Extracellular matrix formation in piecemeal . necrosis: munoelectron mcroscopic study I

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Takahara T, Nakayama Y, Itoh H, Miyabayashi C, Watanabe A, Sasaki H, Inoue K, Muragaki Y, Ooshima A. Extracellular matrix formation in piecemeal necrosis: immunoelectron microscopic study. Liver 1992: 12: 368-380. 0 Munksgaard 1992 Abstract: Immunolocalization of Type I, Type I11 and Type IV collagens, laminin and prolyl hydroxylase (PH), a key enzyme in collagen synthesis, was examined to clarify the fibrotic process in chronic, active liver disease. In piecemeal necrosis of chronic, active hepatitis (CAH) and active liver cirrhosis (LC), fat-storing cells (FSCs) and transitional cells (TSCs), containing abundant rough endoplasmic reticulum (RER), were increased in number and stained intensely for PH. Immunodeposits of extracellular matrix (ECM) components were found in the RER, Golgi apparatus (GA) and vesicles of these cells, especially in cases with marked inflammation. On the other hand, in the periportal areas of chronic, persistent hepatitis (CPH) or inactive LC, immunoreaction of ECM components was seldom found in the RER of FSCs and TSCs. In the portal tract, immunodeposits of ECM components were seldom found in the organelles of fibroblasts, although ECM was increased there. These findings indicate that FSCs and TSCs in piecemeal necrosis might play a role in the production of ECM components in the progression of fibrosis during the development of chronic active liver disease. In addition, ECM component production by FSCs and TSCs is associated with marked inflammation.

Terumi Takahara', Yoshihide Nakayama', Hiroyuki Itoh', Chiharu Miyabayashl', Akiharu Watanabe', Hiroshi Sasaki*, Kyoichi lnoue3, Yasuteru Muragaki4 and Akira Ooshima4 'Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, 'University Hospital, Toyama Medical and Pharmaceutical University, Toyama. 3Third Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, and 4First Department of Pathology, Wakayama Medical College, Wakayama, Japan

Key words: collagen - extracellular matrix - fatstoring cell - fibrosis - immunoelectron microscopy - transitional cell Dr Terumi Takahara, Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama. 930-01 Japan Received 25 February, accepted for publication 18 June 1992

Liver fibrosis has been regarded as a crucial event in the transition from chronic hepatitis to cirrhosis (1). In the process of fibrosis, extracellular matrix (ECM; consisting of interstitial and basement membrane collagens, glycoproteins and proteoglycans), is markedly increased (2), and the immunohistochemical distribution of ECM has been successfully demonstrated in the rat (3-7) and human liver tissues (8-1 1). Previous studies using culture systems showed that hepatic parenchymal and nonparenchymal cells have the potential to produce ECM components (12-18). Among these cells, fat-storing cells (FSCs) are reported to synthesize a large quantity of ECM components (15). In order to determine the cell type responsible for ECM production in vivo, morphological and immunohistochemical studies have been conducted using experimental fibrosis following CCl, intoxication (6,19), swine serum injection (19,20) or alcohol feeding (21-23), and these studies have indicated that FSCs were involved in collagen synthesis. 368

Recently, DNA or RNA probes, complementary to collagen mRNA, have provided quantitation of collagen gene expression in the liver. Northern analysis using isolated cells (24) and in situ hybridization (25) study have shown that mRNA of ECM components were strongly expressed in FSCs after CCl, intoxication. However, in human chronic active liver disease, the situation is quite different. ECM deposition generally occurs adjacent to the periportal areas (26), starting from the portal tracts in contact with abundant inflammatory cells. In contrast to experimentally induced liver fibrosis, the cells producing ECM components in chronic active liver disease have not yet been clarified. In the present study, we used immunoelectron microscopy to investigate the cell type involved in ECM components production in chronic liver disease and to gain possible insight into the mechanism and process of fibrogenesis in chronic active liver disease.

Extracellular matrix formation in piecemeal necrosis

The procedures of both light and electron microscopy were described in the previous paper (6).

described previously (28). Briefly, BALB/C mice were immunized with each collagen extracted from human placenta. The spleen cells were hybridized with myeloma cells. After HAT selection, positive hybrids were screened by enzyme-linked immunosorbent assay. The specificity of each antibody was determined by an immunoblot or inhibition enzyme-linked immunosorbent assay. Cross-reaction was not found between the antibodies. Polyclonal anti-laminin antibody (Gibco Lab., New York, USA) was commercially obtained. Anti-laminin antibody also showed no cross-reaction with Type I, 111, IV, V, and VI collagens or fibronectin. For direct immunoperoxidase staining, each antibody was fractionated into Fab' fragments and conjugated with peroxidase. Monoclonal anti-PH antibody, which recognized the p subunit of prolyl 4-hydroxylase, (anti-hPH(P), Fab'-HRP (29)) was kindly provided by the Fuji Chemical Ind., Ltd., Takaoka, Japan.

lmmunohistochemistry

Control study

The direct immunoperoxidase method was used in this study. Briefly, liver specimens (1 x 5 x 5 mm) were fixed in periodate lysine-paraformaldehyde solution (27) for 4 h. After inhibition of endogenous peroxidase with periodic acid, 6-pm-thick cryosections were incubated with the respective antibody. After fixation in 1% glutaraldehyde for 7 min, the peroxidase activity was detected with diaminobenzidine. For immunoelectron microscopy, the sections were further postfixed with 2% OsO,, dehydrated, and embedded in Epon 812. Ultrathin sections were prepared and observed without staining.

