Departments of 1Internal Medicine and 2Medical Physiology, 5Division of Research and Education, Scott & White Clinic and The Texas A&M University System Health Science Center, College of Medicine, and 3Central Texas Veterans Health Care System, Temple, Texas 76504; 4Division of Gastroenterology, Tohoku University School of Med, Aobaku, Sendai 980-8574, Japan; and 6Division of Gastroenterology, University of Rome, "La Sapienza," Rome 00185, Italy
Submitted 25 October 2002 ; accepted in final form 10 March 2003
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ABSTRACT |
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bile flow; intrahepatic biliary epithelium; proliferation; secretin
In normal physiological conditions, cholangiocytes are mitotically quiescent (3, 32) but undergo proliferation/loss in response to injury/toxins such as bile duct ligation (BDL) (3, 5, 19, 30), partial hepatectomy (32), or acute carbon tetrachloride (CCl4) administration (34). Cholangiocyte proliferation [e.g., after BDL (3, 5, 19, 30) or partial hepatectomy (32)] is associated with increased secretin-stimulated ductal secretion (3, 5, 20, 30, 32), whereas cholangiocyte loss (e.g., after acute CCl4 administration) is associated with a loss of secretin-induced cholangiocyte secretion (34). Cholestasis, defined as impaired bile flow, can be due to functional impairment of either hepatocyte or cholangiocyte function (5, 6, 13). Chronic cholestasis is a feature of many diverse chronic liver diseases such as biliary strictures (52), sclerosing cholangitis (27), and biliary (51) or pancreatic (11) malignancies. Recent studies have provided insights into the role of impaired canalicular membrane transport function as well as the effects of impaired bile flow and bile salt accumulation on hepatocyte injury (21, 28, 29, 53). Although cholangiocyte injury and dysfunction may also contribute to cholestasis, the mechanisms of cholangiocyte injury during chronic cholestasis remain unclear.
Death receptors such as TNF- are members of a super family
characterized by intracellular domains that mediate death in response to
extracellular stimuli. TNF-
plays a critical role in epithelial cell
injury as well as in immune-mediated cholangiocyte injury
(44). Immune mediated injury
has been implicated in the pathogenesis of chronic cholestatic diseases
affecting the biliary tract such as primary biliary cirrhosis or primary
sclerosing cholangitis (6).
Systemic levels of TNF-
are increased following biliary obstruction in
mice (12). Furthermore,
TNF-
(in combination with other inflammatory cytokines) inhibits
cholangiocyte secretory function in vitro
(50). However, the
contribution of TNF-
and the role of death receptor signaling in
cholangiocyte injury and their effect on cholangiocyte function during chronic
cholestasis are unknown.
The aim of our study was to assess the role played by the death receptor
TNF- in mediating cholangiocyte injury in normal physiological
conditions and during extrahepatic cholestasis induced by BDL. We asked the
following questions: Does death receptor-mediated signaling enhance cell death
in cholangiocytes during experimental biliary tract obstruction? Does
TNF-
modulate cholangiocyte growth or apoptosis in vivo during
experimental bile duct ligation? Does TNF-
contribute to functional
impairment of ductal bile secretion in vivo? What are the cellular mechanisms
by which cholangiocytes are susceptible to death receptor-mediated
cytotoxicity?
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MATERIALS AND METHODS |
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Reagents were purchased from Sigma Chemical (St. Louis, MO) unless
otherwise indicated. Porcine secretin was purchased from Peninsula
Laboratories (Belmont, CA). RIA kits for the determination of intracellular
cAMP levels were purchased from Amersham (Arlington Heights, IL). The
substrate for -glutamyltranspeptidase (
-GT),
N-(
-L-glutamyl)-4-methoxy-2-naphthylamide, was
purchased from Polysciences (Warrington, PA). The mouse anti-cytokeratin 19
(CK-19) antibody was purchased from Amersham. The monoclonal mouse antibody
against proliferating cellular nuclear antigen (PCNA) was purchased from Dako
(Kyoto, Japan). The tetrazolium bioreduction assay was purchased from Promega
(Madison, WI). Recombinant TNF-
was purchased from R&D
(Minneapolis, MN). The monoclonal mouse antibodies against the TNF-
R-1
receptor were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
monoclonal mouse antibody against the full-length human caspase-3 protein was
purchased from Oncogene Research Products (San Diego, CA).
