Alcoholic Liver Disease Parenchyma to Stroma Relationship in Fibrosis and Cirrhosis as Revealed by Three-dimensional Reconstruction and Immunohistochemistry

Severe ethanol-induced liver damage is characterized byfibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of 'parenchymalpillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates. The other bile duct, typical CI, namely CK 19, was only detectable in few hepatocytes. However, the expression of CK 7 and/or CK 19 was less frequent in hepatocytes that were closely associated with bile ductules. CK 7 and CK 19 were also found in some, but not all, Mallory bodies. These observations indicate that the expression of these two CKs is neither related to a transformation of hepatocytes to bile duct-like structures ("ductal metapla-sia") nor to the formation of Mallory bodies. Further-more, double immunofluorescence studies revealed small groups of hepatocytes and bile ductules that were encircled by basement membrane material, thus suggesting the formation of "secretory units." (Am J Pathol 1992, 141:69-83) Ethanol-induced chronic liver injury characterized by a broad spectrum of morphologic alterations. As a rule, steatosis the liver cells the first sign of alcoholic damage, by fibrosis, and

tion,1013 and cirrhosis seems to develop without preceding alcoholic hepatitis.13 In humans, development of cirrhosis follows episodes of alcoholic hepatitis, although alcoholic hepatitis does not seem to be an absolute requirement.14 Alcoholic hepatitis leads to liver damage with enlargement of hepatocytes, liver cell necroses, and inflammation with a predominance of granulocytes and formation of cytokeratin (CK)-containing cytoplasmic inclusions, known as Mallory bodies or alcoholic hyalin.'5 Necrosis of liver cells in alcoholic hepatitis does not seem to be a direct consequence of Mallory body formation. 16 Mallory bodies seem to be indicators of severe hepatocellular injury. The activity of alcoholic hepatitis is a marker of a rapid progression of the disease to cirrhosis.41718 However, ethnologic differences may also ex-ist. For example, in Japan alcoholic hepatitis is rare and hepatic fibrosis of the pericellular type without apparent hepatic necrosis and inflammation is the most common histologic feature of alcoholic liver disease.1921 In cases with development of alcoholic liver cirrhosis without preceding alcoholic hepatitis, the extent of the centrolobular (perivenular) fibrosis shows a good correlation with progression to cirrhosis.822 In fully developed lesions, severe fibrotic dissociation of the liver cell plates is also seen in the periportal region and single hepatocytes and small groups of hepatocytes, sometimes arranged in rosettes, are surrounded by fibrotic strands. The dissociation of hepatocytes by fibrosis could impair liver function by interference with exchange processes between hepatocytes and blood, and eventually, bile secretion and flow.
We focused on areas of fibrotic dissociation of the liver parenchyma ("chicken wire fibrosis"). By serial recon-Supported by Fonds zur Forderung der wissenschaftlichen Forschung (grant P 7628-Med to H.D. and K.Z). structions and immunohistochemistry, we investigated the consequences of the fibrotic parenchymal dissociation with respect to the functionally relevant organization of the hepatocytes, particularly hepatocyte-hepatocyte, hepatocyte-stroma, and hepatocyte-bile ductule relationships. Moreover, we analyzed a possible correlation between disturbance of the liver architecture and changes of cytokeratin expression.

Materials and Methods
Liver samples from seven autopsy cases (four males and three females, aged between 48 and 72 years with a mean age of 61 years; obtained 2-16 hours postmortem) were collected. Four livers displayed different stages of ethanol-induced liver injury ranging from alcoholic hepatitis with fibrosis to fully developed cirrhosis. Three livers without pathologic changes were used as controls. From each case at least three tissue blocks were snapfrozen and stored in liquid nitrogen for immunofluorescence microscopy. In addition, samples were fixed in 10% phosphate-buffered formaldehyde solution (pH 7.0) and were, after dehydration in graded ethanol, embedded in methyl-methacrylate for reconstructive studies. Furthermore, four liver biopsy samples (from three males and one female, aged between 30 and 55 years with a mean age of 42 years, showing different stages of ethanolinduced liver injury ranging from severe fibrosis to fully developed cirrhosis in association with alcoholic hepatitis), originally embedded in paraffin, were deparaffinized and re-embedded in methyl-methacrylate for reconstruction (cases 8-11).

