Ultrastructural sinusoidal changes in extrahepatic cholestasis. Light and electron microscopic immunohistochemical localization of collagen type III and type IV

Acta histochem. (lena) 98, 271-283 (1996) Gustav Fischer Verlag lena' Stuttgart· New York

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Ultrastructural sinusoidal changes in extrahepatic cholestasis. Light and electron microscopic immunohistochemical localization of collagen type III and type IV Maya Vladova Gulubova Department of Pathology, Medical Institute, Armeiska str. 16-E-2, BG-6003 Stara Zagora, Bulgaria Accepted 23 February 1996

Summary Extrahepatic cholestasis causes excessive extracellular matrix formation perisinusoidally. Ito cells, transitional and endothelial cells are considered to be a source of extracellular matrix proteins in experimental cholestasis. The localization of collagens type III and type IV in human liver in extrahepatic cholestasis was investigated immunohistochemically in the present study. Immersion fixation was used after modification to be applied to surgical biopsies with commercially available kits. Sinusoidal changes were observed that indicated excessive collagen and matrix formation. Light microscopically, increased immunostaining with the two collagen antibodies was found perisinusoidally and portally. Ultrastructurally, collagen type III positive fibres were found beneath basement membranes of vessels, in collagen bundles and as a fibrillar network in the space of Disse. Collagen type IV immunostaining was located in portal tracts and near hepatocyte microvilli. Intracellular staining with collagen type IV was detected in the rough endoplasmic reticulum of some transitional cells. Immunostaining was located around transitional cells, Ito cells or endothelial cells mainly. Our study indicates that Ito cells, transitional and endothelial cells are the main source of collagens type III and IV in the space of Disse in extrahepatic cholestasis in humans. Key words: collagen type III - collagen type IV - extrahepatic cholestasis - ultrastructural immunohistochemistry - Ito cells

Introduction Hepatic fibrosis is associated with a variety of chronic liver diseases. Extrahepatic cholestasis is one of them (Aronson et ai., 1988; Abdel-Aziz et ai., 1990). Necrosis and inflammation are generally not extensive during extrahepatic cholestasis and fibrosis does not directly emerge from injured areas (Clement et ai., 1986). Extracellular matrix proteins were studied immunohistochemically mainly in experimental bile duct ligated rats (Abdel-Aziz et ai., 1990, 1991; Milani et ai., 1990). In previous work (Gulubova and Popov, 1996) we studied light microscopically the distriCorrespondence to: M. V. Gulubova

