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From the Liver Research Laboratories,*
Department of
Medicine, School of Biochemistry,
and the
Department of Pathology,¶
Children’s
Hospital,||
University Hospital, Birmingham, United
Kingdom; the Department of Medical and Surgical
Sciences,
Clinica Medica I, Università
di Padova, Padova, Italy; and the Istituto di Clinica Medica
II,§
Policlinico S. Orsola, Università di
Bologna, Bologna, Italy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,11
nitric oxide,12
and parathyroid hormone-related peptide,13
which is
probably involved in paracrine communications with mesenchymal and
inflammatory cells. Currently, four types of ductular reaction are described: typical, atypical, cholangiolar, and oval cell.14,15 Atypical ductular reaction is characterized by an anastomosing network of ductules, with poorly defined lumina, lined by flattened cells with a scant cytoplasm; atypical ductules are most often localized at the peripheral zone of the portal space. This occurs in most forms of chronic cholestatic liver injury, such as in primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and chronic extrahepatic biliary obstruction. Available data indicate that whereas typical reactive ductules derive from proliferation of the preexisting well-differentiated biliary epithelial cells,16 atypical ductules arise from ductular metaplasia of hepatocytes14,17 and/or from activation of a facultative bipotential stem cell,14,18 the actual identity of which in humans has not yet been determined. These cells, capable of differentiating into biliary epithelial cells and hepatocytes, are thought to be located in close proximity to the terminal bile ductules and to sustain liver regeneration under conditions such as fulminant hepatic failure that compromise the proliferative capability of hepatocytes and cholangiocytes.
The study of the biological characteristics of reactive ductular cells may lead to a better understanding of both the dynamics of epithelial cell populations in the liver and some basic liver pathophysiological mechanisms. The phenotypic profile of ductular cells has been partly characterized by histological studies: they are anchored on a basement membrane and can express major histocompatibility complex (MHC) class I proteins19,20 and cell adhesion molecules, such as carcinoembryonic antigen21 and intercellular adhesion molecule-1.22,23 Furthermore, recent data indicate that reactive ductules display neuroendocrine features, including immunoreactivity for chromogranin A and for the neural cell adhesion molecule (NCAM).24 NCAM is a surface glycoprotein, belonging to the immunoglobulin superfamily, that mediates cell-cell (homophilic) and cell-matrix (heterophilic) interactions during the development of the nervous system,25-28 lung, and gut epithelia.29
Given the role played by NCAM in morphogenetic processes, we have investigated its immunohistochemical expression and that of a number of markers relevant to morphogenesis and cell proliferation on the biliary epithelium in a wide variety of chronic hepatobiliary diseases and in fetal livers at different gestational ages. In particular, the B-cell leukemia lymphoma-2 protein (Bcl-2), a protooncogene product localized to the mitochondrial inner membrane,30 is of potential interest: its ability to block apoptosis is required in the developmental processes of new epithelial structures.31,32 Our results indicate that in congenital diseases related to ductal plate malformation and primary diseases of the biliary tree, atypical ductules retain less differentiated immunophenotypic features, such as coexpression of NCAM and Bcl-2, similar to those transiently expressed during the embryonic development of the biliary system. Furthermore, based on a positive selection of cells expressing NCAM, we have devised a method to purify a viable population of atypical ductular cells from cirrhotic human liver; ultrastructural studies of these isolated cells further support the less differentiated biliary phenotypic traits of NCAM-positive cells.
