American Journal ofPathology, Vol. 137, No. 5, November 1990

Copyright © American Association of Pathologists

Rapid Communication Neuroendocrine Features of Reactive Bile Ductules in Cholestatic Liver Disease Tania Roskams, Joost J. van den Oord, Rita De Vos, and Valeer J. Desmet From the Department of Pathology, Laboratory for Histoand Cytochemistry, University Hospital St. Rafael,

Catholic University of Leuven, Leuven, Belgium

Various cholestatic liver diseases are accompanied by a striking increase in the number of bile ductules. This so-called ductular reaction is thought to arise both from ductular metaplasia of hepatocytes and from proliferation of pre-existing bile ductules. Previous studies have shown that these reactive bile ductules differ from their normal counterpart in enzyme and immunohistochemical make-up. Using monoclonal antibodies directed to neuroendocrine markers and immunohistochemistry, wefound that reactive bile ductules in cholestatic liver disease display neuroendocrinefeatures. In all cases ofprimary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), extrahepatic obstruction, and acute hepatitis A, reactive bile ductules expressed the neural cell adhesion molecule (N- CAM) and reacted with monoclonal antibody A2B5. Both N- CAM and the ganglioside, recognized by A2B5, are restricted to neuroendocrine cells and tissues. In all but four of these cases, the same bile ductules expressed chromogranin-A, present in the matrix of neuroendocrine granules. Furthermore, in three cases of longstanding cholestasis, scattered periportal hepatocytes expressed chromogranin-A but not N- CAM. Other neuroendocrine markers such as neuron-specific enolase, synaptophysin, or myelinassociated glycoprotein were lacking from both bile ductules and hepatocytes. The neuroendocrinephenotype of bile ductules and hepatocytes was confirmed on electronmicroscopy, demonstrating various numbers of dense-cored, neuroendocrine granules near the peripheral cell membrane in bile ductules as well as in cells intermediate between hepatocytes and bile ductular cells. In 10 cases of normal liver tissue without ductular reaction, bile

ductules lacked neuroendocrine markers except in two cases in which very weak reactivity for chromogranin-A was observed. Thesefindings illustrate the presence of a new, neuroendocrine cell type that emerges in the liver during cholestasis. Elucidation of the significance of the neuroendocrine substance(s) produced in the dense cored granules of reactive bile ductules awaits their isolation and characterization. We can speculate that this molecule plays an autocrine or paracrine regulatory role in the process of ductular metaplasia of hepatocytes or growth of bile ductules. (Am JPathol

1990, 137:1019-1025) Various cholestatic liver diseases are associated with a reaction of biliary epithelial cells resulting in a striking increase in the number of bile ductules (ductular reaction). This reversible modulation of the intrahepatic biliary tree is most typically seen in bile duct obstruction and creates a labyrinth of small ductular branches that extend around the portal tracts where they add to the development of periportal fibrosis in chronic cholestasis.1 2 Previous studies have shown that reactive bile ductules are phenotypically different from original bile ductules.1 In this study, we present immunohistochemical and electronmicroscopic evidence that reactive bile ductules display neuroendocrine features.

Materials and Methods Sixty-seven liver biopsies formed the basis of this study. Their histologic diagnoses are listed in Table 1. All specimens were received freshly and divided into two or three parts. The largest part was fixed in Bouin's solution or B5fixative and used for routine light microscopy. One part was snap frozen in liquid nitrogen-cooled isopentane, Accepted for publication September 5, 1990. Address reprnt requests to Dr. Tania Roskams, Universitair Ziekenhuis St. Rafa6l, Laboratonum voor Histo- and Cytochemie, Minderbroedersstraat 12, B-3000 Leuven, Belgium.

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Table 1. Expression of LEU- 19 and Chromogranin Number of cases

Histologic diagnosis Cases with Ductular Reaction Primary biliary cirrhosis Extrahepatic obstruction Primary sclerosing cholangitis Liver tissue adjacent to tumor Acute hepatitis A Cases Without Ductular Reaction Chronic hepatitis B Pure (intrahepatic) cholestasis Near-normal liver tissue

