For consideration of publication in American Journal of Physiology ...

Articles in PresS. Am J Physiol Cell Physiol (March 12, 2003). 10.1152/ajpcell.00497.2002
Alpini et al. 1
C-00497-2002.R2
For consideration of publication in American Journal of Physiology February 3, 2003
Increased Susceptibility of Cholangiocytes to Tumor Necrosis Factor-α Cytotoxicity after Bile Duct
Ligation
Gianfranco Alpini, Ph. D. 1, 4, 5
Yoshiyuki Ueno, M. D., Ph. D. 3
Laura Tadlock, M. H. S. M. 2
Shannon S Glaser, M. S. 2
Gene LeSage, M.D. 1
Heather Francis, B. S. 2
Silvia Taffetani, M. D. 2
Marco Marzioni, M. D. 4
Domenico Alvaro, M. D. 6
Tushar Patel, M.D. 1
1 From the Department of Internal Medicine and 4 Medical Physiology, 2 Division of Research and
Education, Scott & White Hospital and The Texas A&M University System Health Science Center,
College of Medicine and 5 Central Texas Veterans Health Care System, Temple, TX 76504, 6 Division
of Gastroenterology, University of Rome, "La Sapienza", Rome, Italy, and 3 Division of
Gastroenterology, Tohoku University School of Med, Aobaku, Sendai, Japan, 6 Div Gastroenterol,
University of Rome, "La Sapienza", Rome, Italy. This work was supported by a grant award to Dr.
Patel, Dr. LeSage and Dr. Alpini from Scott & White Hospital and The Texas A&M University
System, by Grant-in-Aid for Scientific Research (C) (13670488) from JSPS to Dr. Ueno, by Grant
from MURST 40%(MM06215421/2) progetto nazionale 2000 to Dr. Domenico Alvaro, by a NIH
grants DK 02678 and DK 60637 to Dr. Patel, by an NIH grant DK 54208 to Dr. LeSage and by a VA
Merit Award and an NIH grant DK 58411 to Dr. Alpini.
Short Title: TNF-α induction of bile duct injury
Key words: Apoptosis; bile flow; intrahepatic biliary epithelium; proliferation; secretin.
Abbreviations: BDL = bile duct ligation; BSA = bovine serum albumin; cAMP = adenosine 3', 5’monophosphate;
CK-19 = cytokeratin-19; KRH = Krebs Ringer Henseleit; γ-GT = γglutamyltranspeptidase;
MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4sulfophenyl)-2H-tetrazolium,
inner salt; PCNA = proliferating cellular nuclear antigen.
Address Correspondence to Tushar Patel, M.D..
Associate Professor of Internal Medicine and
Division of Gastroenterology
Scott & White Clinic
Texas A&M University Health Science Center
2401 South 31 th Street
Temple, Texas 76502
Phone: 254-724-2237
Fax: 254-724-8276
E-mail: tpatel@medicine.tamu.edu
Copyright (c) 2003 by the American Physiological Society.

Abstract
Alpini et al. 2
C-00497-2002.R2
Tumor necrosis factor (TNF)- α plays a critical role in epithelial cell injury. However, the role of
TNF- α in mediating cholangiocyte injury under physiological or pathophysiological conditions are
unknown. Thus, we assessed the effects of TNF- α alone or following sensitization by actinomycin D
on cell apoptosis, proliferation and basal and secretin-stimulated ductal secretion in cholangiocytes
from normal or bile duct ligated (BDL) rats. Cholangiocytes from normal or BDL rats were highly
resistant to TNF- α alone. However, pre-sensitization by actinomycin D increased apoptosis in
cholangiocytes following BDL, and was associated with an inhibition of proliferation and secretin-
stimulated ductal secretion. Thus, TNF- α mediates cholangiocyte injury and altered ductal secretion
following bile duct ligation. These observations suggest that cholestasis may enhance susceptibility to
cytokine-mediated cholangiocyte injury.

Introduction
Alpini et al. 3
C-00497-2002.R2
Bile flow originates from both hepatocyte and cholangiocyte secretion (5, 41). Cholangiocytes modify
canalicular bile by a series of reabsorptive and secretory events regulated by gastrointestinal hormones
(5, 15, 20, 31, 39) and bile salts (4). The hormone secretin plays an important role by stimulating
ductal secretion (3, 5-8, 24, 25, 39) by interaction with specific receptors on cholangiocytes (9) through
an increase in intracellular adenosine 3', 5’-monophosphate (cAMP) levels (3, 4, 7, 20, 25, 30, 32-34).
The increase in cAMP levels leads to the opening of Cl - channels (16), activation of the Cl - -
/HCO3 exchanger (7) with subsequent secretion of bicarbonate into bile (5).
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 (CCl 4 ) 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 CCl 4
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), as well as 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-α are members of a super-family characterized by intracellular domains
that mediate death in response to extracellular stimuli. TNF- α 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- α are increased
following biliary obstruction in mice (12). Furthermore, TNF- α (in combination with other