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Source of Tissue
Human tissue samples from hepatectomy specimens were snap frozen in liquid nitrogen and stored at -70°C. Normal liver tissue (n = 10) was obtained from graft reductions for pediatric liver transplant recipients. Diseased liver tissue was obtained at the time of the orthotopic liver transplantation; this included parenchymal liver cirrhosis (chronic autoimmune hepatitis, n = 5; cryptogenic cirrhosis, n = 5; alcoholic liver cirrhosis, n = 5), primary immune-mediated cholangiopathies (PBC, n = 12; PSC, n = 8), and congenital cholangiopathies (extrahepatic bile duct atresia, n = 5), congenital diseases of bile ducts related to ductal plate malformation (Caroli’s disease, n = 2; polycystic liver disease, n = 2). Diagnosis was made by clinical, serological, and histological criteria. Fetal livers (n = 10) from human embryos and fetuses from the 10th to 16th weeks of gestation were obtained, after informed consent, from legal voluntary abortions.
Classification of Intrahepatic Bile Ducts
In adult livers, intrahepatic biliary epithelium was categorized according to the method of Ludwig.33 Major ducts were accompanied by a hepatic artery branch and comprised both interlobular ducts (luminal diameter between 50 and 100 µm, lined by cuboidal cells) and septal ducts (luminal diameter greater than 100 µm, lined by a columnar epithelium). Bile ductules were the smallest conduits (less than 50 µm in diameter), were lined by cuboidal epithelial cells, and were not accompanied by the small branches of portal vein and hepatic artery. Typical or atypical reactive ductules were respectively classified according to the following histological criteria: lumen patency (discernible or no discernible lumen), morphology of the lining epithelium (cuboidal or flattened), and ductule profile (well-formed or irregular anastomosing plexuses).
In fetal livers development of biliary epithelium was categorized as reported by Van Eyken34 and Shah.35 During the early stages of embryonic development (from 6 to 8 weeks), hepatoblasts adjacent to mesenchyme around the largest portal vein branches close to the liver hilum alter their phenotype toward bile duct-type cells, giving rise to the primordial ductal plate (9–10th weeks).
Alkaline Phosphatase Immunostaining
Serial 5-µm cryostat sections were fixed in acetone at room
temperature, and immunohistochemistry was performed by the alkaline
phosphatase method, using monoclonal antibodies against human
epithelial antigen-125 (HEA-125), NCAM, epithelial membrane antigen
(EMA), and B-cell leukemia lymphoma-2 protein (Bcl-2) (see also Table 1
). The working dilution of each antibody
was determined in preliminary tests. After preliminary blocking with
normal rabbit serum (1:10; Dako), sections were incubated with primary
antibodies for 1 hour at room temperature. After incubation with the
primary antibody, sections were washed three times before incubation
with rabbit anti-mouse immunoglobulins (1:25; Dako) for 45 minutes.
After further washing, sections were incubated with alkaline
phosphatase mouse anti-alkaline phosphatase (APAAP) (1:50; Dako) for 45
minutes. Antibody binding was detected using fast red substrate (Sigma
Chemical Co., Poole, UK), and sections were counterstained with
Mayer’s hematoxylin (Sigma). In controls, primary antibody was
omitted. The sections were assessed by three independent observers (LF,
RJ, SGH) according to the classification based on bile duct morphology
given above.
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Double immunostaining was performed on selected sections with
different combinations of the following antibodies (see Table 1
):
HEA-125, NCAM, EMA, Bcl-2, and Ki-67 (MIB-1 clone, a nuclear antigen
associated with the cell cycle),36
and Texas red and
fluorescein isothiocyanate (FITC) (anti-IgG1 or anti-IgG2a, 1:50;
Europath, Bude, UK) conjugates as secondary fluorescent antibodies.