Animal Model
Male Fisher 344 rats (175200 g) were purchased from Charles River
(Wilmington, MA), maintained in a temperature-controlled environment
(2022°C) with a 12:12-h light-dark cycle, and fed standard rat chow
ad libitum. Rats had free access to drinking water. The in vivo studies on
cholangiocyte apoptosis, proliferation, and secretion were performed in liver
sections and pure cholangiocytes from normal rats and rats that, following BDL
or bile duct incannulation [BDI, for bile collection
(5)] for 7 days, were treated
by a single intraperitoneal injection with 0.9% NaCl, actinomycin D (100
µg/kg body wt), TNF- (50 ng/kg body wt), or actinomycin D (100
µg/kg body wt) + TNF-
(50 ng/kg body wt). Twenty-four hours later,
the animals were used for the selected experiments (e.g., preparation of liver
blocks, isolation of cholangiocytes, or collection of bile). The in vitro
studies were performed in pure cholangiocytes isolated from normal rats and
rats with BDL for 8 days as described below. BDL or BDI were performed as
described (5). Before each
experimental procedure, the animals were anesthetized with pentobarbital
sodium (50 mg/kg body wt ip) according to the regulations of the panel on
euthanasia of the American Veterinarian Medical Association.
Purification of Cholangiocytes
The isolation of pure cholangiocytes from the selected group of animals was
achieved by immunoaffinity separation using a mouse monoclonal IgM antibody
(kindly provided by Dr. R. Faris, Brown University, Providence, RI) against an
unidentified membrane antigen expressed by all rat intrahepatic cholangiocytes
(23). Cell number and
viability was assessed by trypan blue exclusion. Cholangiocyte purity was
assessed by histochemistry for -GT
(48).
In Vivo Effect of Acute Administration of Actinomycin D, TNF-,
or Actinomycin D + TNF-
on Cholangiocyte Apoptosis, Proliferation, and
Ductal Functional Activity
We performed studies to determine whether in vivo administration of
actinomycin D + TNF- induces damage of bile ducts with loss of
proliferative and secretory activity of cholangiocytes of BDL but not normal
rats. Normal rats and rats with BDL or BDI for 7 days were treated by a single
intraperitoneal injection of 0.9% NaCl, actinomycin D (100 µg/kg body wt),
TNF-
(50 ng/kg body wt), or actinomycin D (100 µg/kg body wt) +
TNF-
(50 ng/kg body wt) +. Twenty-four hours later, we evaluated
1) duct damage by TdT-mediated dUTP nick end labeling (TUNEL)
analysis, 2) cholangiocyte proliferation by quantitative measurement
of the number of PCNA- or CK-19-positive cholangiocytes in liver sections
(33) and PCNA protein
expression by immunoblots (19)
in purified cholangiocytes, and 3) ductal functional activity by
measurement of secretin-stimulated bicarbonate-rich choleresis in bile fistula
rats (5) and secretin-induced
cAMP levels in purified cholangiocytes. When injected into the hepatic artery,
secretin induces an increase in bicarbonate of normal rats
(22). However, because
secretin does not induce choleresis in normal rats when administered
intravenously (5,
32), we evaluated the in vivo
effect of secretin on bile and bicarbonate secretion in BDI but not normal
rats.