Alpini et al. 4
C-00497-2002.R2
inflammatory cytokines) inhibits cholangiocyte secretory function in vitro (50). However, the
contribution of TNF- α 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-α 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- α modulate
cholangiocyte growth or apoptosis in vivo during experimental bile duct ligation? Does TNF- α
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?
MATERIALS AND METHODS
Materials
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
γ-glutamyltranspeptidase (γ-GT), N (γ-L-glutamyl)-4-methoxy-2-naphthylamide, was purchased from
Polysciences (Warrington, PA). The mouse anti-cytokeratin 19 (CK-19) antibody was purchased
from Amersham (Arlington Heights, IL). 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- α was
purchased from R&D (Minneapolis, MN). The monoclonal mouse antibodies against the TNF- α 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
Alpini et al. 5
C-00497-2002.R2
Male Fisher 344 rats (175-200 gm) were purchased from Charles River (Wilmington, MA),
maintained in a temperature-controlled environment (20-22°C) with a 12:12-hour light-dark cycle, and
fed ad libitum standard rat chow. 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 IP injection with: (i) 0.9% NaCl; (ii) actinomycin D
(100 µg/Kg body weight)]; (iii) TNF-α (50 ng/Kg body weight); or (iv) actinomycin D (100 µg/Kg
body weight) + TNF- α (50 ng/Kg body weight). 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 anaesthetised with pentobarbital sodium (50 mg/kg weight,
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 gMantibody (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 γ-GT (48).
In Vivo Effect of Acute Administration of Actinomycin D, TNF-α or Actinomycin D + TNF-
α on Cholangiocyte Apoptosis, Proliferation and Ductal Functional Activity
We performed studies to determine if in vivo administration of actinomycin D + TNF- α induces
damage of bile ducts with loss of proliferative and secretory activity of cholangiocytes of BDL but not

Alpini et al. 6
C-00497-2002.R2
normal rats. Normal rats and rats with BDL or BDI for 7 days were treated by a single IP injection of:
(i) 0.9% NaCl; (ii) actinomycin D (100 µg/Kg body weight)]; (iii) TNF- α (50 ng/Kg body weight);
or (iv) TNF- α (50 ng/Kg body weight) + actinomycin D (100 µg/Kg body weight). Twenty four
hours later, we evaluated: (i) duct damage by TUNEL analysis; (ii) 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 (iii) 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, since secretin does
not induces 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.
a. 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 by a
commercially availbale kit. TUNEL analysis was also performed using a commercially available kit
(Wako Chemicals, Tokyo, Japan). Following counterstaining with Hematoxylin solution, sections
were examined by light microscopy with an Olympus BX-40 microscope equipped with a CCD
camera. Approximately 200 cells per slide were counted in a coded fashion in seven non-overlapping
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 minutes,
incubated in lysis buffer on ice for 10 minutes, and centrifuged at 10,000 g for 10 minutes. Following
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 2x Reaction Buffer.
Caspase activity was measured by proteolytic cleavage of the caspase-3 like substrate, DEVD-pNA.
The assay is based on the photometric detection of the chromophore p-nitroanilide (pNA) after
cleavage from the substrates. The pNA light emission was quantified using a microtiter plate reader at
406 nm. The quantitative protein expression of caspase 3 in purified cholangiocytes was evaluated by

Alpini et al. 7
C-00497-2002.R2
immunoblots. Proteins (10 µg/lane) were resolved by SDS-7.5% polyacrylamide gel electrophoresis
and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated
overnight at 4 o C with a mouse antibody against the full-length human caspase-3 protein (1:500)
(Oncogene Research Products) followed by incubation with an HRP-Goat-Anti-Mouse IgG + A+M
(1:2000) (Zymed, San Fransisco, CA). Following several washes, the membrane was visualized using
chemiluminescence (ECL Plus kit, Amersham Life Science, Little Chalfont, Buckinghamshire,
England). The intensity of the bands was determined using the ChemiImager TM 4000 low light
imaging system (Alpha Innotech Corp., 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 Optical Co., BX
40, Tokyo, Japan). Approximately 200 cells per slide were counted in a coded fashion in seven non-
overlapping 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%
polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. After blocking,
the membrane was incubated overnight at 4 o C with a rabbit anti-PCNA antibody (1:200) followed by
incubation with an anti-rabbit biotinylated anti-mouse immunoglobulin (ECL kit, Amersham Life
Science, Little Chalfont, Buckinghamshire, England) diluted 1:100,000 with TBST. Following several
washes, the membrane was visualized using chemiluminescence (ECL Plus kit, Amersham Life
Science, Little Chalfont, Buckinghamshire, England). The intensity of the bands was determined
using the ChemiImager TM 4000 low light imaging system (Alpha Innotech Corp.).
b. 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. Following purification, cholangiocytes were incubated for 1 hour at 37 o 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 minutes at 22°C (3, 7, 19, 20, 23, 25,

Alpini et al. 8
C-00497-2002.R2
30, 32-34). Following ethanol extraction, cAMP levels were measured by RIA using a commercially
available kit (Amersham) (3, 7, 19, 23, 25, 30, 32-34).
Following anaesthesia, rats were surgically prepared for bile collection (5). When steady-state bile
flow was achieved, secretin (100 nM) was infused for 30 minutes followed by a final infusion of
Krebs Ringer Henseleit (KRH) for 30 minutes. Bile was collected at 10-minute intervals, placed in
pre-weighed tubes and immediately stored at -70 o C before determining bicarbonate concentration.
Bicarbonate concentration (measured as total CO 2 ) in bile from the selected group of animals was
determined by a Natelson microgasometer apparatus (Scientific Industries, Bohemia, NY).
In Vitro Effect of TNF- α on Apoptosis of Purified Cholangiocytes from Normal and BDL
Rats
Next, we performed in vitro experiments in purified cholangiocytes aimed to: (i) demonstrate that
actinomycin D + TNF- α in vivo effects on cholangiocyte apoptosis are due to a direct interaction with
cholangiocytes rather than an effect on other liver cells; and (ii) elucidate the mechanisms by which
actinomycin D + TNF- α induces bile duct damage. Freshly isolated cholangiocytes from normal rats
and rats with BDL for 8 days were treated for 18 hours with varying concentrations of TNF- α (0.1-
100 ng/ml) in the presence or absence of preincubation with the RNA synthesis inhibitor, actinomycin
D (1 µM) for 30 minutes. 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- α 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% sodium dodecyl sulfate (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% polyacrylamide gel

