed to be deposited progressively from the vicinity of the fibroblasts towards the epithelia. These collagen fibrils were smaller in diameter (about 300 A wide) than those elsewhere. They appeared loosely arranged, being intermingled with the microfibrils and occasional fragments of the dense material. These fibrils seemed to correspond to the reticular fibers in light microscopy. The periductular collagen deposition did not reach the basal surface of the ductular epithelium so that the dense material remained just beneath the epithelium as a continuous thin layer (lamina densa) (Fig. 7). Thus, presumably mature basement membrane appeared to fuse with the stromal microfibrils and collagen fibrils without distinct demarcation.
BASEMENT
MEMBRANE
OF
BILE
DUCTULES
23
With collagen deposition, in the periductular regions the stromal cells, presumably fibrocytes, were scant and appeared in a given plane of section mostly as thin strands of cytoplasm scattered among the collagen fibrils that were well oriented in small bundles. At a distance from the ductules and further distal from the area where collagen fibrils were in bundles, however, the interstitium had numerous fibroblasts and chronic inflammatory cells (Figs. 3, 6, and 7). What appeared to be fully developed basement membrane was associated with the ductular epithelium that had cytoplasmic structures similar to those in the normal liver (Schaffner and Popper, 1961; Steiner and Carruthers, 1961). The cytoplasm contained small mitochondria and narrow strands of rough-surfaced endoplasmic reticulum. Free ribosomes appeared as a few scattered clusters. Occasionally lysosomes and lipid droplets were observed. The basal surface resting on the basement membrane was smooth. DISCUSSION It was a noticeable finding in this study that the basement membrane did not surround the ductules where inflammatory cells were near them, or where fibroblasts and histiocytes were in direct contact with them. In this region the ductular epithelium was relatively rich in free ribosomes in the cytoplasm
FIG. density, collagen
6. Concomitant with the reduction of areas occupied by the diffuse and amorphous which results in a more defined dense zone like basement membrane, a narrow layer of fibrils (Cf) appears on the luminal side of the periductular fibroblasts (Fb). ~20,000.
24
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AND
,KAKIHARA
FIG. 7. Well-organized basement membrane (Bm) is separated from the ductular epithelium (De) by the lamina lucida. The amorphous density (Gs) around the process of fibroblast (Fb) appears partly continuous with the basement membrane. In the right corner a portion of fibroblast with wide cytoplasm is seen. The microfibrils (Mf) are often seen close to the fibroblasts. X~O,OOO.
and showed occasionally cytoplasmic projections at the basal surface. These features may reflect immaturity of the ductular epithelium. In cirrhotic areas, however, the ductules which had apparently similar cytoplasmic structure, were surrounded by the basement membrane-like material. Thus the absence of the basement membrane in the region of ductular cell reaction does not appear to be owing to immaturity of the epithelial cells, but rather to environmental conditions which seem to be controlled by the underlying connective tissue,; we are inclined to think that the inflammatory cells could not provide a suitable environment for the formation of the basement membrane. Our observation showed that a dense material was deposited in the space between the ductular epithelium and subjacent fibroblasts. The origin of the material is not clear. One is tempted to speculate that the fibroblasts may produce some components of the material, since it appears structurally very similar to the material (presumably the stromal ground substance) around the fibroblasts which are located freely in the stroma, and since these two materials are continuous in places.
BASEMENT
MEMBRANE
OF
BILE
DUCTULES
25
On the other hand, autoradiographic study by Hay (1964) has indicated that the epithelial cells secrete proline-rich protein which apparently contributes to the formation of their own basement membrane. Similarly, on the basis of chemical and immunological studies, Pierce et al. (1963, 1964), and Mukerjee et al. (1965) concluded that the basement membrane was an epithelial secretion and differed from the connective tissue elements. Since these studies seem to be indicate quite convincingly that the epithelial cells do secrete the basement membrane material, one could not rule out the possibility that the dense material beneath the ductular epithelium, at least in part, may be of epithelial origin. Chemical studies (Bruckhausen and Merker, 1965; Mukerjee et al., 1965) have indicated that the basement membrane contains polysaccharides and collagen as do the products of fibroblasts. The fibroblasts and epithelial cells may produce common materials which are concerned with the formation of the basement membrane. For the moment, it seems reasonable to assume that the deposits in the space between the epithelium and subjacent fibroblasts are occupied by a mixture of materials which are produced by both cells. The dense material beneath the epithelium appeared to be converted into the basement membrane, concurrent with collagen deposition which was most likely induced by the underlying fibroblast (Figs, 4-7). Grobstein and gallman (1965) proposed the view that the epithelial surface provides a favorable site for collagen polymerization. Our observation, however, indicated that the collagen fibrils in the periductular regions were smaller in number and diameter than those in the stroma on the opposite side of the underlying fibroblasts. This suggests that the fiber formation may be inhibited in the periductular regions. The reason for this is unknown, but it is assumed that the epithelial cells exert influence to the surrounding connective tissue, by which collagen polymerization or fibroblastic activity may be affected. Wood (1960) demonstrated that mucopolysaccharides could alter the rate of collagen polymerization in vitro. The basement membrane material is known to be relatively rich in mucopolysaccharides and these would seem to inhibit further polymerization of collagen produced by the fibroblasts. In addition it should be considered that collagen synthesis by fibroblasts may be reduced in the periductular region, since the fibroblasts in this region appeared to be rapidly transformed into fibrocytes. We do not feel that the situation is as simple as to categorically state that the proliferating ductular epithelium stimulate fibroblastic proliferation and collagen deposition (Popper et al., 1960, 1961; Carruthers et al., 1961; Grishman and Hartroft, 1961; Hutterer et al., 1961), and that the newly formed basement membrane merely provides an environment conducive to collagen deposition (Thomas, 1964). Further investigation seems required to understand the precise nature of interaction between the epithelial cells and fibroblasts. From the findings presented in this paper, however, we concluded that the epithelial basement membrane is probably elaborated by a combined effort of the epithelial cells and the underlying fibroblasts.
26
KAJIKAWA
AND
KAKIHARA
SUMMARY
An electron microscopic study was made on hepatic cirrhosis of rats fed a diet containing 3’methyl-diaminoazobenzene. Special attention was focused on the possible role of the fibroblast in the organization of basement membrane of proliferated bile ductules. It was noted that no basement membrane was seen where the ductular cell reaction was evident, and that the dense material deposited beneath the ductules appeared to be converted into the basement membrane concurrent to collagen deposition which was most likely induced by the underlying fibroblasts. Our results suggest that the epithelial cells exert some influence over polymerization of collagen and differentiation of fibroblasts in the juxtaepithelial regions. Data obtained in this study strongly suggest that the epithelial basement membrane is elaborated by cooperation of the epithelial cells and fibroblasts. REFERENCES
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