Measurement of cholangiocyte apoptosis and proliferation. Cholangiocyte apoptosis was evaluated by TUNEL analysis in liver sections (33) and measurement of caspase-3 protein expression by immunoblots and caspase-3 activity in purified cholangiocytes using a commercially available kit. TUNEL analysis was also performed using a commercially available kit (Wako Chemicals, Tokyo, Japan). After being counterstained with hematoxylin solution, sections were examined by light microscopy with an Olympus BX-40 microscope (Olympus Optical, Tokyo, Japan) equipped with a charge-coupled device camera. Approximately 200 cells per slide were counted in a coded fashion in seven nonoverlapping fields. The activity of caspase-3 in purified cholangiocytes was measured as follows. Pure cholangiocytes from the selected group of animals were centrifuged at 1,500 rpm for 10 min, incubated in lysis buffer on ice for 10 min, and centrifuged at 10,000 g for 10 min. After centrifugation, the supernatant, containing the cytosolic fraction, was transferred to a clean tube. For each sample, 100 µg of proteins or BSA (negative control) were added to 50 ml of 2 x reaction buffer. Caspase activity was measured by proteolytic cleavage of the caspase-3-like substrate DEVD-p-nitroanilide (pNA). The assay is based on the photometric detection of the chromophore pNA after cleavage from the substrates. The pNA light emission was quantified by using a microtiter plate reader at 406 nm. The quantitative protein expression of caspase-3 in purified cholangiocytes was evaluated by immunoblots. Proteins (10 µg/lane) were resolved by SDS-7.5% PAGE and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated overnight at 4°C with a mouse antibody against the full-length human caspase-3 protein (1:500; Oncogene Research Products) followed by incubation with a horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:2,000; Zymed, San Francisco, CA). After several washes, the membrane was visualized using enhanced chemiluminescence (ECL Plus kit; Amersham Life Science, Little Chalfont, UK). The intensity of the bands was determined using the ChemiImager 4000 low-light imaging system (Alpha Innotech, San Leandro, CA).
To detect the number of PCNA- or CK-19-positive cholangiocytes, we performed immunohistochemistry in liver sections (n = 3) from the selected group of animals (33). Sections were counterstained with hematoxylin and examined under a light microscope (Olympus BX-40). Approximately 200 cells per slide were counted in a coded fashion in seven nonoverlapping fields. Immunoblots for PCNA in purified cholangiocytes from the selected group of animals was performed as described (19). Proteins (10 µg/lane) were resolved by SDS-7.5% PAGE and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-PCNA antibody (1:200), followed by incubation with an anti-rabbit biotinylated anti-mouse Ig diluted 1:100,000 with Tris-buffered saline containing Tween 20 (TBST). After several washes, the membrane was visualized using the ECL Plus kit (Amersham Life Science). The intensity of the bands was determined using the ChemiImager 4000 low-light imaging system (Alpha Innotech).
Measurement of ductal functional activity. Basal and secretin-stimulated intracellular cAMP levels [an index of cholangiocyte proliferation and cholangiocyte secretion (3, 19, 30)] were determined in pure cholangiocytes from the selected group of animals. After purification, cholangiocytes were incubated for 1 h at 37°C to restore surface proteins damaged by treatment with proteolytic enzymes and subsequently stimulated with 0.2% BSA (basal) or secretin (100 nM) in the presence of 0.2% BSA for 5 min at 22°C (3, 7, 19, 20, 23, 25, 30, 32, 33, 34). After ethanol extraction, cAMP levels were measured by RIA using a commercially available kit (Amersham) (3, 7, 19, 23, 25, 30, 32, 33, 34).
After anesthetization, rats were surgically prepared for bile collection (5). When steady-state bile flow was achieved, secretin (100 nM) was infused for 30 min, followed by a final infusion of Krebs-Ringer-Henseleit for 30 min. Bile was collected at 10-min intervals, placed in preweighed tubes, and immediately stored at 70°C before bicarbonate concentration was determined. Bicarbonate concentration (measured as total CO2) in bile from the selected group of animals was determined with a Natelson microgasometer apparatus (Scientific Industries, Bohemia, NY).