Alpini et al. 9
C-00497-2002.R2
electrophoresis (PAGE) and transferred onto a nitrocellulose membrane (BioRad, Hercules, CA). The
membrane was blocked using a 5% solution of nonfat dry milk in Tris-buffered saline (TBST). The
membrane was then incubated overnight with rotation at 4 o C anti-TNF-α R-1 receptor antibody diluted
1:500 with TBST-5% milk. The membrane was then washed five times with TBST and incubated for
1 hour with rotation at room temperature with anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa
Cruz, CA) diluted 1:2,000 with TBST-5% milk. The comparability of the protein used was assessed
by immunoblots for beta-actin, the internal control (8). The membrane was washed again three times
with TBST and proteins were visualized using chemiluminescence (ECL kit, Amersham Life Science,
Little Chalfont, Buckinghamshire, England). The intensity of the bands was determined using the
ChemiImager TM 4000 low light imaging system, (Alpha Innotech Corp.).
Statistical Analysis
All data are expressed as mean ± SEM (standard error). The differences between groups were
analysed by Student's t-test when two groups were analysed or analysis of variance (ANOVA) if
more than two groups were analyzed.
RESULTS
In Vivo Acute Administration of Actinomycin D + TNF- α Increases Apoptosis and Inhibits
Cholangiocyte Proliferation and Ductal Functional Activity of BDL but not Normal Rats
a. Cholangiocyte Apoptosis and Proliferation
To assess the role of TNF- α induced cholangiocyte injury in vivo, we measured cholangiocyte
apoptosis by TUNEL analysis in liver sections from normal and 1 week BDL rats 24 hours after the
administration of a single dose of NaCl, actinomycin D, TNF-α, or TNF- α with actinomycin D. A
single dose of NaCl, actinomycin D, TNF- α or actinomycin D + TNF- α did not alter cholangiocyte
apoptosis of normal rats (Figure 1 a). However, a significant increase in cholangiocyte apoptosis was
observed in liver sections from 1 week BDL rats treated with actinomycin D + TNF- α as compared to
liver sections from BDL rats treated with NaCl (Figure 1 b). When administered alone, neither

Alpini et al. 10
C-00497-2002.R2
actinomycin D nor TNF- α affected cholangiocyte apoptosis in liver sections from 1 week BDL rats
(Figure 1 b). Although TNF-α is increased in serum following BDL in experimental animals (12),
there may be considerable variation in levels. The temporal discordance between the actual TNF-α
levels and the administration of actinomycin D may account for the lack of significant effects
following a single IP administration of actinomycin D in these studies.
To begin to understand the intracellular mechanisms by which TNF-α 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-α to BDL rats
induces bile duct apoptosis, we found increased activity (Figure 2 a) and protein expression (Figure 2
b) for caspase 3 in purified cholangiocytes from these animals compared to cholangiocytes isolated
from BDL rats treated with sodium chloride.
Consistent with the concept that normal cholangiocytes are mitotically quiescent (3, 32), there were no
PCNA-positive cholangiocytes in liver sections from normal rats (Figure 3 a). Administration of
actinomycin D, TNF- α or actinomycin D + TNF- α to normal rats did not alter the number of
PCNA- (Figure 3 a) and CK-19-positive cholangiocytes (Figure 4 a) and PCNA protein expression
(Figure 5 a) as compared to normal rats treated with NaCl. The number of PCNA and CK-19 positive
cholangiocytes assessed from actinomycin D + TNF- α treated rats was decreased as compared to
liver sections from BDL rats treated with NaCl (Figures 3 b and 4 b). Similarly, we found that a single
injection of actinomycin D + TNF- α significantly decreased PCNA protein expression in purified
cholangiocytes compared with cholangiocytes isolated from 1 week BDL rats treated with a single
dose of NaCl (Figure 5 b). When administered alone, neither actinomycin D nor TNF- α altered the
number of PCNA or CK-19 positive cholangiocytes (Figures 3 b and 4 b) or PCNA protein
expression (Figure 5 b) of 1 week BDL rats.
b. Measurement of Ductal Functional Activity
Basal cAMP levels of normal and BDL control rats were similar to that of previous studies (Figure 6
a-b) (19, 20, 32). Intracellular basal cAMP levels of cholangiocytes from BDL rats were significantly

Alpini et al. 11
C-00497-2002.R2
(p < 0.05) higher than those of normal cholangiocytes (Figure 6 a-b). Consistent with the concept that
actinomycin D + TNF- α 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- α
or actinomycin D + TNF- α (Figure 6 a). Secretin increased cAMP levels of cholangiocytes purified
from 1 week BDL rats treated in vivo with NaCl, actinomycin D or TNF- α alone (Figure 6 b). Basal
and secretin-stimulated cAMP levels of cholangiocytes from BDL rats treated with a single IP
injection of actinomycin D or TNF- α were similar to that of cholangiocytes from BDL rats treated
with NaCl (Figure 6 b). Secretin did not increase cAMP levels in cholangiocytes purified from 1 week
BDL rats treated with a single IP injection of actinomycin D + TNF- α (Figure 6 b).
Basal bile flow, bicarbonate concentration and secretion of BDL control rats were similar to that of
previous studies (Table 1) (5, 20). Basal bile flow, bicarbonate concentration and secretion of 1 week
BDL rats treated with actinomycin D, TNF- α or actinomycin D + TNF- α was similar from that of 1
week BDL rats treated with NaCl (Table 1). Secretin increased bile flow and bicarbonate
concentration and secretion of 1 week BDL rats treated with actinomycin D or TNF- α alone (Table
1). Secretin failed to increase bile flow and bicarbonate concentration and secretion of 1 week BDL
rats treated with a single IP injection of actinomycin D + TNF- α (Table 1).
In Vitro Studies in Purified Cholangiocytes from Normal and BDL Rats
a. Actinomycin D Sensitized Cholangiocytes from BDL (but not Normal) Rats to TNF-
Mediated Toxicity
The in vitro cytotoxicity of TNF- α 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- α over a wide concentration range (Figure 7 a). Pre-incubation
with actinomycin D did not sensitize normal cholangiocytes to TNF- α toxicity (Figure 7 a). Similar
to normal cholangiocytes, cholangiocytes from BDL rats were resistant to TNF- α mediated toxicity
(Figure 7 b). However, pre-incubation with actinomycin D sensitized cholangiocytes from BDL rats
to TNF- α mediated toxicity (Figure 7 b).