When primary antibodies belonged to different subclasses (such as
HEA-125 and NCAM, HEA-125 and EMA, HEA-125 and Ki-67, NCAM and Bcl-2)
conventional simultaneous labeling could be used: the two primary
antibodies were mixed, incubated together for 30 minutes, and then
visualized with a mixture of Texas red and FITC conjugates. When
primary antibodies shared the same subclass (such as HEA-125 and Bcl-2,
NCAM and EMA, NCAM and Ki-67), staining was performed using the reverse
sequential indirect method, in which the sequence of addition of
primary antibodies in duplicate immunostaining experiments is
reversed.37
FITC-conjugated human IgG from a liver kidney
microsome-1 (LKM-1)-positive patient (0.1 mg/ml in phosphate-buffered
saline (PBS)) was used as a hepatocellular marker, because it
homogeneously and specifically decorates the hepatocyte smooth
endoplasmic reticulum by reacting with a cytochrome P-450 monooxygenase
isoform.38-41
LKM-1 was used in combination with
HEA-12542
and NCAM,24
taken as biliary cell
markers. Sections were mounted in Diazabicyclo(2.2.2)octane (DABCO) to
retard the fading of the fluorochromes. In controls reactivity of each
conjugate with each primary antibody was tested to exclude the
possibility of subclass cross-reactivity.
Sections were analyzed by with a laser scanning confocal microscopy MCR-500 system, equipped with a krypton-argon laser (Bio-Rad), for coincident sites of reactivity. Fluorescence images were collected and analyzed in the dual-channel mode, with the laser lines at 488 and 568 nm. Under these conditions, single labeling appears red (Texas red) and green (FITC), whereas coincident labeling appears yellow. To measure the relative fluorescence intensity of antigen expression on reactive ductules, the fluorescence within a defined area occupied by biliary ductular cells was defined in bands of increasing intensity (from 0 to 255), where the highest values were shown in red (219–255) and the lowest appeared in blue (37–72); intensity below 36 was black and indicated unstained areas.
Isolation of NCAM-Positive Ductular Cells from Human Liver
Source of Tissue
Normal liver (n = 12) from the pediatric segmental transplantation procedures and cirrhotic liver (n = 12) from patients undergoing liver transplantation for PBC (n = 9) and PSC (n = 3) were used for cell isolation studies. Donor organs, perfused with University of Wisconsin surgical preservation fluid, and slices of cirrhotic liver, in tissue culture medium, were stored for up to 24 hours at 4°C before cell preparation.
Purification of Cells
Approximately 30 g of liver tissue was dissected, finely diced, and incubated at 37°C with 50 ml collagenase type 1A (1 mg/ml; Sigma Chemical Company). After 1–2 hours of incubation the digest was filtered through fine mesh (Sigma screen cup; Sigma Chemical Company), and the filtered tissue pieces were further diced to enhance the release of cells. Digested tissue pieces and filtrate were recombined and incubated for an additional 30 minutes to 1 hour at 37°C. After a total of 2–3 hours of digestion the tissue pieces were again filtered through fine mesh and the filtrate was washed three times in PBS. After the final wash the volume of filtrate was adjusted to 24 ml. Three milliliters of filtrate was overlaid on Percoll gradients, each composed of 3.5 ml 1.04 g/ml Percoll underlaid by 3.5 ml 1.09 g/ml Percoll and then centrifuged at 800 x g for 30 minutes at room temperature. After centrifugation, five separate fractions were evident. Each fraction was harvested separately, washed three times in PBS, and centrifuged at 800 x g for 30 minutes. Cell smears from the final cell pellet derived from each fraction were stained with the biliary specific HEA-125 and cytokeratin 19 (CK-19) antibodies, to establish where atypical cholangiocytes could be harvested. In addition, from each fraction, cells were immunopurified using HEA-125 as already described43,44 and further characterized by immunocytochemistry for CK-19, NCAM, and EMA. The supernatant and pellicle (fraction 1) that floated as a layer on top of the gradient were composed mainly of cell debris and hepatocytes. Some HEA-125/CK-19-positive cells were present, but cells in this fraction had very low viability (<20%). Cells equilibrating in 1.04 g/ml Percoll (fraction 2) and as a layer on top of 1.09 g/ml Percoll (fraction 3) were composed of single cells and cell aggregates, which were positive for HEA-125 and CK-19 and were >95% viable. In particular, fraction 2 was composed of single cells or small aggregates of 2–10 cells each. All were positive for CK-19 and, in