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M. V. Gulubova

bution of collagen type IV and fibronectin in cholestatic human liver. Ultrastructural identification of the major types of collagen and different patterns of distribution have been demonstrated in man (Grimaud et al., 1980; Clement et al., 1986; Burt et aI., 1990; Takahara et aI., 1992) and rats (Hernandez, 1984, 1985; Abdel-Aziz et aI., 1990, 1991). Collagen type III is found in portal tracts, around central veins and along sinusoids (Grimaud et al., 1980; Clement et al., 1986; Abdel-Aziz et al., 1991; Takahara et al., 1992). Collagen type IV is normally present in vascular and peri biliary basement membranes and in the space of Disse in fibrotic liver (Hahn et aI., 1980; Hernandez, 1985; Abdel-Aziz et aI., 1990, 1991; Takahara et aI., 1992; Gulubova and Popov, 1996). It was shown by in situ hybridization with [35 S]-labeled RNA probes that procollagen gene transcripts are produced by mesenchymal, endothelial and bile duct epithelial cells and not by hepatocytes in adult human and rat liver (Milani et aI., 1990). However, ultrastructural immunohistochemical studies showed that under different inflammatory, neoplastic and toxic stimuli in vivo and in vitro hepatocytes might be a source of extracellular matrix proteins such as fibronectin and different types of collagen (Diegelmann et aI., 1983; Hernandez, 1985; Clement et aI., 1986; Albrechtsen et aI., 1988). It is now generally accepted that Ito cells and their transformed forms, i.e., transitional cells (Mak and Lieber, 1988; Takahara et aI., 1992), or myofibroblast-like cells (Bhathal, 1972; Callea et aI., 1982; Enzan et al., 1994) are responsible for the overproduction of extracellular matrix proteins, e. g. collagen type III, collagen type IV and laminin in different chronic liver diseases (Albrecht sen et al., 1988). Normally the space of Disse has an indistinct matrix with discontinuous deposits of fibronectin and occasionally thin bundles of type I collagen (Hernandez, 1984; Burt et aI., 1990) and only rare discontinuous deposits of collagen type IV (Hernandez, 1984, 1985; Burt et aI., 1990; Burt, 1993). In fact hepatocytes are normally the only epithelial cells that are not separated from plasma by at least a basement membrane. In liver injury, an excessive and disorganized deposition of extracellular matrix proteins occurs perisinusoidally (Burt et aI., 1990; Burt, 1993). In the present work, we have studied the extracellular and intracellular distribution of collagen type III and type IV in human liver with extrahepatic cholestasis that lasted more than 14 days. We have applied a general procedure for immunoelectron microscopy with commercially available antibodies and detection systems to human liver samples. Material and Metbods Wedge surgical biopsies were taken from 6 patients (patients 4 - 9) with extrahepatic cholestasis caused by bile stones in the common bile duct. The duration of cholestasis was more than 14 days (15 - 37). One control patient underwent explorative laparotomy because of a large pelvic tumor (myoma). Two control patients had hepatic haemangioma. All patients had no history of previous liver diseases, alcohol consumption or diabetes mellitus. The main serum parameters are presented in Table 1. Informed consent was obtained from each patient. Routine histology. Paraffin sections were stained with haematoxylin-eosin and Van Gieson. Electron microscopy. Liver tissue samples were perfusion-fixed as was described previously (BioulacSage et aI., 1986) in 4070 glutaraldehyde in 0.1 M phosphate buffer pH 7.4 and postfixed in 1070 osmium tetroxide in the same buffer. The semithin sections were stained by the Hamphrey and Pittman procedure for assessment of fibrosis and Ito cell counting. Ultrathin sections were cut from periportal zones of liver lobules. Sections were contrasted with uranyl acetate and lead citrate. Light microscopic immunohistochemistry. 5 11m cryostat sections were fixed in cold acetone, incubated with normal goat serum for 20 min and then incubated with primary antibodies for 24 h. Afterwards they were reacted with goat anti-mouse antibodies and then with mouse peroxidase-anti peroxidase complex. The used antibodies were monoclonal antibodies against human collagen III (MAI67-5C) and collagen IV (MA079-5C) and the detection system was a mouse peroxidase antiperoxidase kit (HPOOO-5M) (BioGenex Laboratories, San Ramon, USA). The used chromogen was 3-amino-9-ethylcarbazole.

Collagen immunohistochemistry in extrahepatic cholestasis

273

Electron microscopic immunohistochemistry. 3 x 4 x 4 mm samples were immersion-fixed in a mixture of 4% paraformaldehyde, 0.08070 glutaraldehyde and 0.\5070 picric acid in 0.\ M phosphate buffer, pH 7.4 for 4 h at 4°C, and then washed overnight at 4 °C in 0.1 M phosphate buffer, pH 7.4 with 20070 sucrose. Free-floating cryostat sections (20 ~m) were dipped in 0.1 M phosphate buffer, pH 7.4 with 20070 sucrose, and rinsed in the same buffer overnight. Afterwards the sections were washed in 0.1 M phosphate buffered saline (PBS), pH 7.4 for 15 min, incubated with 1.2070 hydrogen peroxide in methanol, rinsed in PBS and incubated with normal goat serum. After this initial treatment, the sections were incubated with the primary antibodies for 24 h at room temperature, rinsed in PBS, incubated with goat anti-mouse antibodies for 60 min, rinsed in PBS, reacted with mouse peroxidase-anti peroxidase for 60 min, rinsed in PBS and in 0.05 M Tris-HCL buffer, pH 7.54 for 10 min. Finally, peroxidase activity was demonstrated by using a mixture of 3 mg diaminobenzidine in 15 ml 0.05 M Tris-HCL buffer, pH 7.54 and 36 ~I 1070 hydrogen peroxide for 10 min, and rinsed in 0.1 M phosphate buffer, pH 7.4. Then sections were post fixed with 1070 osmium tetroxide for 30 min, rinsed in phosphate buffer, dehydrated with graded concentrations of ethanol and propylene oxide, and flat-embedded with Durcupan. Ultrathin sections were counterstained with uranyl acetate only and examined and photographed with a Zeiss EM-109 electron microscope at 50 kV. The procedures were carried out immediately after biopsy taking. Controls were first incubated with non-immune sera. Cell counting. Ito cells were counted in periportal zones of liver lobule using semi thin sections. Five fields of vision from each case were taken at a magnification of 400. For statistical analysis, the Student's t test was used.