Reconstructive Studies
Twenty to forty-five consecutive serial sections of 2 pLm thickness from the methacrylate-embedded liver samples were cut with a Reichert-Jung microtome (K-Mikrotom, Polycut S, Reichert-Jung, Vienna, Austria). These sections were stained by the chromotrope-aniline blue (CAB) method. Several histologic motives were consecutively followed up and photographically documented. The contours of the hepatocellular and bile duct epithelial borders were transcribed to transparent foils, digitized with a computer-assisted image-analyzing system (IBAS, Fa Zeiss, Oberkochen, FRG) and reconstructed to provide a view of the three-dimensional architecture.

Controls
The specificity of the immunoreactions was controlled by replacement of the primary antibodies by PBS or nonimmune sera. Moreover, the results obtained in fibrotic and cirrhotic livers were compared with normal livers.

Three-dimensional Reconstruction
A section through a severely fibrotic or cirrhotic liver gives the impression of many isolated hepatocytes, which are surrounded by connective tissue (Figure 1 A). However, as demonstrated by three-dimensional reconstructions, these hepatocytes are still in contact with other hepatocytes or bile ductules. Two different situations could be detected: 1) A "single" hepatocyte, seen in one section (e.g., .A_6 Figure 1. Reconistruction ofa pillar of liver parenchyma from 16 serial sections qf2 p.m thickness in a cirrhotic liver. At sectioni level Nr. 1, a single hepatocy,te is surrounded by connective tissue (arrouw, A). At the level of section Nr. 106, this liver cell is aissociated uwith a group of hepatocytes displaying bile canaliculi (B). Three-dimenzsional reconistructioun model on the ba.sis of 16 serial sections reveals the sinigle hepatocyte in section Nr. 1 as the enidpoint ofa parenchymalpillar (C). In section level Nr. 16, three bile canaliculi can be seen (arrouws, C).
2) Isolated hepatocytes were directly connected with bile ductules (Figure 3). This was particularly frequent in areas with chicken wire-like fibrosis. This situation was best revealed in double IIF with antibodies to different CKs by which hepatocytes were clearly distinguished from bile duct and ductular epithelium (Figures 4, 5).
Completely "isolated" hepatocytes (i.e., hepatocytes without connection either to other hepatocytes or to bile ducts) were not found. Therefore, even in areas with severe fibrosis and parenchymal dissociation, a bile draining system via canaliculi between neighboring hepatocytes and bile ductules was maintained.
Immunohistochemical Studies on CK Expression in Bile Duct Epithelia, Hepatocytes, and Mallory Bodies CK expression in cirrhotic livers with alcoholic hepatitis was investigated in frozen sections by double IIF using polyclonal CK antibodies with broad CK reactivity, which recognize bile duct epithelia, hepatocytes, and Mallory bodies, as well as monoclonal antibodies to CKs, which in normal adult human liver are only expressed in bile duct epithelia but not in hepatocytes, i.e., CK 7  immunohistochemically, bile ductules contained CK 7 tocytes lacked and CK 19, whereas adjacent hepatocytes, even those bodies, howev "transitional" in phenotype between hepatocytes and bile CK 19 ( Figure   Figure 3. Reconstruction model ofcirrhotic liver (8 serial sections) with an isolated hepatocyte (asterisk) lying in close proximity to proliferated bile ductules. This single hepatocyte has no connection to a neighboring parenchymal plate (upper right). n ( Figure 5B, D), were unstained by these )wever, some hepatocytes mostly found in )derate fibrosis but still organized in cell hout association with bile ductules reacted , to CK 7 ( Figure 6). Although CK 7-positive vere preferentially found in periportal re-,ere not strictly confined to these areas.