The control studies were as follows: (a) incubation with PBS to check the evaluation of endogenous peroxidase activity; (b) incubation with normal mouse Fab-POD to examine nonspecific binding with sections; (c) blocking test by preincubation with unconjugated antisera. The intensity of the staining was clearly reduced.

Material and methods Samples

In a retrospective study, we analysed the needle biopsy specimens from 48 patients: 22 with CAH, 8 with CPH, 5 with CAH with bridging necrosis, and 13 with LC. Serologically, 18 patients had evidence of hepatitis B virus infection, 24 had evidence of hepatitis C virus infection, 3 had autoimmune hepatitis, and 3 patients had neither evidence of virus infection nor autoimmune disease. Four surgical specimens, in which no pathological alteration was detected, were used as controls. None of the patients had a history of alcoholism. The clinical summary is shown in Table 1. Methods

Antibodies

Monoclonal antibodies against human Type I, Type 111, and Type IV collagens were used for this study. Preparation of the antibodies has been

Results Control liver: immunohistochemistry

Light microscopy. The major sites of immunodeposition of Type I, Type I11 and Type IV collagens in human liver were the portal tracts and the central veins, as previously reported (Fig. la, b) (8-11). The bile ducts and blood vessels were surrounded by strongly stained reaction products of Type IV collagen (Fig. lb) and laminin. In the lobular region, each antibody stained in different forms.

Table 1. Clinical summary of the samples. Correlation between history and etiology of chronic liver diseases Etiology Histological diagnosis

HBV

HCV

Autoimmune

Unknown

Total ~

CPH CAH CAH with BN LC active inactive

n=3

n=5

n=O

n=O

n=8@J n =2'bI n =2@) n=3

n=12('J

n=2

n=O

n=8 n=22

n=2 n=3'd' n=2

n =1(CI n=O n=O

n=O n=2 n=l

n=5 n=7 n=6

Total

n=18

n=24

n=3

n=3

n=48

The letters in parentheses show which samples are used for the figures. (a): Fig. 4a. (b): Fig. 4b, Fig. 4d. (c): Fig. 4c. (d): Fig. 5, Fig. 6b. (e): Fig. 6a. (1): Fig. 7.

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Fig. 1. Immunolight microscopy in human liver. a. Type Ill collagen in normal liver. Type Ill collagen is observed in the portal tract (PT), around the central vein (CV) and along the sinusoidal walls. b. Type IV collagen in normal liver. The distribution pattern of Type IV collagen is similar to the Type I l l collagen. Type IV collagen is also intensely stained around vessels and bile ducts in the portal tract. c. Type I collagen in CAH. lmmunodeposits are increased in the PT. d. Type Ill collagen in LC. Type 111 collagen is intensely stained in the fibrotic septa. e. Laminin in LC. Laminin is clearly stained around the vessels and bile ducts and on the border of the septa. In the lobule, immunoreaction of laminin is heterogeneously increased along the sinusoidal walls (arrows). f. PH in CAH with bridging necrosis. Reacted PH is diffusely observed in hepatocytes. Intensely stained cells (short arrows) are found in the area of piecemeal necrosis and in the lobule. Note several cells containing fat droplets (long arrows). Magnification: la, b: x80, c: x50, d, e: x30, f: x330.

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Extracellular matrix formation in piecemeal necrosis

Fig. 2. lmmunoelectron microscopy of normal liver. a. Type I collagen fibers in longitudinal-(long arrow) and cross-sections (short arrow) are stained beneath the endothelial cells (END) around the central vein (CV). Periodicity is seen on the longitudinal fibers. b. Type IV collagen in the portal tract. Type IV collagen is intensely stained along the basement membrane of bile duct (BD) and vessel, and on the surface of hepatocytes (H). c. Laminin in the lobular region. Immunoreaction of laminin is moderate in the RER of fat-storing cell (FSC), but weak in the Disse space, mainly on the surface of hepatocyte microvilli, and in the RER (arrow heads) of H. d. Stained PH is seen in the RER of FSC, H and END. F; fat droplet. S; sinusoid. Co; collagen fibers. K; Kupffer cell. Magnification: 2a: x 6500. b: x 3500, c: x 6100, d: x 6500.