Results Histology. The patients showed different degrees of cholestasis from mild to moderate and different degrees of parenchymal deterioration. There was an increased perisinusoidal Van Gieson staining in periportal zones (Table 1). The number of Ito cells was increased significantly (p < 0.05) in periportal zones of cholestatic patients (mean 4.8 ± 2.5) as compared with the controls (mean 2.4 ± 0.7). Electron miscroscopy. Disse space was slightly to moderately enlarged and filled with many collagen bundles and amorphous matrix. Discontinuous basement membrane material was found between sinusoidal endothelial cells and Ito cells (Fig. 1) and sometimes between Ito cells and hepatocytes. Ito cells transformed mainly in transitional cells with decreased number of lipid droplets, increased amounts of filaments with dense bodies, pinocytic vesicles and collagen around them (Figs. 2 - 3). They had well developed Golgi apparatus and rough endoplasmic reticulum (RER). Ito cells transformed also into myofibroblast-like cells. The latter had crenated nuclei with prominent nucleoli, many cytoplasmic processes and intermediate filaments in their cytoplasm with dense bodies, RER and Golgi apparatus and were surrounded by basement membrane material. Light microscopical immunohistochemistry. In control patients (patients 1 - 3) collagen type III was localized mainly in portal tract and to a lesser extent around central veins. Its deposition in the space of Disse was very faint and discontinuous (Fig. 4). Collagen type IV was found in the basement membranes of vessels, bile ducts and perineurally in the portal tract and around central veins. Faint and discontinuous immune staining was detected perisinusoidally (Fig. 6). Cholestatic patients. Collagen type III showed more intensive immunostaining in collagen bundles in portal tracts and septa, and less around central veins. Along sinusoids it appeared as a continuous and intensively stained layer, mainly in periportal and midzonal areas (Fig. 5). Collagen type IV increased in portal tracts and septa, around hepatocytes showing feathery degeneration and lytic necrosis. Intense and continuous immunostaining was present in the space of Disse (Fig. 7; Table 2). Electron microscopic immunohistochemistry. Control patients. Collagen type 111positive and negative bundles were found in portal tracts (Fig. 8). Osmium black deposits

Patients Patient 1 (control)

Serum biochemistry Total serum bilirubin SGar SGPT

to 50 cells

Ly, Macro

Liver tissue without histological changes

Ly, Macro

Liver tissue without histological changes

normal normal normal

to 50 cells

normal normal normal

m 48

Patient 2 (control

-+

-+

Slight to moderate cholestasis

++

+

+ + (around PT) + + + + (zone 1, 3) +

-+

Moderate cholestasis

++ +

++ + ++

++

++

+

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Ly, Macro, Neu

to 50 cells

44mkm/l 76 VII 121 VII

m 80 37 days

Patient 6

+

Ly, Macro, Neu

to 50 cells

143.6mkm/l 76 VII 120 VII

m 54 24 days

Patient 5

+

Ly, Macro

to 50 cells

42mkm/l 12 VII 30 VII

m 54 19 days

Patient 4

Liver tissue Slight without cholestasis histological changes

Ly, Macro

to 50 cells

normal normal normal

f 59

Patient 3 (control)