cirrhotic liver, many (>50%) were positive for NCAM and negative for EMA. In contrast, HEA-125-positive cells equilibrating in fraction 3 were predominantly small to large aggregates of around 10–100 cells each. These cells were positive for CK-19 and EMA (>95% positive) but were largely negative for NCAM (<5% positive). Very few cells were present in 1.09 g/ml Percoll (fraction 4), whereas the pellet (fraction 5) appeared to be composed predominantly of erythrocytes. These preliminary studies indicate that atypical ductular cells fractionate predominantly in 1.04 g/ml Percoll, whereas mature cholangiocytes pass through this fraction and can be harvested from the layer of cells floating on 1.09 g/ml Percoll. Therefore, fractions 2 and 3 were pooled together for further sequential immunomagnetic separation of biliary cells expressing NCAM and HEA-125. Pooled fractions 2 and 3 were incubated with anti-NCAM (37°C for 30 minutes) followed by bead-conjugated rat anti-mouse IgG2 (Dynal, Oslo, Norway) (4°C for 30 minutes). After magnetic harvest of NCAM-positive cells, a subset of biliary epithelial cells was purified from the NCAM-negative population by immunomagnetic separation using HEA-125, as previously reported.43,44 The two purified biliary epithelial cell populations (NCAM-positive versus NCAM-negative, respectively) were characterized for immunocytochemistry and transmission electron microscopy (TEM).
Characterization of Purified Cells
Immunostaining: Smears of freshly isolated cells were immunostained with anti-HEA-125 (1:10), anti-CK19 (1:50, IgG2a isotype; Dako), anti-NCAM (1:10), and anti-EMA (1:50) monoclonal antibodies. Sites of primary antibody binding were visualized with sheep anti-mouse IgG2a (CK19, NCAM and EMA) conjugated to FITC (1:50). In controls primary antibody was omitted. Cells were counterstained with propidium iodide.
Transmission Electron Microscopy: After they were washed three times, purified cells were resuspended in 1 ml of PBS in 1.5-ml Eppendorf tubes. Magnetic beads and attached cells were concentrated for 2 minutes, using a Dynal multiEppendorf magnetic particle concentrator (Dynal). Once the cells had concentrated, PBS was replaced with 1 ml of 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer for 2 hours. Without removing the tubes from the magnet, so as not to disturb the concentrated cells, the fixed cells were washed gently three times in cacodylate buffer, osmicated in 1% OsO4 for 4 hours, dehydrated through graded alcohols, and embedded in Epon resin. Sections showing gold interference color (approximately 70 nm) were cut and mounted on Formvar/carbon-coated slot grids. They were stained with uranyl acetate and lead citrate and were examined using a Jeol 100x TEM.
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Normal Livers
Distribution of HEA-125, EMA, NCAM, and Bcl-2 alkaline phosphatase
immunostaining in normal liver tissue is summarized in Table 2
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Fetal Livers
The results of the immunostaining in fetal liver are shown in
Figure 1
. In fetal livers from 10–12
weeks of gestation (Figure 1a)
, ie, during the early stages of bile
duct development, NCAM was positive in most cells forming the
single-layered ductal plates surrounding a large portal vein branch. As
the ductal plate developed and the single layer was duplicated by a
second layer (14 weeks of gestation, Figure 1b
), NCAM immunoreactivity
began to disappear from those segments in which a lumen could be
recognized and started incorporating into mesenchyma (16 weeks of
gestation, Figure 1c
). In contrast, EMA was never seen on
single-layered ductal plates and gave a faint staining only in the
duplicated segments forming tubular structures (not shown). Double
immunostaining using NCAM (Texas Red conjugate) and Bcl-2 (FITC)
demonstrated that NCAM-positive cells forming a ductal plate also
expressed Bcl-2, as shown by dismerged images (Figure 1d)
. These cells
were also positive for HEA-125. Finally, NCAM-positive ductal plate
cells (Texas Red conjugate) showed no evidence of active proliferation,
as judged by the negative double immunolabeling with Ki-67 (FITC) (not
shown). A widespread, strong Ki-67 expression was instead noticed on
the surrounding parenchymal and hemopoietic cells. These findings
indicate that NCAM and Bcl-2 positivity identifies immature biliary
cells at an early and transient developmental stage, which
is also characterized by a low rate of proliferation. A focal
positivity for Ki-67 could be observed in NCAM-negative cells lining
bile ducts with a clear tubular morphology.