Immunohistochemical Characterization of Mesenchymal Cells and of the Extracellular Matrix
In livers with alcoholic fibrosis numerous a1l -smooth muscle actin-positive cells were seen in areas with chicken wire-type fibrosis ( Figure 8A). In cirrhotic livers, the pseudolobules were surrounded by fibrotic strands rich in al-smooth muscle actin-positive cells ( Figure 8B). Coexpression of al -smooth muscle actin and vimentin could be demonstrated in many cells within the fibrotic strands, but with different cytoplasmic distribution ( Figure  9). For comparison, in normal human livers portal tracts contained many vimentin-positive cells (Figure 1 OA), whereas al -smooth muscle actin-positive cells were mostly restricted to vessel walls (Figure 1 OB). In normal human livers and in livers with chicken wirelike fibrosis and cirrhosis, only few desmin-positive cells were present (Figure 11), most of them within vessel walls. In the intact liver lobule, but also in the pseudolobule in cirrhosis, desmin-positive cells were not observed.
The simultaneous decoration of the CK cytoskeleton of hepatocytes and bile duct epithelia with a polyclonal pan-CK antibody ( Figure 12A, C), and of basement membrane constituents with a monoclonal antibody to laminin ( Figure 122B, D), was performed in alcoholic cirrhosis. Small groups of hepatocytes lying in direct contact with bile ductules were visualized by staining with pan-CK antibodies ( Figure 12A, C). Bile ducts and ductules and, partly, hepatocytes were surrounded by a laminin-containing layer ( Figure 122B, D). The association of small groups of hepatocytes with their bile duct draining system in the form of ductules and the "encapsulation" by basement membrane constituents may indicate a "secretory unit." This is in agreement with the ultrastructural observations of Uchida and Peters,26 who found basement membrane-like structures around islands of hepatocytes and ductules. Although hepatocytes were not necessarily completely surrounded by laminin as shown in Figure 12C and D, bile ductules were always completely surrounded. In inactive cirrhosis, the basement membranes of the bile ductules and also of the venules within the fibrotic strands reacted with antibodies to laminin ( Figure 13). Sometimes, at the periphery of the pseudolobules (Figure 13), a strong immunoreaction for laminin was observed at the sinusoidal side of the hepatocytes (thus indicating capillarization of sinusoids). Moreover, "secretory units" were also encircled by laminin-containing material ( Figure 13A, B). In contrast, in the centers of the pseudolobules, no deposition of laminin resembling capillarization of sinusoids was detectable. Normal lobules were absent in accordance with the severe fibrosis and cirrhotic transformation of the liver. Dissociated groups of hepatocytes were also partly surrounded by fibronectin ( Figure 14). Fibronectin was not only distributed around hepatocytes, but was also present within fibrotic areas.

Three-dimensional Reconstruction
Our three-dimensional reconstructions and immunohistochemical studies on the CK-expression pattern of hepatocytes and bile duct epithelia show that apparently isolated hepatocytes in sections from fibrotic or cirrhotic livers always have connections to other hepatocytes or to bile ductules. Therefore, even in cases with severe fibrosis and parenchymal dissociation, a bile draining system  (B) shou s that some hepatocytes react uwith the CK 7 antibodqy. Note that these hepatocvtes are not in association with bile ducts. A, B X 528 where maintenance of bile flow was found, even in endstage cirrhosis.29