In Vitro Effect of TNF- on Apoptosis of Purified
Cholangiocytes From Normal and BDL Rats
We next used purified cholangiocytes to perform in vitro experiments aimed
to 1) demonstrate that actinomycin D + TNF- in vivo effects on
cholangiocyte apoptosis are due to a direct interaction with cholangiocytes
rather than an effect on other liver cells, and 2) elucidate the mechanisms by
which actinomycin D + TNF-
induces bile duct damage. Freshly isolated
cholangiocytes from normal rats and rats with BDL for 8 days were treated for
18 h with varying concentrations of TNF-
(0.1100 ng/ml) in the
presence or absence of preincubation with the RNA synthesis inhibitor
actinomycin D (1 µM) for 30 min. The number of viable cells was then
assessed using the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS) assay
(37).
Death Receptor Expression in Purified Cholangiocytes From Normal and BDL Rats
The expression of TNF- R-1 receptor in pure cholangiocytes from
normal and BDL rats was measured by immunoblots
(19). The cells were sonicated
six times in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5%
deoxycholic acid, 0.1% SDS, 2 mM EDTA, 10 mM NaF, 10 µg/ml leupeptin, 20
µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Proteins (10
µg for each sample) were resolved by SDS-7.5% PAGE and transferred onto a
nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked by
using a 5% solution of nonfat dry milk in TBST. The membrane was then
incubated overnight with rotation at 4°C with anti-TNF-
R-1
receptor antibody diluted 1:500 with TBST-5% milk. The membrane was then
washed five times with TBST and incubated for 1 h with rotation at room
temperature with anti-rabbit IgG-HRP (Santa Cruz Biotechnology) diluted
1:2,000 with TBST-5% milk. The comparability of the protein used was assessed
by immunoblots for
-actin, the internal control
(8). The membrane was washed
again three times with TBST, and proteins were visualized using the ECL kit
(Amersham Life Science). The intensity of the bands was determined using the
ChemiImager 4000 low-light imaging system (Alpha Innotech).
Statistical Analysis
All data are expressed as means ± SE. The differences between groups were analyzed by using Student's t-test when two groups were analyzed or analysis of variance if more than two groups were analyzed.
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RESULTS |
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Cholangiocyte apoptosis and proliferation. To assess the role of
TNF--induced cholangiocyte injury in vivo, we measured cholangiocyte
apoptosis by TUNEL analysis in liver sections from normal and 1-wk BDL rats 24
h after the administration of a single dose of NaCl, actinomycin D,
TNF-
, or TNF-
plus actinomycin D. A single dose of NaCl,
actinomycin D, TNF-
, or actinomycin D + TNF-
did not alter
cholangiocyte apoptosis of normal rats
(Fig. 1A). However, a
significant increase in cholangiocyte apoptosis was observed in liver sections
from 1-wk BDL rats treated with actinomycin D + TNF-
compared with
liver sections from BDL rats treated with NaCl
(Fig. 1B). When
administered alone, neither actinomycin D nor TNF-
affected
cholangiocyte apoptosis in liver sections from 1-wk BDL rats
(Fig. 1B). Although TNF-
is increased in serum following BDL in experimental animals
(12), there may be
considerable variation in levels. The temporal discordance between the actual
TNF-
levels and the administration of actinomycin D may account for the
lack of significant effects following a single intraperitoneal administration
of actinomycin D in these studies.
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To begin to understand the intracellular mechanisms by which TNF-
induces apoptosis of cholangiocytes from BDL rats, we measured, in purified
cholangiocytes, protein expression and activity of caspase-3, which is
considered to be the common caspase end point for apoptotic stimuli
(42). Consistent with the
concept that a single injection of actinomycin D + TNF-
to BDL rats
induces bile duct apoptosis, we found increased activity
(Fig. 2A) and protein
expression (Fig. 2B)
for caspase-3 in purified cholangiocytes from these animals compared with
cholangiocytes isolated from BDL rats treated with NaCl.