Slight cholestasis

+

+

+ +

+

Ly, Macro

to 50 cells

43 mkm/l 15 VII 26 VII

f 49 15 days

Patient 7

+

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++ + +

++ + ++

Slight cholestasis

+

++ + +

+ + (zone 3) + +++ ++

+

++

Ly, Macro, Neu

Ly, Macro

++

to 50 cells

68.3 mkm/l 72 VII 97 VII

20 days

f

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to 50 cells

66.2mkm/l 64 VII 70 VII

f 63 23 days

Patient 8

Abbreviations: f female; m male; Ly lymphocytes; Macro macrophages; Neu neutrophils; PT portal tract; - missing; - + weakly positive to missing; + weakly positive; + + moderately positive; + + + strongly positive.

Histological diagnosis

1.PT a. Number of inflammatory cells b. Cell type c. Bile ductular proliferation 2. Lytic necrosis in the limiting plate 3. Lobular parenchyma a. Cholestasis b. Feathery degeneration 4. Fibrosis a. Portal periductular periportal b. Septa formation c. Peri sinusoidal zone 1 zone 2 zone 3

B. Histology

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1. Sex f 2. Age 43 3. Duration of cholestasis

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Abbreviations: PT portal tract; BM basement menbrane; CIII collagen type III; CIV collagen type IV; - missing; - + weakly positive to missing; + weakly positive; + + moderately positive; + + + strongly positive.

2. Lobular parenchyma perisinusoidally a. Periportal b. Midzonal c. Peri central

l.PT a. Matrix b. Collagen bundles c. BM of vessels and bile ducts d. Septa

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Table 2. Light microscopical localisation of collagen type III and IV in livers with extrahepatic cholestasis

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Fig. 9. Collagen type III positive fibers and fibrillar matrix in the space of Disse. x20000. Fig. 10. Collagen type III positive immunodeposits beneath a vessel in the portal tract. Fig. 11. Collagen type IV immunostaining in the basement membrane of a vessel next to a smooth muscle cell. x20000. Fig. 12. Collagen type IV immunostaining between hepatocyte microvilli. x 30000.

Collagen immunohistochemistry in extrahepatic cholestasis

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13

Fig. 13. Faint collagen type IV immunoreaction in the space of Disse (control patient). x20000. Fig. 14. Collagen type IV immunostaining in the rough endoplasmic reticulum of a transitional cell. x 30 000. Fig. 15. Intensive collagen typeiV immunoreaction in the space of Disse. x12000. Fig. 16. Collagen type IV immunoreaction around a transitional cell. x 20000.

280

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were localized on typical collagen fibers with periodicity of 60 - 70 nm and in particular on a thin network of fibrillar material present in the extracellular matrix. Thin extrafibrillar osmium black deposits surrounded the labeled fibers. Labeled fibrillar material surrounded discontinuously basement membranes of vessels and bile ducts in portal tracts. A positive fibrillar network was present in Disse space between endothelial cells and hepatocyte membranes. It surrounded Ito cells, outlined endothelial cells and was present there in combination with negative collagen bundles. Fibroblast-like cells in portal tracts were negative for collagen type III, but were surrounded by positive collagen bundles. In liver parenchyma, Ito cells did not show intracellular immunostaining for collagen type III. Hepatocytes and endothelial cells were negative for collagen type III. Collagen type IV positivity was found in basement membranes around vessels and bile ducts in portal tracts (Fig. 11) and discontinuously in close relationship to microvilli of hepatocytes in the space of Disse (Figs. 12 -13). Intracellular immunostaining of collagen type IV was not present in RER of Ito and endothelial cells. Cholestatic patients. Collagen type III labeling pattern and localization was identical with controls. However, the amount of collagen deposit was increased in portal tracts and septa and in Disse space. In liver parenchyma collagen was particularly well developed around transitional cells (Fig. 9). Immunoreactive deposits around basement membranes of vessels and bile ducts in portal tracts were increased (Fig. 10). Large amounts of labeled fibrillar material surrounded fibroblasts in portal tracts, but there was no intracellular staining there. Immunostaining of collagen type IV was increased in portal tracts and appeared in the extracellular matrix between collagen fibres and in vesicles of some bile duct cells. In the sinusoids some Ito cells and transitional cells showed immunostaining in RER for collagen type IV (Fig. 14). Collagen type IV appeared continuously distributed along sinusoids (Fig. 15), beneath endothelial cells, around Ito cells and transitional cells (Fig. 16) and between hepatocyte microvilli. In Kupffer cells vesicles positive for collagen type III and IV were observed, but clear staining of RER could not be established.