CK Expression in Hepatocytes and Bile Duct Epithelia
Van Eyken et al30'31 reported that some hepatocytes, at least in pathologic situations, express CK 7 and CK 19 in addition to CK 8 and CK 18, which was regarded as "ductular metaplasia."27,28,31 We could also demonstrate that, especially in areas with fibrosis, hepatocytes which were still organized in cell plates without any evidence of metaplastic changes expressed CK 7 and occasionally also CK 19 ( Figure 6). But, we did not find CK 7 and CK 19 expression in cells transitional in phenotype between hepatocytes and bile duct epithelia or in hepatocytes adjacent to bile duct epithelium. In our cases of ethanolinduced fibrosis and cirrhosis, we could not find a predominance of the hepatocellular expression of CK 7 and/ or CK 19 in a distinct acinar region. This is in line with van Eyken et a130 who did not find a characteristic distribution of these CKs in severely ethanol-damaged livers, whereas in earlier lesions (fatty liver with or without alcoholic hepatitis) they reported a predominance of CK 7-positive hepatocytes in the perivenular (acinar zone 3) region. In ethanol-damaged livers, no apparent continuity of CK 7-positive ductules with hepatocytes was found by van Eykens et al.30 However, in severe lesions associated with chronic cholestatic liver disease a close association could be demonstrated.31 Evidence in favor of "ductular metaplasia of hepatocytes" was also provided by ultrastructural and histochemical observations by Uchida and Peters26 who demonstrated markers of hepatocytes, such as glycogen and glucose-6phosphatase activity in cells of proliferated bile ductules. During embryonal development and in regenerating liver, cells with characteristics of bile duct epithelium and/or hepatocytes develop from a common stem cell. 32 In the liver, the so-called "oval cells" might represent such a stem cell reservoir. In a recent study using in situ hybridization, immunohistochemical, histochemical, and morphologic methods, Shiojiri et a132 showed that intrahepatic bile ducts originated from cells morphologically resembling oval cells and expressing hepatocyte and bile duct markers. The occasional expression of CK 7 and CK 19 in Mallory bodies does not justify the notion of a causal relationship between CK 7 and CK 19 expression and Mallory body formation. Our observation is, therefore, in line with the results of van Eyken et al,30 who stated that the shift in CK gene expression towards bile duct-type CKs is not directly related to Mallory body formation.
Although the monoclonal antibodies AE1 and AE3, raised against epidermal CKs, recognize a rather broad Figure 7. Double immunofluorescence microscopy u'ith apolyclonalpan-CKantibody decorating hepatoc-rtes and Alallorv bodies (A,C) and with a monoclonal antibody to CK 19 in (B) and a monoclonal antibody to CK 7 in (D). Note that tu'o Mtallorp bodies are not recognized by the antibody to CK 19 in (B) (arrous) and that most Mallorn bodies in (D) remzain unistained b the antibody, to CK 7 (arrouws). A-D x 528. range of CK polypeptides (molecular weights between 50 kd and 67 kd33) in our hands they only stained bile duct epithelia but not hepatocytes in human liver, in agreement with Preisegger et al. 25 This is in contrast to Katsuma et al' who reported that AE3 alone reacted with normal hepatocytes in addition to bile duct epithelia, whereas AED only reacted with bile duct epithelia and a few pathologically altered hepatocytes. The reason for this discrepancy is presently unknown, but may be due to differences in methods.    Figure 13. Double immunofluorescence microscopy. Hepatocytes and bile duct epithelia are stained by a polyclonalpan-CK antibody (A), and basement membranes byI a monoclonal antibodv to laminin (B) in inactive liver cirrhosis; (A) shows two pseudolobules separated by a broadfibrous strand All hepatocvtes and some proliferated bile ductules are stained by the pan-CK antibody. In (B), laminin is demonstrable along sinusoids near the margins of the pseudolobules reflecting "capillarization" of the sinusoids. Furthermore, within thefibrous strand (asterisk) basement membranes are stained by the antibody to laminin not onlyj around the proliferated ductules (arrowheads) but also in the vessel uwalls (arrou). A,B x320. desmin (compare Figure 8 with Figure 11). Sappino et a139 discussed development and evolution of myofibroblasts from normal stromal fibroblasts in reparative processes. They provided evidence for a continuous change of the intermediate filament and microfilament composition within the heterogeneous group of myofibroblasts. In this context, our cells in areas of fibrotic changes seem to represent VA-myofibroblasts containing vimentin and o1 -smooth muscle actin as described by Sappino et al. 39 Ito cells and myofibroblasts are expected to contribute to the collagen deposition in the Disse space in alcoholic liver injury.12,14 4041 In the rat, stimulated Ito cells are also regarded to represent cells "transitional" between Ito cells and myofibroblasts.11,4243 They show, in addition to desmin expression, overexpression of actin. 36 Myofibroblasts are a major cell type in pathologic processes associated with fibrosis and architectural remodeling, such as liver cirrhosis. 44 They are heterogeneous in their content of actin isoforms and intermediate filament proteins but consistently express high amounts of alsmooth muscle actin. 45 Therefore, (x -smooth muscle actin could serve as marker of an active fibrotic process. The recently reported observation that mRNA for laminin was detectable by in situ hybridization only in bile duct and ductular epithelia but not in hepatocytes46 is morphologically reflected by our observation that the encirculation of hepatocytes by laminin is mostly incomplete (see, however, the observations by Clement et al47 on laminin synthesis by hepatocytes in vitro). Therefore, it is most likely that laminin is produced by bile duct epithelia, and its deposition extends to the adjacent surface of the hepatocytes which finally could result in a complete encapsulation. As shown in previous reports41'47-52 fibronectin and laminin, besides collagen, are produced in large amounts in liver fibrosis53 leading to "capillarization"54 of liver sinusoids. A deposition of these glycoproteins along the sinusoids and around hepatocytes might build up a diffusion barrier inhibiting exchange processes between blood and hepatocytes with adverse effects on several hepatocytic metabolic activities.55 A disturbance of assimilatory functions of the hepatocytes could result in cell atrophy and degeneration. This is in line with our observation that several "isolated" hepatocytes within severely fibrotic areas or at the endpoints of parenchymal pillars revealed degenerative changes. The consecutive death and dropout of "isolated" hepatocytes would lead to a progressive loss of parenchyma with further deterioration of organ structure, even without alcoholic hepatitis.