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Consistent with the concept that normal cholangiocytes are mitotically
quiescent (3,
32), there were no
PCNA-positive cholangiocytes in liver sections from normal rats
(Fig. 3A).
Administration of actinomycin D, TNF-, or actinomycin D + TNF-
to normal rats did not alter the number of
PCNA-(Fig. 3A) and
CK-19-positive cholangiocytes (Fig.
4A) or PCNA protein expression
(Fig. 5A) compared
with normal rats treated with NaCl. The number of PCNA- and CK-19-positive
cholangiocytes assessed from actinomycin D + TNF-
-treated rats was
decreased compared with liver sections from BDL rats treated with NaCl (Figs.
3B and
4B). Similarly, we
found that a single injection of actinomycin D + TNF-
significantly
decreased PCNA protein expression in purified cholangiocytes compared with
cholangiocytes isolated from 1-wk BDL rats treated with a single dose of NaCl
(Fig. 5B). When
administered alone, neither actinomycin D nor TNF-
altered the number
of PCNA- or CK-19-positive cholangiocytes (Figs.
3B and
4B) or PCNA protein
expression (Fig. 5B)
of 1-wk BDL rats.
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Measurement of ductal functional activity. Basal cAMP levels of
normal and BDL control rats were similar to those of previous studies
(Fig. 6, A and
B) (19,
20,
32). Intracellular basal cAMP
levels of cholangiocytes from BDL rats were significantly (P <
0.05) higher than those of normal cholangiocytes
(Fig. 6, A and
B). Consistent with the concept that actinomycin D +
TNF- induces damage of cholangiocytes from BDL but not normal rats,
secretin increased cAMP levels of cholangiocytes from normal rats treated with
NaCl, actinomycin D, TNF-
, or actinomycin D + TNF-
(Fig. 6A). Secretin
increased cAMP levels of cholangiocytes purified from 1-wk BDL rats treated in
vivo with NaCl, actinomycin D, or TNF-
alone
(Fig. 6B). Basal and
secretin-stimulated cAMP levels of cholangiocytes from BDL rats treated with a
single intraperitoneal injection of actinomycin D or TNF-
were similar
to those of cholangiocytes from BDL rats treated with NaCl
(Fig. 6B). Secretin
did not increase cAMP levels in cholangiocytes purified from 1-wk BDL rats
treated with a single intraperitoneal injection of actinomycin D + TNF-
(Fig. 6B).
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Basal bile flow and bicarbonate concentration and secretion of BDL control
rats were similar to those of previous studies
(Table 1)
(5,
20). Basal bile flow and
bicarbonate concentration and secretion of 1-wk BDL rats treated with
actinomycin D, TNF-, or actinomycin D + TNF-
were similar to
those of 1-wk BDL rats treated with NaCl
(Table 1). Secretin increased
bile flow and bicarbonate concentration and secretion of 1-wk BDL rats treated
with actinomycin D or TNF-
alone
(Table 1). Secretin failed to
increase bile flow and bicarbonate concentration and secretion of 1-wk BDL
rats treated with a single intraperitoneal injection of actinomycin D +
TNF-
(Table 1).
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In Vitro Studies in Purified Cholangiocytes from Normal and BDL Rats
Actinomycin D sensitized cholangiocytes from BDL (but not normal) rats
to TNF--mediated toxicity. The in vitro cytotoxicity of
TNF-
on isolated cholangiocytes was evaluated by measuring the number
of viable cells using the MTS assay. Similar to previous reports
(38), cholangiocytes from
normal rats were resistant to TNF-
over a wide concentration range
(Fig. 7A).
Preincubation with actinomycin D did not sensitize normal cholangiocytes to
TNF-
toxicity (Fig.
7A). Similar to normal cholangiocytes, cholangiocytes
from BDL rats were resistant to TNF-
-mediated toxicity
(Fig. 7B) However,
preincubation with actinomycin D sensitized cholangiocytes from BDL rats to
TNF-
-mediated toxicity (Fig.