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The results of the immunostaining in a variety of chronic liver
diseases are reported in Table 2
. NCAM and EMA immunoreactivity was
compared to the biliary epithelial cell marker HEA-125 (Figure 2a–f)
; whereas HEA-125 homogeneously
stained all biliary epithelial structures (Figure 2
, a and d), EMA and
NCAM showed an inverse pattern of distribution, similar to that
reported above for fetal livers. EMA (Figure 2
, b and e) was positive
in major ducts and typical ductules that were negative for NCAM,
whereas NCAM (Figure 2
, c and f) was consistently positive in ductules
with atypical morphology that were negative for EMA.
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. Major ducts and typical ductules,
showing focal NCAM/Bcl-2 double positive cells, were observed only
occasionally (5–10%) (not shown).
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, b–f, atypical ductular cells coexpressing NCAM
and Bcl-2 were seen particularly in immune-mediated cholangiopathies
(PBC and PSC), in biliary atresia, and in congenital diseases related
to ductal plate malformations (Caroli’s disease, polycystic liver
disease). In a number of cases representative of the range of liver
diseases studied (biliary atresia: n = 2; PBC:
n = 2; PSC: n = 1; cryptogenic
cirrhosis: n = 2; autoimmune hepatitis:
n = 1; alcoholic cirrhosis: n = 1), we
calculated the ductular area positive for NCAM as a percentage of the
total ductular area recognized by HEA-125. As summarized in Table 3
, the most extensive immunoreactivity
for NCAM was found in biliary atresia and PBC; in PSC the proportion of
NCAM-positive ductules was still higher than in cryptogenic cirrhosis,
alcoholic cirrhosis, and autoimmune hepatitis. It is noteworthy that
whereas in cholestatic liver diseases NCAM and Bcl-2-positive atypical
ductules were present both at the center and at the margin of portal
tracts (Figure 3
, c–f, yellow), in parenchymal liver cirrhosis NCAM
and Bcl-2 coincident staining was restricted to a small proportion of
ductules, at the margin between the regenerative hepatocyte nodule and
the fibrous septa (Figure 3b
, yellow).
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To study the histological relationship between hepatocytes
and NCAM-positive cholangiocytes, double immunostaining for the biliary
markers NCAM or HEA-125 and the hepatocellular marker LKM-1
was performed for a range of liver diseases (Caroli’s disease:
n = 2; polycystic liver disease: n = 2;
PBC: n = 4; PSC: n = 1, cryptogenic
cirrhosis: n = 3; autoimmune hepatitis:
n = 2; alcoholic cirrhosis: n = 3). In
all types of liver diseases studied, NCAM-positive cholangiocytes (red)
formed periportal sprouting cords organized into irregularly shaped and
poorly formed ductules that directly anastomosed with periseptal
LKM-1-positive hepatocytes (Figure 4a
,
green). Double labeling of NCAM with LKM-1 revealed no sites of NCAM
coexpression on the plasma membrane of LKM-1-positive hepatocytes;
however, some discrete intracytoplasmic granules positive for NCAM
could be observed in periseptal hepatocytes that were positive for
LKM-1. In contrast, double labeling for HEA-125 (red) and LKM-1 (green)
showed that periportal hepatocytes, which were negative for NCAM, as
judged in serial consecutive sections (Figure 4b)
, began to express
HEA-125 plasma membrane immunoreactivity (Figure 4c
, yellow). This
finding was most commonly observed in parenchymal liver cirrhosis,
particularly in alcoholic liver disease.