Alpini et al. 12
C-00497-2002.R2
b. The Expression of TNF- Receptor Increases in Purified Cholangiocytes following
BDL
Immunoblot analysis shows that following BDL, there was an increase in TNF- α receptor protein
expression in purified cholangiocytes as compared to normal cholangiocytes (Figure 8).
DISCUSSION
In this novel study we have shown that a single intraperitoneal injection of actinomycin D + TNF- α
induces a marked increase in cholangiocyte apoptosis in BDL, but not normal rats. In BDL rats, the
increase in cholangiocyte apoptosis was associated with decreased cholangiocyte proliferation and
inhibition of secretin-stimulated ductal secretion. By in vitro experiments, we found that
cholangiocytes from normal and BDL rats were resistant to TNF- α. Co-incubation with actinomycin
D sensitized cholangiocytes from BDL (but not normal) rats to TNF- α toxicity. These observations
suggest that during chronic cholestasis, the increased proliferative and secretory activities of
intrahepatic cholangiocytes are highly sensitive to the toxic effects of TNF- α.
TNF- α is a multifunctional cytokine that plays a critical role in both hepatic regeneration and injury
(26). Cholangiocytes are known to express TNF- α, and biliary levels of TNF- α are increased in
patients with cholangitis following biliary tract obstruction (47). Cholangiocytes are the primary
epithelial source of TNF- α (35), a key mediator of hepatic regeneration following partial hepatectomy
in rats (35). However, there is considerable variability in target cell responses to TNF- α, and the
effects of increased local or circulating TNF- α on cholangiocyte growth or function have not
previously been reported. Our results show that cholangiocytes are sensitized to TNF- α 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 markedly proliferate in response
to pathological maneuvers including BDL (3-6, 8, 19, 30-34). 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

Alpini et al. 13
C-00497-2002.R2
mechanisms of action by which TNF- α modulates cholangiocyte apoptosis, proliferation and
secretion is based on: (i) 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- α
treatment; (ii) cholangiocytes from BDL rats retain normal phenotypes of biliary lineage (1); (iii)
secretin induces no choleresis in normal rats when infused intravenously (5); (iv) following 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- α, which has been linked with severity of liver damage; in that TNF- α 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- α and
related receptors occurs in cholangiocytes but their role in mediating cholangiocyte injury have been
not yet elucidated. With this background we evaluated the effect of TNF- α 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- α impairs cholangiocyte
proliferative and secretory activities and activated apoptosis but only when an inhibitor of protein
synthesis (i.e., actinomycin D) was administered together with TNF- α. This indicates that: (i)
proliferating cholangiocytes, typical of obstructive cholestasis, are more sensitive to TNF- α mediated
cell injury and this is consistent with the increased expression of TNF- α receptor and basal caspase
activities of cholangiocytes from BDL rats; (ii) the increased sensitivity to TNF- α mediated injury is
blunted by a parallel activation of unidentified rescue mechanisms and indeed only when protein
synthesis is blocked by actinomycin D, activation of apoptosis, inhibition of cell proliferation and
secretion was evident.
A balance between apoptosis/proliferation regulates intrahepatic ductal mass in a number of chronic
cholestatic liver diseases (6). Associated with TNF- α induced damage of bile ducts of BDL rats,

Alpini et al. 14
C-00497-2002.R2
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-α−induced cholangiocyte apoptosis is consistent with our previous studies
showing 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
CCl 4 induces cholangiocyte apoptosis in bile ducts of BDL rats, an event that was associated with
decreased cholangiocyte proliferation and number of ducts (34). Gastrin inhibits cholangiocarcinoma
growth through Ca 2+ - and PKC-α-regulated activation of cholangiocyte apoptosis (24). Following
cessation of α-naphthylisothiocyanate feeding, regression of ductal hyperplasia is associated with
increased apoptosis (33). Treatment of BDL rats with the anti-estrogens, tamoxifen or ICI 182,780,
inhibited cholangiocyte growth and induced overexpression of Fas antigen and apoptosis in
cholangiocytes (10). We next evalauted if the TNF- α increase in cholangiocyte apoptosis and
decrease in cholangiocyte proliferation are associated with inhibition of secretin-stimulated ductal
secretion. Secretin receptor expression and secretin-stimualted ductal secretion are important
physiological markers of cholangiocyte proliferation and bile duct integrity (3-6, 8, 9, 19, 20, 25, 30-
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 (2-6, 8, 9, 19, 20, 25, 30-34). Consistent with these previous
studies, concomitant with increased ductopenia and inhibition of cholangiocyte proliferation, we found
that a single administration of actinomycin D + TNF- α 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 HCO 3 and secretin