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Takahara et al. Type I and Type I11 collagens were found along the sinusoidal wall, and Type IV collagen was continuously observed there. Laminin was very faintly present (data not shown). PH, a key enzyme in collagen synthesis, was diffusely observed, mainly in the cytoplasm of hepatocytes, as previously shown in the rat (6) (data not shown). Electron microscopy. Type I and Type III collagens: Extracellularly, Type I and Type I11 collagen fibers were found in the portal tracts, subendothelial spaces around the central veins and in Disse’s space. In the longitudinal section, immunoreactive fibers were selectively stained at typical periodicities of 60-70 nm (Fig. 2a). Intracellular observation showed very faint staining for both Type I and Type I11 collagens in the RER of hepatocytes, FSCs and endothelial cells (data not shown). Type IV collagen: Amorphous reaction products of Type IV collagen were intensely and continuously found on the basement membrane around the bile duct and vessels and in between collagen fiber bundles (Fig. 2b). In the Disse space, strong staining was observed. Intracellular immunodeposits of Type IV collagen were present in some vesicles of FSCs,

endothelial cells and bile duct cells, although they were seldom found in the RER of those cells (data not shown). Laminin: Laminin was intensely stained on the basement membrane around bile ducts and vessels (data not shown), similar to Type IV collagen. In the Disse space, amorphously but faintly stained immunodeposits were observed, mainly on the surface of the hepatocyte microvilli or endothelial cell membrane (Fig. 2c). In contrast, moderately stained deposits were present in the RER of FSCs, bile duct cells, and endothelial cells, while only a very faint reaction was seen in the hepatocytes (Fig. 2c). PH: PH was present in the RER and lumen of the nuclear membrane of most of the cell types of the liver, i.e., hepatocytes, FSCs, endothelial cells, Kupffer cells, fibroblasts (Fbts) and bile duct cells, as shown in the rat (6) (Fig. 2d). Chronic liver disease

In order to facilitate the consideration of the process of fibrosis in chronic liver disease, the liver was divided into three parts for the present study: periportal area, portal area and the lobular region.

Fig. 3. Conventional electron microscopy of CAH. In piecemeal necrosis, fat-storing cells (arrow) and transitional cells (TSC) are increased in number and both contain dilated RER. Necrotic hepatocyte (NH) is found. L; lymphocyte. P; plasma cell. H; hepatocyte. END; endothelial cell. Magnification: 3: x 2700.

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Extracellular matrix formation in piecemeal necrosis

Fig. 4. Immunoelectron microscopy in piecemeal necrosis. a. Type I collagen is increased in the area of piecemeal necrosis in the CAH. The arrangement of the increased fibers is not uniform. Type I collagen is also found in the RER (arrow) and GA (arrow head) of fat-storing cells (FCS) (Case: Table I-a). b. Type IV collagen is increased in the Disse space, near necrotic hepatocytes (H). Intracellularly, Type IV collagen is observed in the cytoplasm of a transitional cell (TSC), and vesicles (arrows) of a capillary endothelial cell (END) (Case: Table I-b). c. PH is intensely stained in the dilated RER of FSC and TSC, in the area of piecemeal necrosis, in active liver cirrhosis. PH is also present in hepatocytes (H) (Case: Table I-c). d. Higher magnification of Fig. 4b in an ultrathin serial section. Type IV collagen is observed in the RER (arrow), GA and vesicles (arrow heads) close to the plasma membrane of TSC, exhibit an exocytic formation of Type IV collagen (Case: Table I-b). F; fat droplet. BDC; bile duct cell. N; nucleus. L; lymphocyte. Magnification: 4a: x 3700, b: x 4100, c: x 2800, d: x 20000.

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Takahara et al. Electron microscopy. Periportal area: In piecemeal necrosis of CAH and active LC, the borders of portal tracts were complicated by extending fibers, inflammatory cells, proliferated vessels and bile ducts. Characteristically two kinds of cells were increased in number, especially, in cases with marked cell infiltration and necrosis (Fig. 3). One kind was transitional cells (TSCs), which were found on the side of the portal tract. They contained abundant and dilated RER including flocculent materials. However, no dense area or basement membrane was found around the TSCs. The other cell type was FSCs. According to the general definition, FSCs were located in the Disse space. Fat droplets were decreased in number or absent from the cytoplasm, although RER and GA were developed and dilated, and contained flocculent materials in the cisterna. On the other hand, TSCs were not often found in the periportal area of CPH or inactive LC. Portal tract: In the portal tract and completed septa, Fbts and infiltrating cells, such as lymphocytes, macrophages and plasma cells, were

surrounded by increased collagen fibers. The Fbts were elongated in shape and had few organelles. Lobular region: In the lobular region, FSCs were also changed ultrastructurally in active cases. They were enlarged with dilated RER and GA and contained few fat droplets (data not shown). However, in cases of CPH or inactive LC, the morphological appearance of FSCs was nearly normal in both the periportal area and lobular region. Immunohistochemistry: light microscopy. In cases of CAH or CPH, immunodeposits of Type I collagen were increased in the portal tract (Fig. lc), and similar increases in immunoreactivity corresponding to Type I11 and Type IV collagens were observed (data not shown). In cases of LC, immunoreaction of these collagens was markedly increased in the septa (Fig. Id). Laminin and Type IV collagen were intensely stained around the proliferated bile ducts and vessels (Fig. lc). Laminin was also found on the border of the septa. PH was diffusely present in the liver, except in the fibrous