23 19 3 5 3

4 2 8

stored at -700C, and used for immunohistochemistry. In four cases, small parts were fixed in glutaraldehyde, postfixed in Os04, and used for electron microscopy. For immunohistochemistry, 5-Mm semiserial cryostat sections were dried overnight at room temperature, fixed in acetone for 10 minutes, and stained with a three-step indirect immunoperoxidase method using the mouse monoclonal antibodies listed in Table 2. Monoclonal antibodies to cytokeratins 7 and 19 also were used because these antibodies have been found to decorate bile ductular structures in liver tissue.8 Incubation with primary monoclonal antibody was followed by peroxidase-labeled rabbit anti-mouse and peroxidase labeled swine anti-rabbit immunoglobulins (1g), respectively. All secondary and tertiary antisera were obtained from Dakopatts (Copenhagen, Denmark) and diluted in phosphate-buffered saline (PBS) pH 7.2 containing 10% normal human serum. All incubations were carried out for 30 minutes at room temperature and followed by a wash in three changes of PBS, pH 7.2 for 15 minutes. The reaction product was developed with the use of 3-amino-9-ethylcarbazole and H202. Controls, which revealed only inflammatory cells with endogenous peroxidase, consisted of replacement of priTable 2. Monoclonal Antibodies Used in Working Monoclonal antibody dilution Anti-Chromogranin-A 1:10 (clone LK2H10) Anti-synaptophysin 1:20 (clone SY38) Leu 19 1:160 NKH-1

Leu 7 HNK-1

A2B5 Anti-NSE (clone MIGN3) Anti-cytokeratin 7 Anti-cytokeratin 19

Chromogranin+

LEU 19+

19 19 3 5 3

23 19 3 5 3

0 0 2

0 0 0

mary antibody by nonimmune mouse ascites (Cappel Laboratories, Cochranville, PA). Results In normal liver tissue, small bile ductules were hardly visible on routine haematoxylin and eosin staining; immunostain-

ing for cytokeratins 7 and 19 revealed very few bile ductules that lacked reactivity for any of the other antibodies, except in two cases in which weak chromogranin A reactivity was observed. In all liver diseases without ductular reaction, reactivity for Leu-19 and NKH-1 was restricted to nerves in larger portal tracts and scattered nerve fibers and mononuclear cells in the lobular parenchyma; occasional chromograninA-positive cells with a triangular shape were found in the otherwise unreactive epithelium lining medium-sized and larger interlobular bile ducts. In liver specimens with ductular reaction, reactivity for Leu-1 9 and NKH-1 was observed as an intense membranous staining on proliferating bile ductules (Figure 1 A); in all but four of these cases, the same bile ductules also

the Present Study Reactivity Chr. A in matrix of NE granules

Source Clonatec

NE vesicle membranes

Biotest

Neural cell adhesion molecule

Becton-Dickinson

5, 11

Coulter Immunology

5, 11

Reference 3 4

(N-CAM) 1:10

1:2.5 1:10 1:100

Neural cell adhesion molecule

(N-CAM)

1:10

Myelin associated glycoprotein Myelin associated glycoprotein Ganglioside displayed by neurons and NE cells and tissues Glycolytic isoenzyme of enolase

Becton-Dickinson Monosan Dr. Eisenbarth Boston, USA Monosan

1:5 1:10

Cytokeratin 7 Cytokeratin 19

Amersham International Amersham International

6 6 7 17 8 8

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E.

Figure 1. Bile ductular reaction in a case of PBC. N-CAM (A) as well as chromogranin-A (B) are expressed by reactive bile ductules. Three-step indirect immunoperoxidase with (A) Leu- 19; (B) LK2H10; counterstained with Harris'haematoxylin. Original

magnification,

X800.

expressed chromogranin-A in the form of a granular, cytoplasmic staining (Figure 1 B). Both anti-chromograninA and anti-NCAM antibodies occasionally revealed reactivity only in part of the ductular cells in areas of ductular reaction. Serial sections revealed that the chromogranin and NCAM-positive structures corresponded to bile ductules because they also contained cytokeratins 7 and 19. In the cases in which serial sections were stained for synaptophysin, HNK-1, Leu-7, NSE, and A2B5, the proliferating bile ductules were reactive with monoclonal antibody A2B5 only. A diffuse cytoplasmic positivity for chromogranin-A was observed in scattered hepatocytes in three cases of PBC and in one case of long-standing extrahepatic obstruction (Figure 2). These hepatocytes were preferentially situated

in the periportal regions and lacked Leu-19 immunoreactivity. Electronmicroscopy, performed in three cases of PBC and one case of liver tissue adjacent to tumor, revealed lightly stained cells in the portal tract, arranged in small ductules and cords (Figure 3). These cells presented a round nucleus with a thin peripheral rim and small clumps of heterochromatin. Their cytoplasm contained few organelles: round to oval mitochondria, small short cisternae of the granular endoplasmic reticulum, very small smooth vesicles, and some small lysosomes located at the apical pole. In addition, these cells presented a few small dense cored granules that preferentially showed a peripheral localization. Bundles of intermediate filaments were scattered throughout the cytoplasm. Epithelial cells presenting