Discussion It has been established that the number of Ito cells in periportal zones is 2.4 ± 0.6 (Sztark et aI., 1986). We have found an increased number of these cells in cholestatic patients. Therefore, it can be concluded that cholestasis is a cause for proliferation of Ito cells. Ultrastructural changes in the space of Disse in cholestatic human liver were described earlier (Gulubova and Popov, 1996). We found mainly transitional cells and less myofibroblast-like cells in periportal zones. It was already reported that Ito cells transform into transitional cells (Mak and Lieber, 1988; Hines et aI., 1993) or myofibroblast-like cells (Bhathal, 1972; Callea et aI., 1982; Enzan et aI., 1994) during liver fibrosis. Hines et al. (1993) showed that during chronic cholestasis, Ito cells increase in number reaching a plateau by day 14, and that they were mainly of the transitional type, whereas during progression of the disease, the myofibroblast type also appeared. Collagen formation in extrahepatic cholestasis was investigated mainly experimentally (Kountouras et aI., 1984; Abdel-Aziz et aI., 1990, 1991), and only occasionally in humans (James et aI., 1989; Gulubova and Popov, 1996). In the present study collagen type III and IV immunostaining was stronger perisinusoidally, mainly in periportal and midzonal areas as compared with the controls. Collagen type IV increased also between hepatocytes with feathery degeneration, and collagen type III and IV together - in portal tracts and septa. Similar expression of collagen type III and IV in portal tracts and