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Differential Immunomagnetic Purification of Immature versus Mature Biliary Epithelial Cells and Their Characterization by TEM
Anti-NCAM antibody was used to purify atypical ductular cells from
the Percoll fractions 2 and 3, which were combined to increase the cell
yield. The harvested cells were single cells or small aggregates
composed of ~2–10 cells each and were positive also for HEA-125 and
CK19, but were negative for EMA (not shown). Whereas NCAM-positive
cells could be harvested from cirrhotic liver
(103
to 104
NCAM-positive
cells/g of cirrhotic liver), none could be isolated from normal liver,
thus reflecting the immunohistochemical finding. TEM demonstrated that
these cells were characterized by a rounded shape and a scant
cytoplasm, showed only few short microvilli, had a poor intracellular
organization, and lacked polarity (Figure 5a)
, consistent with an immature
epithelial phenotype.
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The normal biliary epithelium was homogeneously stained by HEA-125 and EMA, but was consistently negative for NCAM. In our hands Bcl-2 was not expressed on small ducts from normal livers, which is consistent with early reports from Frommel,49 Arora,50 and Gapany.51 These findings are at odds with those reported by Charlotte52 and Skopelitou.53 Differences in the source of control tissue are likely the cause of these different findings. In Charlotte’s study normal tissue was taken from hepatectomies performed for liver cancer, a source that may not be considered appropriate.54 However, any uncertainty about whether bile ducts in normal liver are positive for Bcl-2 does not detract from our observation that NCAM-positive ductular cells in fetal and diseased liver were also Bcl-2-positive.
In fact, in contrast with normal biliary epithelium, NCAM and Bcl-2 were transiently expressed on immature biliary cells at a very early developmental stage of the intrahepatic biliary tree (10th week); these cells, which were positive for HEA-125 but negative for EMA and Ki-67, were organized into a single-layered ductal plate, still lacking a lumen. Over the following weeks, when ductal plates duplicated and began to assume the shape of a tubular structure lining a slit-like circular lumen, they gradually lost NCAM and Bcl-2 expression, began to express EMA, and showed a proliferation activity. These data are consistent with those of Terada and Nakanuma,55 who reported a relatively high rate of proliferation and apoptosis when ductal plates began to be incorporated into the mesenchyma (remodeling ductal plate). Conversely, the single-layered ductal plate showed poor evidence of active proliferation in Terada’s study, as well as in our own. Negativity of ductal plate for proliferation markers at the 12th week has also been reported by Cocjin.56 An intense proliferation activity was instead observed in hepatoblasts, consistent with the hypothesis that ductal plate is formed via a phenotypic shift from hepatoblasts to biliary cells.17,34 Similar to the progressive expression of additional cytokeratin types (first CK-19 and then CK-7), NCAM, Bcl-2, and EMA may thus be considered as differentiation markers along the biliary cell lineage.