Fig. 5. Immunoelectron microscopy of larninin in piecemeal necrosis of active LC. Laminin is intensely stained on the basement membrane of the vessels (V) and the bile duct cells (BDC) at Hering’s canal. This is also present in the RER of BDC, FSC and TSCs. C; collagen fiber (Case: Table I-d). S; sinusoid. L; lymphocyte. H; hepatocyte. Magnification: 5 : x6000.

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Extracellular matrix formation in piecemeal necrosis tissue. In piecemeal necrosis, especially, in cases of severe cell infiltration and necrosis, cells intensely stained by PH were observed (Fig. If). They sometimes contained fat droplets in their cytoplasm. In such active cases, intensely PH stained cells were also present in the lobular region (Fig. If). Electron microscopy. Periportal area: In piecemeal necrosis of CAH and active LC, the extending fibers stained for Type I and Type I11 collagen antibodies were increased (Fig. 4a). The arrangement of the increased fibers was not uniform. Immunodeposits of Type IV collagen and laminin were also prominent around the proliferated vessels and bile duct cells (Fig. 4b, 5). Intracellularly, intense PH staining was seen in the dilated and abundant RER of FSCs and TSCs in piecemeal necrosis (Fig. 4c). Intracellular deposit of the ECM components was also prominent in FSCs and TSCs especially, in most of the highly active cases (Fig. 4a, b, d, 5). Positive case numbers of intracellular staining of each ECM components are shown in Table 2. ECM components were sometimes present in the RER, GA, and vesicles close to the plasma membrane of these cells (Fig. 4d), demonstrating an exocytic process of ECM component production. On the other hand, at the periportal area of CPH, there were few fibers extending from the portal tracts and the margin of the portal tracts was clearly observed, staining of Type IV collagen and laminin, on the outer layer of hepatocytes (data not shown). Immunodeposits of Type I, Type 111, and Type IV collagens were very faintly found in the RER of FSCs or TSCs, at the periportal area, in CPH or inactive LC. Portal tract: In the portal tract and septa, intensely stained immunodeposits of Type I and Type I11 collagens were observed in almost all fiber bundles (Fig. 6a). These bundles were loosely ar-

ranged in random fashion, where infiltrating cells were numerous. Type IV collagen and laminin were present in the space between fiber bundles and on the basement membrane of bile ducts and vessels, similar to that in the normal portal tract (Fig. 6b). Intracellular, faint immunodeposits of PH were found in the RER of slender Fbts, bile duct cells, endothelial cells and infiltrating cells (data not shown). Laminin was also present in the RER of slender Fbt (Fig. 6b). However, Type I, Type I11 and Type IV collagens were seldom found in the RER of Fbts (Fig. 6a). Lobular region: Generally, there were increased amounts of immunodeposits of the ECM components in the Disse space (Fig. 7a), although they were heterogeneously distributed. Immunodeposits of Type I, Type I11 collagens and laminin were specifically increased in small nodules of LC. Prominent intracellular deposits of PH were present in the dilated RER of FSCs, especially in cases with marked inflammation (data not shown). Intracellular immunostaining of ECM components was sometimes present in the RER, GA, and vesicles close to the plasma membrane of the FSC (Fig. 7b). The intracellular staining of the components was intense in cases with active inflammation. However, in the hepatocytes, intracellular reaction products of the ECM components were very faintly present in most cases, although immunodeposits of PH were diffusely found in the RER of the hepatocytes. A summary of the intracellular immunoreaction of the ECM components is given in Table 2. Discussion

Increased deposition of ECM is a characteristic change in chronic active liver disease (1). The ECM deposition occurs near the periphery of the lobule,

Table 2. Summary of intracellular immunoreaction of antigens in chronic liver diseases Portal activelinactive n=34 n=14 Antigens

Fibroblast

Periportal activelinactive n=34 n=14 Transitional cell

Type I collagen

-1-

++I+ (31) (11)

Type 111 collagen

-1-

++I+ (32) (10)

Type IV collagen

-1-

++I(18)

Laminin

+I+ (34) (14)

Fat-storing cell

lntralobular activelinactive n=34 n=14 Fat-storing cell

Hepatocyte

Endothelial cell

++I+ (34) (14)

-: no staining. +: faint. ++: marked. The numbers of positive cases are shown in parentheses.