Collagen immunohistochemistry in extrahepatic cholestasis

281

perisinusoidally was obtained after experimental bile duct ligation for 14 days (AbdelAziz et aI., 1990, 1991). Aronson et aI. (1988) detected an increase of portal and perisinusoidal collagen 2, 4 or 6 weeks after bile duct ligation in rats. Immunostaining of collagen type III and IV in portal tracts and peri sinusoidally was observed in alcoholic liver (Hahn et aI., 1980), in chronic active liver disease of viral origin (Takahara et aI., 1992), and in different fibrotic and cirrhotic human livers (Grimaud et aI., 1980; Clement et aI., 1986). Our study demonstrates that collagen type III and IV is also increased after extrahepatic cholestasis in the human. Our ultrastructural finding of type III positive deposits on collagen fibres, on fibrillar material in portal matrix Disse space and beneath basement membranes of vessels confirms other results (Grimaud et aI., 1980; Abdel-Aziz et aI., 1991; Takahara et aI., 1992). Collagen type IV immunostaining in basement membranes in portal tracts and close to hepatocyte microvilli are also well known (Hernandez, 1984, 1985; Clement et aI., 1986; Abdel-Aziz et aI., 1991; Takahara et aI., 1992). Intracellular struc~ures involved in protein synthesis, such as RER, Golgi apparatus and transport vesicles were found to be heavily stained for collagen type III and IV in Ito cells and fibroblasts in bile-duct ligated rats (Abdel-Aziz et aI., 1991), in Ito and fibroblast-like cells in fatty and fibrotic human liver (Clement et aI., 1986), in transitional cells and Ito cells in chronic active liver disease (Takahara et aI., 1992) and in Ito cells in CCl 4-treated rats (Hernandez, 1985). Staining was not uniform, but preferentially present along RER membranes of Ito cells in normal human liver (Clement et aI., 1986). In chronic non-active liver disease very faint staining for collagen type III and IV was observed in the RER of Ito cells and transitional cells, and fibroblasts in portal tracts were negative for collagen type III and IV intracellularly (Takahara et aI., 1992). It was found that sinusoidal endothelial cells stain for collagen type III and IV; however, the major part of staining was present in vesicles of various sizes and not in RER of normal and fibrotic human liver (Clement et aI., 1986) and in bile duct ligated rats (Abdel-Aziz et aI., 1991). Very faint to absent staining for collagen type III and IV was found in sinusoidal endothelial cells in chronic active and inactive liver disease (Takahara et aI., 1992). Compared with rat liver (Abdel-Aziz et aI., 1991) the intense staining of RER only of some Ito cells and the intense extracellular staining around them as found in the present study is probably a result of the used method of fixation by immersion with Bouin's fixative. Our method allows the ultrastructural demonstration of extracellular matrix proteins in human liver samples, appling monoclonal antibodies and ready-to-use detections systems. We utilized a universal fixative and fixation time suitable for preservation of the antigenicity and to a certain extent for preservation of the structure. By this method we avoided the imperfection of tissue freezing which destroyed partly the antigenicity and obscured the ultrastructure. The sinusoidal cells' changes from the perfusion pressure were avoided by the immersion technique. Grimaud et aI. (1980) using immersion-fixation for human liver biopsies with fibrosis and cirrhosis did not observe intracellular immunostaining for extracellular matrix components in transitional and endothelial cells. The immunostaining of collagen type III and type IV was intensive around Ito cells and endothelial cells. Using the same technique Takahara et aI. (1992) observed intracellular immunostaining for extracellular matrix components in Ito cells, transitional cells and in some hepatocytes only in the area of piecemeal necrosis. In all other cases with inactive liver disease very faint to missing immunostaining was observed in perisinusoidal cells. Clement et aI. (1986) used a short term fixation by perfusion with 4070 paraformaldehyde and observed intensive intracellular immunostaining in mesenchymal cells. Hernandez (1984) used a longer fixation time and visualized no intracellular immunostaining.

282

M. V. Gulubova

In conclusion, using fixation by immersion with Bouin's fixative for 4 h at 4°C the immunostaining of extracellular collagen type III and IV was comparatively better expressed and intracellular staining weaker. In our opinion to label intracellular structures for extracellular matrix proteins is necessary to use perfusion-fixation, cryostat sections not thick, than 20 /lm; and to collect samples from patients suffering from liver diseases with marked parenchymal deterioration (inflammation, necrosis) which stimulate intensive extracellular matrix formation. There are many immunohistochemical studies demonstrating Ito cells to be actively involved in the production of collagens in CCl 4-treated rats (Hernandez, 1985), in normal and alcoholic human livers (Clement et aI., 1986) and in bile-duct ligated rats (Abdel-Aziz et aI., 1991). By means of in situ hybridization of a [35 S]-labelled 1(1), 2(1), 1(III), 1(IV) procollagen anti-sense RNA, Milani et ai. (1990) showed that localization of cellular transcripts in fibrotic liver was mainly present in mesenchymal cells (Ito cells, transitional cells; fibroblasts). There are several indirect arguments suggesting that the pattern of extracellular matrix formation by Ito cells is altered during liver injury. Interestingly, the rate of collagen synthesis in Ito cells isolated from CCl4-treated rats is 4- to 6-fold increased when compared to their normal counterparts (Shiratori et aI., 1987). We cannot conclude from immunohistochemical observations only whether Ito cells express differently matrix protein genes in cholestatic patients as compared with controls. However, the fact that collagen type IV immunostaining was found intracellularly in transitional cells and that collagen type III and IV immunostaining was found surrounding these cells, suggest that they are good candidates for synthesis of these collagens in extrahepatic cholestasis in humans. Our finding that in cholestasis collagen bundles, amorphous matrix and basement membrane formation on one side and collagen type III and IV immunodeposits on the other increased perisinusoidally, lead us to propose that this process is directed to protect hepatocytes from injury but at the expense of hindering systemic functions.