7B).
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Expression of TNF- receptor increases in purified
cholangiocytes following BDL. Immunoblot analysis shows that following
BDL, there was an increase in TNF-
receptor protein expression in
purified cholangiocytes compared with normal cholangiocytes
(Fig. 8).
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DISCUSSION |
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TNF- is a multifunctional cytokine that plays a critical role in
both hepatic regeneration and injury
(26). Cholangiocytes are known
to express TNF-
, and biliary levels of TNF-
are increased in
patients with cholangitis following biliary tract obstruction
(47). Cholangiocytes are the
primary epithelial source of TNF-
(35), a key mediator of
hepatic regeneration following partial hepatectomy in rats
(35). However, there is
considerable variability in target cell responses to TNF-
, and the
effects of increased local or circulating TNF-
on cholangiocyte growth
or function have not previously been reported. Our results show that
cholangiocytes are sensitized to TNF-
cytotoxicity following biliary
tract obstruction. These observations are highly germane to the
pathophysiology of cholestasis in inflammatory cholangiopathies or during
biliary tract obstruction.
In normal rat liver, cholangiocytes are mitotically dormant
(3,
32), but they markedly
proliferate in response to pathological maneuvers including BDL
(3,
4,
5,
6,
8,
19,
30,
31,
32,
33,
34). The BDL rat model is
widely used for studying the mechanisms of cholangiocyte hyperplasia,
secretin-stimulated ductal secretion, and cholangiocyte injury
(3,
5,
6,
19,
20,
30). The rationale for using
the BDL model for evaluating the mechanisms of action by which TNF-
modulates cholangiocyte apoptosis, proliferation, and secretion is based on
the findings that 1) ductal hyperplasia after BDL is devoid of
apoptosis (30), which allows
for a precise evaluation of the changes in cholangiocyte apoptosis and/or
proliferation following TNF-
treatment; 2) cholangiocytes from
BDL rats retain normal phenotypes of biliary lineage
(1); 3) secretin
induces no choleresis in normal rats when infused intravenously
(5); and 4) after BDL
there is a marked increase in basal and secretin-stimulated ductal secretion
(3,
5,
6,
9,
19,
20,
30), which allows for better
evaluation of the changes in basal and secretin-stimulated cholangiocyte
secretion. During intrahepatic cholestasis, there is an increased production
of basal and/or endotoxin-induced TNF-
, which has been linked with
severity of liver damage, in that TNF-
is considered a crucial mediator
in inducing and processing the inflammatory cascade
(43,
49). During experimental
cholestasis (49) as well as in
primary biliary cirrhosis
(17), an increased expression
of TNF-
and related receptors occurs in cholangiocytes, but their role
in mediating cholangiocyte injury have not yet been elucidated. With this
background we evaluated the effect of TNF-
administration on the
proliferative, apoptotic, and secretory activities of cholangiocytes in BDL
rats. Our findings demonstrate that in BDL but not normal rats, a single dose
of TNF-
impairs cholangiocyte proliferative and secretory activities
and activated apoptosis, but only when an inhibitor of protein synthesis
(i.e., actinomycin D) is administered together with TNF-
. This finding
indicates that 1) proliferating cholangiocytes, typical of
obstructive cholestasis, are more sensitive to TNF-
-mediated cell
injury, consistent with the increased expression of TNF-
receptor and
basal caspase activities of cholangiocytes from BDL rats; and 2) the
increased sensitivity to TNF-
-mediated injury is blunted by a parallel
activation of unidentified rescue mechanisms and, indeed, only when protein
synthesis is blocked by actinomycin D, was activation of apoptosis and
inhibition of cell proliferation and secretion evident.