In reactive ductules from diseased livers, we have observed a pattern
of expression similar to that found in fetal livers. In these
conditions immunoreactivity for NCAM and Bcl-2 and negativity for EMA
identified ductules with an atypical morphology. Instead, typical
reactive ductules were positive for EMA, a marker of terminally
differentiated biliary cells,57
further indicating that
typical ductules retain the phenotypic properties of their normal
counterparts.58
Ductules coexpressing NCAM and Bcl-2 were
particularly represented in diseases of ductal plate malformation, such
as polycystic liver disease and Caroli’s disease. In these
developmental liver diseases, ductal plate remodeling is arrested
during the embryonic development, causing the persistence of an excess
of bile ductules in a ductal plate configuration with immunophenotypic
features of immaturity.59
Similar to ductal plate
malformations, in extrahepatic bile duct atresia and in immune-mediated
cholangiopathies (PBC and PSC), most reactive ductules were positive
for NCAM and Bcl-2 and were distributed throughout the portal tracts,
whereas in parenchymal liver cirrhosis ductules that reacted with NCAM
and Bcl-2 were much less represented and were confined to the interface
between the portal space and the regenerative nodule. The different
pattern of extension of NCAM reactivity on reactive ductules between
cholestatic and parenchymal liver diseases (see Table 3
and Figures 2 and 3
), particularly their wider distribution throughout the portal
space in primary cholangiopathies, suggests that retention of bile
constituents is a strong stimulus for atypical ductular reaction.
Interestingly, in contrast with experimental bile duct ligation, where
proliferation of preexisting biliary epithelial cells gives rise to
well-differentiated bile ducts, under these conditions ductular
structures appeared unable to form mature ducts.
The colocalization of NCAM with Bcl-2 in atypical ductules from diseased livers may be related to the role of these two proteins in morphogenesis. NCAM is known to mediate the establishment of cell-cell and cell-matrix interactions26,27,60 ; homophilic and heterophilic interactions through NCAM are crucial to regulate the migration of cells and the differentiation of tissues in the embryo. For example, when neural crest cells stop producing NCAM and N-cadherin and start displaying integrin receptors, they can separate and begin to migrate on the extracellular matrix.61 In a similar manner, in fetal livers, NCAM may play a role in mediating the phenotypic shift from hepatoblasts to ductal plate cells,17,34 induced by the mesenchyme that surrounds the portal vein branches.62 On the other hand, Bcl-2 may favor survival and clustering of immature cells that are committed to the formation of more differentiated structures.31 Bcl-2 is known to be expressed in several fetal tissues (skin, tracheobronchial tree, kidney), at sites characterized by inductive interactions between epithelial and mesenchymal structures, and it may play a role not only in blocking apoptosis (a major mechanism in tissue remodeling) but also in the commitment process of cells engaged in the formation of new structures.31,32 Thus coexpression of NCAM and Bcl-2 on "atypical" ductules after chronic liver damage is likely induced by changes in extracellular matrix and identifies immature biliary cells with prolonged survival advantage, because they are involved in active tissue remodeling processes.
Our data indicate that, in contrast to periportal hepatocytes, atypical reactive ductules coexpressing NCAM and Bcl-2 were not actively proliferating, as shown by negative double labeling of NCAM with Ki-67. These expression patterns resemble those seen early in developing ductal plates from 10 to 16 gestation weeks. Early Bcl-2 expression is a well-documented event in the process of metaplasia, as reported in Barrett esophagus63 and gastric intestinal metaplasia.64 In particular, in gastric intestinal metaplasia double staining for Bcl-2 and Ki-67 revealed that Bcl-2 was not expressed by proliferating epithelial cells, but was present in cells undergoing metaplasia,64 as reported here for NCAM-positive atypical ductular cells. By the use of computerized three-dimensional reconstructions of the biliary system, atypical ductules were shown to be in communication with liver cell plates but not with preexisting bile ducts and ductules.65 Both the flattened shape and the network arrangement of atypical ductules are difficult to explain as a result of proliferation of preexisting ducts and are more consistent with an origin in liver cell muralia66 or cells located at the canal of Hearing. This hypothesis is further supported by double staining of NCAM with the hepatocellular marker LKM-1. In all types of liver disease studied, NCAM-positive atypical ductules appeared to be in close proximity to LKM-1-positive hepatocytes at the margin of the portal tracts; immunoreactivity for NCAM was present in the cytoplasm of some periportal hepatocytes, but cells coexpressing LKM-1 and NCAM on the plasma membrane were not observed. In parenchymal liver cirrhosis, and particularly in alcoholic liver disease, double staining with HEA-125 and LKM-1 revealed a coincident labeling in some periportal hepatocytes, but these structures were negative for NCAM, indicating that NCAM expression is associated with loss of hepatocellular phenotype.