Alpini et al. 15
C-00497-2002.R2
shows a lack of secretin response, which is restored by ursodeoxycholate treatment (45, 46).
TNF- α has been implicated in many different physiological processes, and subserves multiple cell-
type or tissue-specific functions (36, 40, 53). Cytotoxicity from TNF- α 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- α expresses cytoprotective 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- α activates both cytoprotective and cytotoxic cellular responses
in rat cholangiocytes. In normal cholangiocytes, the former predominates and cholangiocytes are
resistant to TNF- α toxicity. Following BDL, there is a shift towards 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 by increased oxidative stress,
which may overwhelm protective cellular antioxidant defenses. Additional studies to evaluate the role
of bile acids in mediating TNF- α cytotoxicity as well as cellular oxidative stress in vivo and in vitro,
and the effect of antioxidants is thus warranted.
REFERENCES
1. Alpini, G, Aragona E, Dabeva M, Salvi R, Shafritz DA, and Tavoloni N. Distribution
of albumin and alpha-fetoprotein mRNAs in normal, hyperplastic, and preneoplastic rat liver. Am J
Pathol 141: 623-632, 1992.
2. Alpini, G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H, Baiocchi L, Stati T,
Barbaro B, Phinizy JL, Mauldin J, and LeSage G. Bile acid depletion and repletion regulate
cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats.
Gastroenterology 123: 1226-1237, 2002.
3. Alpini, G, Glaser S, Ueno, Pham L, Podila PV, Caligiuri A, LeSage G, and LaRusso
NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. Am J
Physiol 274: G767-G775, 1998.

Alpini et al. 16
C-00497-2002.R2
4. Alpini, G, Glaser S, Ueno Y, Rodgers R, Phinizy JL, Francis H, Baiocchi L, Holcomb
L, Caligiuri A, and LeSage G. Bile acid feeding induces cholangiocyte proliferation and secretion:
evidence for bile acid-regulated ductal secretion. Gastroenterology 116: 179-186., 1999.
5. Alpini, G, Lenzi R, Sarkozi L, and Tavoloni N. Biliary physiology in rats with bile
ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest
81: 569-578, 1988.
6. Alpini, G, Prall RT, and LaRusso NF. The pathobiology of biliary epithelia. In: The Liver:
Biology & Pathobiology (4th ed.), edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W,
Schachter D, and Shafritz DA. Philadelphia, PA: Lippincott, Williams & Wilkins, 2001, p. 421-435.
7. Alpini, G, Roberts SK, Kuntz SM, Ueno Y, Gubba S, Podila P, LeSage G, and
LaRusso NF. Morphological, molecular and functional heterogeneity of cholangiocytes from normal
rat liver. Gastroenterology 110: 1636-1643, 1996.
8. Alpini, G, Ueno Y, Glaser S, Marzioni M, Phinizy JL, Francis H, and LeSage G. Bile
acid feeding increased proliferative activity and apical bile acid transporter expression in both small
and large rat cholangiocytes. Hepatology 34: 868-876, 2001.
9. Alpini, G, Ulrich II C, Phillips J, Pham L, Miller L, and LaRusso NF. Upregulation of
secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol 266:
G922-G928, 1994.
10. Alvaro D, Alpini G, Onori P, Perego L, Baroni-Svegliati G, Franchitto A, Baiocchi L,
Glaser S, Le Sage G, Folli F, and Gaudio E. Estrogens stimulate proliferation of intrahepatic
biliary epithelium in rats. Gastroenterology 119: 1681-1691, 2000.
11. Arguedas, MR, Heudebert GH, Stinnett AA, and Wilcox CM. Biliary stents in malignant
obstructive jaundice due to pancreatic carcinoma: a cost-effectiveness analysis. Am J Gastroenterol 97:
898-904, 2002.
12. Bemelmans, MH, Gouma DJ, Greve JW, and Buurman WA. Cytokines tumor necrosis
factor and interleukin-6 in experimental biliary obstruction in mice. Hepatology 15: 1132-1136, 1992.

Alpini et al. 17
C-00497-2002.R2
13. Beuers, U, Nathanson MH, Isales CM, and Boyer JL. Tauroursodeoxycholic acid
stimulates hepatocellular exocytosis and mobilizes extracellular Ca 2+ mechanisms defective in
cholestasis. J Clin Invest 92: 2984-2993, 1993.
14. Bode, C, Zelder O, Goebell H, and Neuberger HO. Choleresis induced by secretin:
distinctly increased response in cirrhotics. Scand J Gastroenterol 7: 697-699, 1972.
15. Cho, WK, and Boyer JL. Vasoactive intestinal polypeptide is a potent regulator of bile
secretion from rat cholangiocytes. Gastroenterology 117: 420-428, 1999.
16. Fitz, JG, Basavappa S, McGill J, Melhus O, and Cohn JA. Regulation of membrane
chloride currents in rat bile duct epithelial cells. J Clin Invest 91: 319-328, 1993.
17. Floreani, A, Guido M, Bortolami M, Della Zentil G, Venturi C, Pennelli N, and
Naccarato R. Relationship between apoptosis, tumour necrosis factor, and cell proliferation in chronic
cholestasis. Dig Liver Dis 33: 570-575, 2001.
18. Fukumoto, Y, Okita K, Yasunaga M, Konishi T, Yamasaki T, Ando M, Shirasawa H,
Fuji T, and Takemoto T. A new therapeutic trial of secretin in the treatment of intrahepatic
cholestasis. Gastroenterol Jpn 24: 298-307, 1989.
19. Glaser, S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno N, Caligiuri A,
Phinizy JL, Chowdhury U, Papa E, LeSage G, and Alpini G. Gastrin inhibits cholangiocyte
growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-
inositol 1,4,5-triphosphate-, Ca( 2+ )-, and protein kinase C alpha-dependent mechanisms. Hepatology
32: 17-25., 2000.
20. Glaser, S, Rodgers R, Phinizy JL, Robertson WE, Lasater J, Caligiuri A, Tretjak Z,
LeSage G, and Alpini G. Gastrin inhibits secretin-induced ductal secretion by interaction with
specific receptors on rat cholangiocytes. Am J Physiol 273: G1061-G1070, 1997.
21. Green, RM, Gollan JL, Hagenbuch B, Meier PJ, and Beier DR. Regulation of hepatocyte
bile salt transporters during hepatic regeneration. Am J Physiol 273: G621-G627, 1997.
22. Hirata, K, and Nathanson MH. Bile duct epithelia regulate biliary bicarbonate excretion in
normal rat liver. Gastroenterology 121: 396-406, 2001.