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Fig. 6. lmmunoelectron microscopy of the portal tract in chronic liver disease. a. Immunodeposit of type 111 collagen in fibrous septum. N o reaction product is found in the RER of the fibroblast (Fb), in contrast to the intense staining of reaction products of extracellular Type I l l collagen (Case: Table I-e). b. Laminin is present on the basement membrane of the bile duct (BD) and the artery (A). Intracellularly, laminin is found in the RER (arrows) of the slender Fb. No immunoreaction is found in the plasma cell (P) (Case: Table I-d). Magnification: 6a: ~ 6 1 0 0 ,b: x3200.

starting from the portal areas in close contact with inflammatory cells (26). However, the predominant cells responsible for most of the ECM production are still unknown. In the present immunoelectron microscopic study, we investigated the localization of Type I, Type 111, Type IV collagens, laminin and PH in order to clarify the cell types participating in ECM production and the process of fibrosis in chronic active liver disease. Compared to rat liver (6), intracellular staining of ECM components was less often detected in the present study. For example, we found very faint intracellular deposits of Type I and Type 111 collagens in normal hepatocytes and FSCs in the present study, though intracellular deposits were clearly detected in normal rat hepatocytes and FSCs. Geerts et al. (7) reported that gold particles reacted with procollagen Type 111 found in GA of normal human hepatocytes. Clement et al. (10) also described clear intracellular deposits of procollagen Type 111 and collagen Type IV in normal human FSCs. One of the reasons for the differences in the intracellular immunostaining might be the fixation method. In the present study of 48 human liver biopsy samples, we could not perform perfusion fixation, but used immersion fixation. Perfusion 376

fixation gives both good preservation of the ultrastructure of liver cells and clear intracellular staining of reaction products. That might be why only limited intracellular staining was detected in the present study. On the other hand, this limited intracellular distribution may reflect the broad but very drastic immunoreactive changes during the process of liver fibrosis. That would be an advantage when considering the main cell types involved in ECM production during liver fibrosis. Table 2 gives a summary of the findings of the present study, concerning the intracellular immunolocalization of ECM components in chronic active liver disease. Both FSCs and TSCs in piecemeal necrosis of CAH and active LC were most intensely stained for PH and ECM components in the RER in most cases. These cells, containing abundant and dilated RER, were increased in number. These findings suggest that both FSCs and TSCs are actively involved in ECM component production in piecemeal necrosis, and the continuous ECM deposit together with continuous inflammation and necrosis in piecemeal necrosis probably leads to progressive enlargement of the portal tract. This fibrotic process was similar to that in our previous study of rat liver fibrosis

Extracellular matrix formation in piecemeal necrosis

Fig. 7. Immunoelectron microscopy in the lobular region of CAH. a. Reaction products of Type Ill collagen are increased in the Disse space. Type 111 collagen is intensely stained in the RER of FSC (Case: Table 1-0. b. Higher magnification of Fig. 7a reveals intense reaction products with Type 111 collagen in the RER, GA and vesicles (arrows), close to the plasma membrane of FSC, demonstrating an exocytic process of Type 111 collagen. N; nucleus. END; endothelial cell. H; hepatocyte. S; sinusoid. Magnification: 7a: x 12 500, b: x 29 000.

following CCl, intoxication (6), since in both conditions, FSCs and TSCs play important roles after hepatic necrosis, whether caused by a toxic reagent or an immunological reaction. These findings are in agreement with the recent electron autoradiographic study reported by Iwamura et al. (30). They reported that FSCs in piecemeal necrosis showed abundant incorporation of tritium-labeled proline in chronic active liver disease, suggesting an active collagen synthesis in these cells. Nagy et al. (31) recently reported that the main site of the immunodeposits of TGF-f3 was the portal and the periportal area in CAH and active LC. TGF-P, which is released from lymphocytes, platelet cells and macrophages, has been documented to cause an increase in the production of different matrix components in various experimental and in vivo processes (32). Cultured FSCs have also been demonstrated to be stimulated by TGFP to synthesize ECM components (33-35). In vivo study has revealed that the mRNA of TGF-P was increased during the fibrotic process of human chronic inflammatory liver disease (36) and rat CCl, intoxication (25). Furthermore, Nakatsukasa et al. (37) reported that mRNA of TGF-P was

expressed in the FSC in liver fibrosis induced by CCl, intoxication by in situ hybridization, and suggested that FSC was stimulated by TGF-P by an autocrine mechanism. These findings, together with our present study, suggest that FSCs and TSCs synthesize ECM components by stimulation by TGF-P, associated with continuous inflammation and necrosis, in piecemeal necrosis. Besides TGF-P, other types of cytokine such as PDGF, IL-1, IL-6, aqd TNF-a, which are all associated with hepatocytes necrosis and inflammation in the liver, might have a complicated influence on FSCs and TSCs for ECM production and cell proliferation (38). On the other hand, in the portal tract, slender Fbt, in between collagen fibers, showed faint immunostaining for PH, and reaction products of collagens were seldom found in the RER. These Fbts seemed rather static for ECM production. These results could be explained by the decreased rate of collagen catabolism and a low rate of collagen synthesis in the portal tract (39). However, it is not known why immunodeposits of TGF-P were prominently observed in the portal tract (3 1). FSCs and TSCs were distinguished from each other by their location, in this study. However, 377