A balance between apoptosis and proliferation regulates intrahepatic ductal
mass in a number of chronic cholestatic liver diseases
(6). In association with
TNF--induced damage of bile ducts of BDL rats, there was a marked
decrease in cholangiocyte proliferation isolated from BDL rats, as assessed by
a decreased number of PCNA- and CK-19-positive cholangiocytes in liver
sections and inhibition of PCNA protein expression in purified cholangiocytes.
The decrease in cholangiocyte proliferative capacity with TNF-
-induced
cholangiocyte apoptosis is consistent with our previous studies showing that
enhanced cholangiocyte apoptosis induced by vagotomy in BDL rats is associated
with loss of cholangiocyte DNA synthesis and loss of intrahepatic bile ducts
(30). Similarly, a single dose
of CCl4 induces cholangiocyte apoptosis in bile ducts of BDL rats,
an event that has been associated with decreased cholangiocyte proliferation
and number of ducts (34).
Gastrin inhibits cholangiocarcinoma growth through
Ca2+-and PKC-
-regulated activation of
cholangiocyte apoptosis (24).
After cessation of
-naphthylisothiocyanate feeding, regression of
ductal hyperplasia is associated with increased apoptosis
(33). Treatment of BDL rats
with the anti-estrogens, tamoxifen, or ICI-182780 inhibited cholangiocyte
growth and induced overexpression of Fas antigen and apoptosis in
cholangiocytes (10). We next
evaluated whether the TNF-
increase in cholangiocyte apoptosis and
decrease in cholangiocyte proliferation are associated with inhibition of
secretin-stimulated ductal secretion. Secretin receptor expression and
secretin-stimulated ductal secretion are important physiological markers of
cholangiocyte proliferation and bile duct integrity
(3,
4,
5,
6,
8,
9,
19,
20,
25,
30,
31,
32,
33,
34). In a variety of animal
models of ductal injury, cholangiocyte proliferation is associated with
increased secretin-regulated ductal bile secretion, whereas cholangiocyte loss
is coupled with decreased secretin-stimulated ductal secretion
(2,
3,
4,
5,
6,
8,
9,
19,
20,
25,
30,
31,
32,
33,
34). Consistent with these
previous studies, concomitant with increased ductopenia and inhibition of
cholangiocyte proliferation, we found that a single administration of
actinomycin D + TNF-
induced inhibition of secretin-stimulated ductal
secretion.
The concept that secretin and its receptor may be important in the
modulation of cholangiocyte proliferation/loss is also supported by studies in
patients with cholangiopathies
(18). For example, enhanced
secretin-stimulated bile flow and bicarbonate secretion is observed in
patients with ductal hyperplasia induced by hepatic cirrhosis
(14). Furthermore, positron
emission tomography scanning of patients with primary biliary cirrhosis
following administration of labeled
and secretin shows a lack of
secretin response that is restored by ursodeoxycholate treatment
(45,
46).
TNF- has been implicated in many different physiological processes
and subserves multiple cell-type or tissue-specific functions
(36,
40,
53). Cytotoxicity from
TNF-
can involve oxidative stress and result in either cell necrosis or
apoptosis (54). The essential
requirement for de novo protein synthesis for manifestation of TNF
cytotoxicity suggests that TNF-
expresses cyto-protective gene products
in cholangiocytes. Identification of these protective factors in
cholangiocytes may potentially be valuable in reducing the adverse effects of
cholestasis during biliary tract obstruction and inflammation. We propose that
TNF-
activates both cytoprotective and cytotoxic cellular responses in
rat cholangiocytes. In normal cholangiocytes, the former predominates, and
cholangiocytes are resistant to TNF-
toxicity. After BDL, there is a
shift toward the cytotoxic pathway. We hypothesize that this shift may occur
in a variety of ways. BDL is accompanied by alterations in bile acid
concentrations, which may alter intracellular signaling, as well as increased
oxidative stress, which may overwhelm protective cellular antioxidant
defenses. Additional studies to evaluate the role of bile acids in mediating
TNF-
cytotoxicity as well as cellular oxidative stress in vivo and in
vitro and the effect of antioxidants is thus warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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