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Figure 2. In addition to reactive bile ductules in the portal tract, a cluster ofperiportal hepatocytes (arrows) expresses chromograninA. Three-step immunoperoxidase with LK2H10, counterstained with Harris' haematoxylin. Original magnification, X 180.

features of both hepatocytes and bile duct cells in variable proportions were found in the parenchyma in close vicinity to the portal tract (Figure 4). Hepatocyte characteristics included glycogen rosettes, abundant mitochondria with short cristae, and many endoplasmic reticulum cisternae; bile duct characteristics included interdigitations of their adjoining lateral membranes, the presence of basement membranes, and many thick bundles of tonofilaments. These cells occasionally contained one or more small dense cored granules, localized preferentially near the peripheral membrane.

Discussion Using immunohistochemistry and electronmicroscopy, we demonstrated that the bile ductules involved in the ductular reaction in chronic cholestatic liver diseases display a neuroendocrine phenotype. In more than 80% of cases, proliferating bile ductules but not the original portal bile ducts showed immunoreactivity for chromogranin-A. The identification of chromogranin-A-positive structures as bile ductules was confirmed by their expression of cytokeratins

7 and 19, previously found to be specific for bile duct structures.8 Chromogranin-A is a molecule present in the matrix of neuroendocrine granules where it is thought to play a role in the packaging and/or processing of certain peptide hormones and neuropeptides, in stabilizing the granule contents, and/or in decreasing cytosolic calcium. Chromogranin-A may have extracellular roles as well because the intact protein or proteolytic fragments derived thereof exert biologic activities on target cells, eg, as a prohormone of pancreastatin.9 In contrast to other molecules, chromogranin-A has been found to be a highly reliable marker for neuroendocrine cells.9' 10 The neuroendocrine character of reactive bile ductules was furthermore supported by their reactivity with monoclonal antibodies Leu-19, NKH-1, and A2B5. The latter monoclonal antibody has been raised against thymic epithelium and presumably reacts with a ganglioside, displayed by neurons and a number of APUD cells.7 Leu-19 and NKH-1 react with different, although related epitopes on the neural cell adhesion molecule (N-CAM).11 Differential expression of N-CAM on either a temporal or a topographic basis is of fundamental importance to neuro-ontogenesis.

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Figure 3. Electron micrograph of part of a portal tract. A small ductule (arrou') and a ductular cell (arrowhead) is apparent. Magnification, X3680. Inset (higher magnification offramed area in Figure 3) shows dense cored graniule (arrow) in a ductular cell (magnification, X36,800).

Although its cell distribution is widespread in early embryonic life, N-CAM shows a more restricted distribution in adult tissues, as it is limited mainly to neural cells and endocrine cells.12 14 The presence of N-CAM on reactive bile ductules is in line with their neuroendocrine phenotype; furthermore this molecule may play a role in modulating the extent of bile ductules in portal connective tissue because N-CAM has been shown to be involved in cellmatrix interactions.15 Other less sensitive neuroendocrine markers, displayed by some, but not all neuroendocrine cells, were lacking. Reactive bile ductules did not react with monoclonal antibodies Leu7 and HNK-1, which identify the myelin-associated glycoprotein. Similarly no immunoreactivity was observed for synaptophysin, a molecule present in synaptic vesicles. Although both antigens are found in many neuroendocrine cells and tissues, their absence does not argue against a neuroendocrine differentiation.16 NSE is a glycolytic enzyme found in neural cells as well as in the cells of the diffuse neuroendocrine system. Although of limited specificity, NSE is used as a broad-range marker that reacts with most neuroendocrine cells and neoplasms. In a previous immunohistochemical study, proliferating bile

ductules were found to express NSE 17; our negative findings need further study but are probably due to technical reasons (eg, type of fixation and antibody). The immunohistochemical evidence for a neuroendocrine phenotype was confirmed by the electron microscopic demonstration of dense cored secretory granules in reactive bile ductular cells. Previous electron microscopic studies focused mainly on the effect of biliary constituents on the ultrastructural appearance of proliferating bile ductules but have failed to identify dense cored granules in these cells.18 This may be due to the relative scarcity of these granules. In addition to reactive bile ductules, scattered hepatocytes in four cases of longstanding cholestasis were found to express chromogranin-A; electronmicroscopy in three of these cases confirmed the presence of neuroendocrine granules in their cytoplasm. This hitherto unrecognized endocrine differentiation of hepatocytes is presumably related to their tendency in chronic cholestasis to undergo a phenotypic switch toward bile ductular cells.' Previous immunohistochemical studies have shown that proliferating bile ductules differ from normal bile ductules in their enhanced expression of alkaline phosphatase,