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they were morphologically similar, because both cell types contained abundant, dilated RER, and fat droplets were seldom found in the FSC in the periportal area. In addition, the Disse space was often collapsed. Therefore, it was difficult to clarify the cellular origin of TSC, since FSC was not stained by anti-desmin antibody, in this study (data not shown). On the other hand, it is well known that FSCs are transformed to fibroblastlike figures, in association with hepatic fibrosis, following CC14 intoxication in rats (3, 4, 6, 19, 40). In an extensive study of liver fibrosis in alcohol-fed baboons, FSC was also reported to change to transitional cells, which are considered to be cells intermediate between FSC and Fbt (21). TSC might originally be FSC in the periportal area and become fibroblast-like, together with the progressive enlargement of the portal tract. There has so far been no report distinguishing human FSC, although desmin staining is well known to be a valuable marker for FSC in the rat (41, 42). Therefore, further studies are needed to clarify the features of FSC and TSC in chronic liver disease. In the present study, immunolocalization of PH was investigated to estimate the total amount of collagen produced in the cell (6). PH, a key enzyme in hydroxylation of proline to hydroxyproline in collagen synthesis, is a tetramer of c1 and p subunits (a,Pz), which acts as an active enzyme (43). In agreement with our previous study in rats (6), PH was extensively demonstrated in the RER of hepatocytes, FSCs, endothelial cells, Kupffer cells, bile duct cells and Fbts in normal human liver, even though collagen was only faintly found in the organelles of these cells. This discrepancy might be explained as follows. First of all, the cDNA sequence for the p subunit of human PH has recently been reported to be highly homologous to those determined for rat protein disulfide isomerase, which is regarded as the in vivo catalyst for disulfide bond formation in the biosynthesis of various secretory proteins (43, 44). Moreover, it is reported that the sequence for sulfide isomerase is identical to the human and bovine cellular thyroid hormonebinding protein (43, 45). These reports suggest that the immunoreaction of the protein of PH might not necessarily reflect the actual PH enzymatic activity. However, Yamada et al. (46) showed the same immunostaining pattern in human fibrotic livers using the respective antibody against CI and p subunits. They suggested that the immunoreaction of the j3 subunit reflected PH enzymatic activity on collagen metabolism in human liver. Secondly, collagen is rapidly degraded in hepatocytes (47). Taking the intracellular local378

ization of collagens and PH into consideration, it has been difficult to determine which cell type is the main collagen producer in the normal liver. Clement et al. (10, 11) investigated the localization of Type I, Type 111, Type IV collagens, Type I11 procollagen, fibronectin and laminin in human liver by immunoelectron microscopy. They reported that immunodeposits of ECM components were increased in the hepatocytes in alcoholic liver injury (lo), suggesting the involvement of hepatocytes in the fibrotic process. However, the mechanism of fibrogenesis in alcohol liver injury might differ from that in chronic liver disease because the morphological findings, such as pericellular fibrosis and central sclerosis, are characteristically observed in alcoholic liver injury. On the other hand, previous reports have shown that acetaldehyde (48, 49) stimulates FSC to produce Type I collagen and fibronectin, not hepatocytes. There is still controversy about which cell type is responsible for ECM production in alcoholic liver disease. Hassan et al. (50) reported that plasma cells, localized near the periportal of the lobule, very actively incorporated proline in chronic active liver disease, suggesting the capability of plasma cells to produce collagen. In our present study, we have not detected immunoreaction products of ECM components in plasma cells. Their autoradiological evidence may reflect an active protein synthesis, although the protein is unknown. On the basis of the findings of this immunoelectron microscopic study, it is concluded that both FSC and TSC actively participate in fibrogenesis in chronic active liver disease, and the site of activity was the periportal area. In addition, production of ECM components by FSC and TSC is stimulated, in association with marked inflammation and necrosis. Acknowledgements We wish to thank Dr. Iwata (Fuji Chemical Ind., Ltd.: Takaoka, Japan) for his suggestion about antibody conjugation).