Thus, ductular reactive cells, protected from apoptotic events, but not
actively proliferating, express neuroendocrine features, including
immunoreactivity for NCAM; some of these features are reminiscent of
the early phases of biliary ontogenesis. There are a number of
similarities and differences between ductal plate cells and ductular
cells, and these are emphasized in Table 4
. A major difference is that in ductular
reaction CK7 appears in metaplasic hepatocytes before the expression of
CK19,17,34
a sequence of events opposite that observed
during embryogenesis of biliary epithelium that first expresses CK19
and later CK7. Furthermore, it is unknown whether atypical reactive
ductules express the hematopoietic stem cell markers CD34, CD33, and
c-kit, the expression of which on ductal plate cells during the early
stages of the embryonic development has been reported.67
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Thus the data support the concept that atypical ductular reaction in
chronic liver diseases recapitulates a program similar to the early
stages of biliary ontogenesis, when NCAM-positive ductal plate cells
are formed by hepatoblasts undergoing a phenotypic switch. Thus, as
proposed by Van Eyken,68
ductular reactive cells may
derive from a ductular metaplasia of periportal hepatocytes. In fact,
in addition to the so-called single-step or direct metaplasia,
involving the direct switch of one overtly differentiated cell type to
another, metaplasia may also occur as a multistep process. This is
characterized by the appearance of intermediate cells, which possess a
less differentiated phenotype but are committed to a different cell
lineage (indirect metaplasia).68-70
NCAM-positive ductal
plate cells in embryogenesis and NCAM-positive ductules in chronic
liver diseases may be the intermediate cells (hepatoblasts 
NCAM-positive ductal plate cells mature cholangiocyte). Our data do
not exclude the presence of liver stem cell or a role for liver stem
cells in generating the cells that ultimately become NCAM-positive
ductules and, later, mature duct cells. From this point of view, the
less differentiated characteristics and the lack of proliferation
activity of the ductular cells coexpressing NCAM and Bcl-2 are
consistent with the possibility they are the descendants of a small
progenitor cell compartment, which could persist in the postnatal liver
in close proximity to the finest ramifications of the biliary tree.
Whether these progenitor cells give rise to periportal hepatocyte-like
cells that may differentiate in hepatocytes and/or in biliary cells, or
whether the progenitor cell itself differentiates directly either into
hepatocytes or bile duct cells cannot be addressed in the present
study. It is interesting to note that, as already reported for
hepatoblasts,71
in diseased livers (see Figure 4c
)
periportal hepatocytes are positive for the cholangiocyte marker
HEA-125, an epithelial cell surface antigen homologous to the matrix
adhesion protein nidogen.72
In chronic cholestatic liver
disease, maturation of NCAM-positive ductular cells seems hampered,
leading to the accumulation of less differentiated atypical ductules
accompanied by the progressive disappearance of mature ducts that
cannot be replaced (as seen, for example, in biliary atresia).
Regardless of their cell origin, the potential of these reactive ductular cells remains of great relevance. In fact, the ability to purify two subpopulations of immature and mature ductular cells is a novel technique that is likely to have an important impact on the study of biliary cell pathophysiology. This method will make it possible to study the role of reactive ductular cells in the production of several paracrine factors involved in regenerative and fibrotic processes, and to clarify the effects of different matrices on the growth properties and differentiation potential of these cells.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Children’s Liver Diseases Foundation (to H.A.C.), Endowment Fund of United Birmingham Hospitals (to J.M.N. and R.J.), and from Telethon (Grants E-430 and E-873) and Cofinanziamento MURST 1998 (Grant 9806210866) (to L.F. and M.S.).
Accepted for publication January 12, 2000.
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