Alpini et al. 18
C-00497-2002.R2
23. Ishii, M, Vroman B, and LaRusso NF. Isolation and morphological characterization of bile
duct epithelial cells from normal rat liver. Gastroenterology 97: 1236-1247, 1989.
24. Kanno, N, Glaser S, Chowdhury U, Phinizy JL, Baiocchi L, Francis H, LeSage G, and
Alpini G. Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of
Ca 2+ -dependent protein kinase C-alpha. J Hepatol 34: 284-291, 2001.
25. Kato, A, Gores GJ, and LaRusso NF. Secretin stimulates exocytosis in isolated bile duct
epithelial cells by a Cyclic AMP-mediated mechanism. J Biol Chem 267: 15523-15529, 1992.
26. Kirillova, I, Chaisson M , and Fausto N. Tumor necrosis factor induces DNA replication
in hepatic cells through nuclear factor kappaB activation. Cell Growth Differ 10: 819-828, 1999.
27. Kowdley, KV. Ursodeoxycholic acid therapy in hepatobiliary disease. Am J Med 108: 481-
486, 2000.
28. Kullak-Ublick, GA, B Stieger, B Hagenbuch, and PJ Meier. Hepatic transport of bile
salts. Semin Liver Dis 20: 273-292, 2000.
29. Lee, J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD, Kramer W, and
Boyer JL. Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in
the rat. Gastroenterology 121: 1473-1484, 2001.
30. LeSage, G, Alvaro D, Benedetti A, Glaser S, Marucci L, Baiocchi L, Eisel W, Caligiuri
A, Phinizy JL, Rodgers R, Francis H, and Alpini G. Cholinergic system modulates growth,
apoptosis, and secretion of cholangiocytes from bile duct-ligated rats. Gastroenterology 117: 191-199,
1999.
31. LeSage, G, Glaser S, and Alpini G. Regulatory mechanisms of ductal bile secretion. Dig
Liver Dis 32: 563-566, 2000.
32. LeSage, G, Glaser S, Gubba S, Robertson WE, Phinizy JL, Lasater J, Rodgers R, and
Alpini G. Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased
secretin-induced ductal bile secretion. Gastroenterology 111: 1633-1644, 1996.
33. LeSage, G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, Kanno N, Phinizy JL, Francis H,
and Alpini G. Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled
with increased apoptosis. Am J Physiol Gastrointest Liver Physiol 281: G182-G190, 2001.

Alpini et al. 19
C-00497-2002.R2
34. LeSage, G, Glaser S, Marucci L, Benedetti A, Phinizy JL, Rodgers R, Caligiuri A,
Papa E, Tretjak Z, Jezequel AM, Holcomb L, and Alpini G. Acute carbon tetrachloride feeding
induces damage of large but not small cholangiocytes from BDL rat liver. Am J Physiol 276: G1289-
G1301, 1999.
35. Loffreda, S, Rai R, Yang SQ, Lin HZ, and Diehl AM. Bile ducts and portal and central
veins are major producers of tumor necrosis factor alpha in regenerating rat liver. Gastroenterology
112: 2089-2098, 1997.
36. Luster, MI, Simeonova PP, Gallucci R, and Matheson J. Tumor necrosis factor alpha and
toxicology. Crit Rev Toxicol 29: 491-511., 1999.
37. Ly, LH, Zhao XY, Holloway L, and Feldman D. Liarozole acts synergistically with
1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking
24-hydroxylase activity. Endocrinology 140: 2071-2076, 1999.
38. Mano, Y, Ishii M, Okamoto H, Igarashi T, Kobayashi K, and Toyota T. Effect of tumor
necrosis factor alpha on intrahepatic bile duct epithelial cell of rat liver. Hepatology 23: 1602-1607,
1996.
39. Marzioni, M, Glaser S, Francis H, Phinizy JL, LeSage G, and Alpini G. Functional
heterogeneity of cholangiocytes. Semin Liver Dis 22: 227-240, 2002.
40. McDermott, MF. TNF and TNFR biology in health and disease. Cell Mol Biol 47: 619-635,
2001.
41. Nathanson, MH, and Boyer JL. Mechanisms and regulation of bile secretion. Hepatology
14: 551-566, 1991.
42. Natori, S, Selzner M, Valentino KL, Fritz LC, Srinivasan A, Clavien PA, and Gores
GJ. Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-
dependent mechanism. Transplantation 68: 89-96, 1999.
43. Neuman, M, Angulo P, Malkiewicz I, Jorgensen R, Shear N, Dickson ER, Haber J,
Katz G, and Lindor K. Tumor necrosis factor-alpha and transforming growth factor-beta reflect
severity of liver damage in primary biliary cirrhosis. J Gastroenterol Hepatol 17: 196-202, 2002.