References 1. POPPERH, UDENFRIEND S. Hepatic fibrosis: correlation of biochemical and morphologic investigations. Am J Med 1970: 49: 707-721. P. The extracellular matrix 2. ROJKINDM, PONCE-NOYOLA of the liver. Coll Relut Res 1982: 2: 151-175. 3. KENTG, GAYS, INOUYE T, BAHUR, MINICK0 T, POPPER H. Vitamin A-containing lipocytes and formation of type 111 collagen in liver injury. Proc Nut1 Acud Sci USA 1976: 73: 3719-3722. 4. MARTINEZ-HERNANDEZ A. The hepatic extracellular matrix. 11. Electron immunohistochemical studies in rats

Extracellular matrix formation in piecemeal necrosis with CCI,-induced cirrhosis. Lab Invest 1985: 53: 166- 186. 5. CLEMENT B, RISSELM, PEYROLS, MAZURIER Y, GRIMAUD J A. GUILLOUZO A. A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver. J Histochem Cytochem 1985: 33: 407-414. 6. TAKAHARA T. KOJIMAT, MIYABAYASHI C et al. Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat. Immunoelectron microscopic observation of type I, type 111 collagens, and prolyl hydroxylase. Lah Invest 1988: 59: 509-521. 7. GEERTSA. GEUZEH J. SLOT J-W et al. Immunogold localization of procollagen 111. fibronectin and heparan sulphate proteoglycan on ultrathin frozen sections of the normal rat liver. Histochemistry 1986: 84: 355-362. 8. GRIMAUD J A, DRUCUETM. PEYROLS, CHEVALIER 0, HERBAGE D, BADRAWY N. Collagen immunotyping in human liver. Light and electron microscope study. J Histodiem Cvtochem 1980: 28: 1145-1 156. R. Distribution of 9. HAHNE, WICKG. PENCEVD, TIMPL basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin. and fibronectin. Gut 1980: 21: 63-71. 10. CLEMENT B, GRIMAUD J A, CAMPIONJ P, DEUGNIER Y, GUILLOUZO A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Heputolog,v 1986: 6: 225-234. 11. CLEMENT B. RESCANP Y, BAFFETG et al. Hepatocytes may produce laminin in fibrotic liver and in primary culture. Heputology 1988: 8: 794-803. 12. GUZELIAN P S, QURESHI G D, DIEGELMAN R F. Collagen synthesis by the hepatocytes: studies in primary cultures of parenchymal cells from adult rat liver. Coll Relat Res 1981: 1: 83-93. 13. DIEGELMANN R F, GUZELIAN P s, GAYR, GAYs. c o l lagen formation by the hepatocyte in primary monolayer culture and in vivo. Science 1983: 219: 1343-1345. 14. DE LEEWA M. MCCARTHY S P, GEERTS A, KNOOKD L. Purified rat liver fat-storing cells in culture divide and contain collagen. Heputology 1984: 4: 392403. 15. FRIEDMAN S L, ROLL F J, BOYLESJ, BISSELLD M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Nut1 Acad Sci USA 1985: 82: 8681-8685. 16. RAMADORI G, RIEDERH, KNITTELT, DIENES H P, MEYER ZUM BUSCHENFELDE K H. Fat storing cells (FSC) of rat liver synthesize and secrete fibronectin. Comparison with hepatocytes. J Hepato1 1987: 4: 190-197. 17. SCHAFER S. ZERRE 0, GRESSNER A M . The synthesis of proteoglycans in fat-storing cells of rat liver. Hqato/oga. 1987: 7: 680-687. 18. MAHERJ J, FRIEDMAN S L, ROLL F J. BISSELD M. Immunolocalization of laminin in normal rat liver and biosynthesis of laminin by hepatic lipocytes in primary culture. Gastroenterology 1988: 94: 1053-1062. 19. SENOOH, WAKEK. Suppression of experimental hepatic fibrosis by administration of vitamin A. Lab Invest 1985: 52: 182-194. 20. BALLARDINI G, DEGLIESPOSTIS, BIANCHIF B et al. Correlation between Ito cells and fibrogenesis in an experimental model of hepatic fibrosis. A sequential stereological study. Liver 1983: 3: 58-63. 21. MAK K M, LEO M A, LIEBERC S. Alcoholic liver injury in baboons: Transformation of lipocytes to transitional cells. Gastroenterology 1984: 87: 188-200. H, TOWNER S J, CIOFALOL M, FRENCH S 22. TSUKAMOTO W. Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology 1986: 6: 8 14822.