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Callea F, Mebis J, and Desmet VJ (1982) Myofibroblasts in focal nodular hyperplasia of the liver. Virchows Arch [AJ 396: 155 -166 Clement B, Grimaud JA, Campion JP, Deugnier Y, and Guillouzo A (1986) Cell types involved in the production of collagen and fibronectin in normal and fibrotic human liver. Hepatology 6: 225 - 234 Diegelmann RF, Guzelian PS, Gay R, and Gay S (1983) Collagen formation by the hepatocyte in primary monolayer culture and in vivo. Science 219: 1343 - 1345 Enzan H, Himeno H, Iwamura S, Onishi S, Saibara T, Yamamoto Y, and Hara H (1994) Smooth muscle actin - positive perisinusoidal stromal cells in human hepatocellular carcinoma. Hepatology 19: 895 -903 Grimaud JA, Druguet M, Peyrol S, Chevalier 0, Herbage D, and Badrawy NL (1980) Collagen immunotyping in human liver: light and electron microscopic study. J Histochem Cytochem 28: 1145-1156 Gulubova M, and Popov A (1996) Electron microscopic investigation on Ito cells and Disse space in patients with extrahepatic cholestasis. Light microscopical immunohistochemistry of collagen type IV and fibronectin in hepatic sinusoids. Gen Diag Pathol 141: 187 -192 Hahn E, Wick G, Pencev D, and Timpl R (1980) Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin and fibronectin. Gut 21: 63 -71 Hernandez AM (1984) The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver. Lab Invest 51: 57 -74 Hernandez AM (1985) The hepatic extracellular matrix. II. Electron immunohistochemical studies in rats with CCl4-induced cirrhosis. Lab Invest 53: 166-186 Hines JE, Johnson SJ, and Burt AD (1993) In vivo responces of peri sinusoidal cells (lipocytes) and macro phages to cholestatic liver injury. Am J Pathol 142: 511-518 James J, Lygidakis NL, van Eyken P, Tanka AKF, Bosch KS, Ramaekers FCS, and Desmet V (1989) Application of keratin immunocytochemistry and sirius red staining in evaluating intrahepatic changes with acute extrahepatic cholestasis due to hepatic duct carcinoma. Hepato-gastroenterol 36: 151 -155P Kountouras J, Billing BH, and Scheuer PJ (1984) Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65: 305 - 311 Mak KM, and Lieber CS (1988) Lipocytes and transitional cells in alcoholic liver disease: a morphometric study. Hepatology 8: 1027 -1033 Milani S, Herbst H, Schuppan D, Kim KY, Riecken EO, and Stein H (1990a) Procollagen expression by nonparenchymal rat liver cells in experimental biliary fibrosis. Gastroenterology 98: 175 -184 Milani S, Herbst H, Schuppan D, Surrenti C, Riecken EO, and Stein H (1990b) Cellular localization of type I, III and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol137: 59-70 Shiratori Y, Ichida T, Greets A, and Wisse E (1987) Modulation of collagen synthesis by fat-storing cells, isolated from CCI 4- or vitamin A-treated rats. Dig Dis Sci 32: 1281- 1289 Sztark F, Dubroca J, Latry P, Quinton A, Balabaud C, and Bioulac Sage P (1986) Perisinusoidal cells in patients with normal liver histology. A morphometric study. J Hepatol 2: 358 - 369 Takahara T, Nakayama Y, Itoh H, Miyabayashi C, Watanabe A, Sasaki H, Inoue K, Muragaki Y, and Ooshima A (1992) Extracellular matrix formation in piecemeal necrosis: immunoelectron microscopic study. Liver 12: 368 - 380