Alpini et al. 20
C-00497-2002.R2
44. Patel, T, Roberts LR, Jones BA, and Gores GJ. Dysregulation of apoptosis as a
mechanism of liver disease: an overview. Semin Liver Dis 18: 105-114, 1998.
45. Prieto, J, Garcia N, Marti-Climent JM, Penuelas I, Richter JA, and Medina JF.
Assessment of biliary bicarbonate secretion in humans by positron emission tomography.
Gastroenterology 117: 167-172, 1999.
46. Prieto, J, Qian C, Garcia N, Diez J, and Medina JF. Abnormal expression of anion
exchanger genes in primary biliary cirrhosis. Gastroenterology 105: 572-578, 1993.
47. Rosen, HR, Winkle PJ, Kendall BJ, and Diehl DL. Biliary interleukin-6 and tumor
necrosis factor-alpha in patients undergoing endoscopic retrograde cholangiopancreatography. Dig
Dis Sci 42: 1290-1294, 1997.
48. Rutemburg, AM, Kim H, Fishbein JW, Hanker JS, Wasserkrug HL, and Seligman
AM. Histochemical and ultrastructural demonstration of γ-glutamyl transpeptidase activity. J
Histochem Cytochem 17: 517-526, 1969.
49. Sheen-Chen, SM, Chen HS, Ho HT, Chen WJ, Sheen CC, and Eng HL. Effect of bile
acid replacement on endotoxin-induced tumor necrosis factor-alpha production in obstructive jaundice.
World J Surg 26: 448-450, 2002.
50. Spirli, C, Nathanson MH, Fiorotto R, Duner E, Denson LA, Sanz JM, Di Virgilio F,
Okolicsanyi L, Casagrande F, and Strazzabosco M. Proinflammatory cytokines inhibit secretion
in rat bile duct epithelium. Gastroenterology 121: 156-169, 2001.
51. Suzuki, S, Kurachi K, Yokoi Y, Tsuchiya Y, Okamoto K, Okumura T, Inaba K,
Konno H, and Nakamura S. Intrahepatic cholangiojejunostomy for unresectable malignant biliary
tumors with obstructive jaundice. J Hepatobiliary Pancreat Surg 8: 124-129, 2002.
52. Theilmann, L, Kuppers B, Kadmon M, Roeren T, Notheisen H, Stiehl A, and Otto G.
Biliary tract strictures after orthotopic liver transplantation: diagnosis and management. Endoscopy 26:
517-522, 1994.
53. Tilg, H. Cytokines and liver diseases. Can J Gastroenterol 15: 661-668, 2001.

Alpini et al. 21
C-00497-2002.R2
54. Xu, Y, Jones BE, Neufeld DS, and Czaja MJ. Glutathione modulates rat and mouse
hepatocyte sensitivity to tumor necrosis factor toxicity. Gastroenterology 115: 1229-1237, 1998.
LEGENDS
Figure 1 Effect of a single IP injection of NaCl, actinomycin D, TNF- α or actinomycin D +
TNF- α on cholangiocyte apoptosis (evaluated by TUNEL analysis in liver sections) of [a] normal
and [b] 1 week BDL rats. [a] In normal rat liver sections, actinomycin D, TNF- α or actinomycin D
+ TNF- α did not alter cholangiocyte apoptosis (that was virtually absent) compared with normal
rats treated with NaCl. Orig. magn., X625. [b] Administration of a single dose of actinomycin D +
TNF- α to 1 week BDL rats induced a significant increase in cholangiocyte apoptosis as compared
to liver sections from 1 week BDL rats treated with NaCl. Actinomycin D or TNF- α alone did not
affect cholangiocyte apoptosis, which remains similar to that of BDL rats treated with NaCl. Data
are mean ± SEM of 3 experiments. *p < 0.05 vs. the number of apoptotic cholangiocytes from 1
week BDL rats treated with a single IP injection of NaCl. Orig. magn., X625.
Figure 2 Measurement of [a] activity and [b] protein expression for caspase 3 in
cholangiocytes from 1 week BDL rats treated with a single injection of NaCl or TNF-α + actinomycin
D. There was increased activity [a] and protein expression [b] for caspase 3 in purified cholangiocytes
from BDL rats treated with a single injection of actinomycin D + TNF-α compared to cholangiocytes
isolated from BDL rats treated with NaCl. Data are mean ± SEM of at least 3 experiments. *p <
0.05 vs. its corresponding basal value.

Alpini et al. 22
C-00497-2002.R2
Figure 3 Immunohistochemistry for PCNA in liver sections from [a] normal and [b] 1 week
BDL rats treated with a single IP injection of NaCl, actinomycin D, TNF- α or actinomycin D +
TNF- α. [a] There were no PCNA-positive cholangiocytes in liver sections from normal rats.
Administration of actinomycin D, TNF- α or actinomycin D + TNF- α to normal rats did not alter
the number of PCNA-positive cholangiocytes. Orig. magn., X1,000. [b] Administration of
actinomycin D + TNF- α induced a significant decrease in the number of PCNA-positive
cholangiocytes in 1 week BDL rats as compared to BDL rats treated with NaCl. Actinomycin D or
TNF- α alone did not affect the number of PCNA-positive cholangiocytes, which remain similar to
that of BDL control rats. Data are mean ± SEM of 3 experiments. *p < 0.05 vs. the number of
PCNA-positive cholangiocytes from 1 week BDL rats treated with a single IP injection of NaCl. Orig.
magn., X1,000.
Figure 4 Immunohistochemistry for CK-19 in liver sections from [a] normal and [b] 1 week
BDL rats treated with a single IP injection of NaCl, actinomycin D or TNF- α or actinomycin D +
TNF- α. [a] Administration of actinomycin D, TNF- α or actinomycin D + TNF- α to normal rats
did not alter the number of CK-19 positive cholangiocytes. Data are mean ± SEM of 3 experiments.
Orig. magn., X125. [b] Administration of actinomycin D + TNF- α induced a significant decrease in
the number of CK-19 positive cholangiocytes in 1 week BDL rats as compared to BDL rats treated
with NaCl. Actinomycin D or TNF- α alone did not affect the number of CK-positive
cholangiocytes, which remain similar to that of BDL control rats. Data are mean ± SEM of 3
experiments. *p < 0.05 vs. the number of CK-19-positive cholangiocytes from 1 week BDL rats
treated with a single IP injection of NaCl. Orig. magn., X125.