23. FRENCH S W. MIYAMOTO K, WONG K , JUI L, BRIEREL. Role of the Ito cell in liver parenchymal fibrosis in rats fed alcohol and a high fat-low protein diet. Am J Pathol 1988: 132: 73-85. 24. MAHERJ J, MCGUIRER F. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest 1990: 86: 1641-1648. 25. NAKATSUKASA H, NAGY P, EVERTSR P, HSIA C C, MARSDENE, THORGEIRSSON S S. Cellular distribution of transforming growth factor-pl and procollagen type I, 111, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis. J Clin Invest 1990: 85: 1833-1843. 26. BIANCHIL, SPICHTINH P, GUDAT F. Chronic hepatitis. In: MacSween R N M, Anthony P P, Scheuer P J, eds. Pathology of the liver. Edinburgh: Churchill Livingston. 1987: 310-341. 27. MCLEANI W, NAKANEP K. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 1974: 22: 1077-1083. 28. OOSHIMAA, MURAGAKIY. Collagen metabolism in atherogenesis. Ann N Y Acad Sci 1990: 598: 582-584. Y, OBATAK, IWATA K, OOSHIMAA. 29. BAI Y, MURAGAKI Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase. J Biochem 1986: 99: 1563-1570. 30. IWAMURA S, ONISHIS, YAMAMOTO Y, ENZANH. Collagen fiber production by Ito cells in human hepatic fibrosis: a light and electron microscopic autoradiographic study. Actu Hepatol Jpn 1988: 29: 643-650. 31. NACYP, SCHAFFZ. LAPISK. Immunohistochemical detection of transforming growth factor-pl in fibrotic liver diseases. Hepatology 1991: 14: 269-273. 32. IGNOTZR A, MASSAGUE J. Transforming growth factor-p stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Cheni 1986: 261: 43374345. 33. DAVISB H. Transforming growth factor p responsiveness is modulated by the extracellular collagen matrix during hepatic Ito-cell culture. J Cell Physiol 1988: 136: 547-553. 34. MATSUOKA M, PHAM N T, TSUKAMOTO H. Differential effects of interleukin 1 alpha, tumor necrosis factor and transforming growth factor beta 1 on cell proliferation and collagen formation by cultured fat storing cells. Liver 1989: 9: 71-78. M-A, CZAJAM J et al. Ito-cell 35. WEINERF R, GIAMBRONE gene expression and collagen regulation. Hepatology 1990: 11: 111-117. 36. CASTILLAA, PRIETOJ, FAUSTO N. Transforming growth factor pf and CL in chronic liver disease: effects of interferon alpha therapy. N Eng/ J Med 1991: 324: 933-940. 37. NAKATSUKASA H, EVARTSR P, HSIAC-C, THORGEIRSSON S S. Transforming growth factor p l and type I procollagen transcripts during regeneration and early fibrosis of rat liver. Lab Invest 1990: 63: 171-180. G. The stellate cell (Ito cell, fat-storing cell, 38. RAMADORI lipocyte, perisinusoidal cell) of the liver: new insights into pathophysiology of an intriguing cell. Virchows Arch B Cell Pathol 1991: 61: 147-158. 39. HUTTERERF, EISENSTADT M, RUBINE. Turnover of hepatic collagen in reversible and irreversible fibrosis. Experientia 1970: 26: 244245. 40. MCGEEJ O’D. PATRICK R S. The role of perisinusoidal cells in hepatic fibrogenesis. An electron microscopic study of acute carbon tetrachloride liver injury. Lab Invest 1972: 26: 429440. 41. YOKOIY, NAMIHISAT, KURODAH et al. Immunocytochemical detection of desmin in fat-storing cells (Ito cells). Hepatology 1984: 4: 709-714.

379

Takahara et al. 42. TSUTSUMI M, TAKADA A, TAKASES. Characterization of desmin-positive rat liver sinusoidal cells. Hepatology 1987: 7: 277-284. 43. KlVlRIKKO K I, MYLLYLA R, PIHLAJANIEMI T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 1989: 3: 1609-1617. 44. EDMAN J C, ELLISL, BLACHER R W, ROTHR A, RUTTER W J . Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature 1985: 317: 267-270. 45. CHENGS-Y, GONGQ-H, PARKINSON C et al. The nucleotide sequence of a human cellular thyroid hormone binding protein present in endoplasmic reticulum. J Siol C h e t ~1987: 262: 11221-11227. 46. YAMADAH, AIDAT, TAGUCHI K, ASANOG. Expression of prolyl 4-hydroxylase, type 111 and IV procollagen mRNAs in fibrotic human liver. Actu Hepatol Jpn 1991: 8: 358-365.

380

47. DIECELMANN R F, COHENI K, GUZELIAN P S. Rapid degradation of newly synthesized collagen by primary cultures of adult rat hepatocytes. Biochem Siophys Res Commun 1980: 97: 819-826. 48. MOSHAGE H, CASINIA, LIEBER C H. Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes. Heptology 1990: 12: 511-518. M, ROJKINDM. LIEBERC H. 49. CASINIA, CUNNINGHAM Acetyldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism. Hepatology 1991: 13: 758-765. 50. HASSANG, STEFANINI S, BARGAGLI M, AUTUORI F. Proline-incorporating cells in chronic active liver diseases. Hepatology 1989: 9: 3749.