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X 1XE~~~~ Figure 4. Llectron micrograph of lwzer parcnchj ma in close i'icinit)' to the portal tract. Epithelial cells presenting features of both hepatocytes and bile duct cells containl some dense cored granules (arrow); B, basement membrane; G, glycogen; F, filaments (magnification, X28.750)9

leucine aminopeptidase, alpha-fetoprotein, and bloodgroup H antigen.' The present study adds chromograninA and N-CAM to their phenotype and thereby identifies a neuroendocrine population of cells that emerges in chronic cholestatic liver disease, and that represents a new member of the diffuse neuroendocrine system. Apart from scattered triangular cells in the epithelium of medium-sized and larger bile ducts, normal liver tissue lacked significant chromogranin-A and N-CAM reactivity. This finding correlates with previous studies using immunohistochemical, immunoblotting, and in situ hybridization techniques.19 20 The scattered chromogranin-Aand N-CAM-positive cells in medium-sized and larger bile ducts have been described previously and found to synthesize various hormones.21 The existence of a well-developed peribiliary capillary plexus led to the hypothesis that biliary epithelial cells subserve an endocrine function and that these cells might release biologically active peptides in the surrounding liver tissue or in the general circulation.22 In two of eight normal liver specimens, weak chromogranin-A reactivity was found in the cytoplasm of a very few bile ductules. Although this may be due to the inclusion of two cases with minimal ductular reaction in the group of cases of nearnormal liver, this finding might also indicate that bile duc-

tules already express a neuroendocrine phenotype under normal conditions and that these characteristics are upregulated during the process of ductular reaction. Ductular reaction is currently believed to be caused by proliferation of pre-existing bile ductules on the one hand, and metaplasia of hepatocytes toward bile ductular cells on the other hand.'23 It is possible that a hitherto unknown molecule, present in the neuroendocrine granules of bile ductules, somehow regulates this process of ductular reaction. Because biliary epithelial cells are required for the longterm survival of hepatocytes in culture,24 this molecule also may be involved in the growth and metabolic function of hepatocytes. Identification of the exact nature of the stored molecule may reveal more insight in the origin and function of ductular structures involved in the ductular reaction in cholestatic liver diseases.

References 1. Desmet VJ: Modulation of biliary epithelium. In Reutter W, Popper H, Arias IM et al, eds. Modulation of Liver Cell Expression. Lancaster, England, MTP Press Limited, 1987, pp 195-214 2. Desmet VJ: Current problems in diagnosis of biliary disease and cholestasis. Semin Liver Dis 1986, 6:233-245

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3. Lloyd RV, Wilson BS: Specific endocrine marker defined by a monoclonal antibody. Science 1983, 222:628 4. Wiedenmann B, Franke WW: Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38000 characteristic of presynaptic vesicles. Cell 1985, 41: 1017-1028 5. Lanier LL, Testi R, Bindl J, Phillips JH: Identity of Leu-19 (CD 56) leukocyte differentiation antigen and neural cell adhesion molecule. J. Exp Med 1989, 169:2233-2238 6. Lipinski M, Braham K, Caillaud JM, Carlu C, Turz T: HNK-1 antibody detects an antigen expressed on neuroectodermal cells. J Exp Med 1983, 158:1775 7. Eisenbarth GS, Shimizu K, Conn M, Miutler B, Wells S: Monoclonal antibody Fl 2 A2B5: Expression on neuronal and endocrine cells. Monoclonal antibodies to neural antigens. Cold Spring Harbor Symposium, 1981, pp 209-218 8. Van Eyken P, Sciot R, Van Damme B, De Wolf-Peeters C, Desmet VJ: Keratin immunohistochemistry in normal human liver. Cytokeratin pattern of hepatocytes, bile ducts and acinar gradient. Virchows Arch A 1987, 412:63-72 9. Simon JP, Aunis D: Biochemistry of the chromogranin A protein family. Biochem J 1989, 262:1-13 10. Wiedenmann B, Huttner WB: Synaptophysin and chromogranins/secretogranins-Widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis. Virchows Arch (Cell Pathol) 1989, 58:95-121 11. Pietsch T, Hadam MR: Epitope analysis of the N-CAM/Leu19 molecule (CD 56) using mAb T-1 99. In Knapp W, Dorken B, et al, eds. Leucocyte Typing IV. Oxford, Oxford University Press, 1989, p 704 12. Crossin KL, Chuong CM, Edelman GM: Expression sequences of cell adhesion molecules. Proc Natl Acad Sci USA 1985, 82:6942 13. Murray BA, Owens GC, Prediger EA, Crossin KL, Cunningham BA, Edelman GM: Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA

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