Alpini et al. 23
C-00497-2002.R2
Figure 5 Measurement of PCNA protein expression in pure cholangiocytes from [a] normal rats
and [b] 1 week BDL rats treated with a single IP injection of NaCl, actinomycin D, TNF- α or
actinomycin D + TNF- α. [a] Administration of NaCl, actinomycin D, TNF- α or actinomycin D +
TNF- α to normal rats did not alter PCNA protein expression. Immunoblots were quantified by
densitometry. Data are mean ± SEM of 4 experiments. [b] A single injection of actinomycin D +
TNF- α decreased PCNA protein expression in purified cholangiocytes compared with cholangiocytes
isolated from 1 week BDL rats treated with a single dose of NaCl. Actinomycin D or TNF- α alone
did not affect PCNA protein expression, which remains similar to that of BDL control rats.
Immunoblots were quantified by densitometry. Data are mean ± SEM of 6-26 experiments. *p <
0.05 vs. PCNA protein expression of cholangiocytes from 1 week BDL rats treated with a single IP
injection of NaCl.
Figure 6 Mesaurement of cAMP levels in pure cholangiocytes from [a] normal rats and [b] 1
week BDL rats treated with a single IP injection of NaCl, actinomycin D + TNF- α, actinomycin D or
TNF- α. [a] Secretin increased cAMP levels of cholangiocytes from normal rats treated with NaCl,
actinomycin D, TNF- α or actinomycin D + TNF- α. Data are mean ± SEM of at least 3
experiments. *p < 0.05 vs. corresponding basal value. [b] Intracellular basal cAMP levels of
cholangiocytes from BDL rats were significantly higher than those of normal cholangiocytes. Secretin
increased cAMP levels of cholangiocytes purified from 1 week BDL rats treated with NaCl,
actinomycin D or TNF- α. Basal and secretin-stimulated cAMP levels of cholangiocytes from BDL
rats treated with a single IP injection of actinomycin D or TNF- α were similar to that of
cholangiocytes from BDL rats. Consistent with the concept that TNF induces duct damage, secretin
did not increase cAMP levels in cholangiocytes purified from 1 week BDL rats treated with a single IP
injection of actinomycin D + TNF- α. Data are mean ± SEM of at least 5 experiments. *p < 0.05 vs.
corresponding basal value. #p < 0.05 vs. basal cAMP levels of normal cholangiocytes.

Alpini et al. 24
C-00497-2002.R2
Figure 7 Freshly isolated cholangiocytes from [a] normal and [b] BDL rats were treated for 18
hours with varying concentrations of recombinant TNF- α in the presence or absence of 1 µM
actinomycin D for 30 minutes. The number of viable cells was assessed using the MTS assay. [a]
Normal cholangiocytes were resistant to TNF- α over the concentration range studied. Furthermore,
pre-incubation with actinomycin D did not sensitize normal cholangiocytes to TNF- α toxicity. Data
are mean ± SEM of at least 3 experiments. [b] Cholangiocytes from BDL rats were resistant to TNF-
α mediated toxicity. However, pre-incubation with actinomycin D sensitized cholangiocytes from
BDL rats to TNF- α mediated toxicity. *p < 0.05 vs. corresponding value of cholangiocytes treated in
vitro with TNF- α. Data are mean ± SEM of at least 3 experiments.
Figure 8 Western blot analysis for TNF-R1 receptor in pure cholangiocytes from normal or
BDL rats. Following BDL, there was an increase in TNF- α receptor protein expression. *p < 0.05
vs. corresponding value of normal cholangiocytes. Data are mean ± SEM of 3 experiments.

Table 1 Effect of actinomycin D, TNF-alpha or actinomycin D + TNF-alpha administration on bile flow, bicarbonate concentration and secretion
of BDI rats.
Bicarbonate secretion
Bicarbonate secretion
Bicarbonate
concentration
Bicarbonate
concentration
Bile flow
Bile flow
Treatment
Secretin
( Eq/min/Kg body
weight)
Basal
( Eq/min/Kg body
weight)
Secretin
(mEq/Liter)
Basal
(mEq/Liter)
Secretin
( l/min/Kg body
weight)
Basal
( l/min/Kg body
weight)
105.76 (± 10.34) 145.01 (± 16.02)a 38.14 (± 2.12) 55.52 (± 2.18)b 4.15 (± 0.65) 8.15 (± 1.12)c
BDL +
NaCl
118.21 (± 7.64) 155.92 (± 8.95)a 43.28 (± 2.05) 52.9 (± 3.58)b 5.18 (± 0.50) 8.06 (± 0.64)c
BDL +
actinomycin D
98.20 (± 5.79) 139.14 (± 7.58)a 41.25 (± 3.67) 62.58 (± 5.73)b 4.15 (± 0.65) 8.79 (± 1.15)c
BDL +
TNF-alpha
112.40 (± 9.67) 121.29 (± 8.77)ns 50.25 (± 3.64) 57.67 (± 2.55)ns 5.77 (± 0.97) 7.15 (± 0.89)ns
BDL +
actinomycin D +
TNF-alpha
Data are mean ± SE of at least 3 rats. a p < 0.05 vs. corresponding value of basal bile flow. b p < 0.05 vs. corresponding value of basal bicarbonate concentration. cp <
0.05 vs. corresponding value of basal bicarbonate secretion. ns =not significative vs. its corresponding basal value. Data are mean ± SEM of at least 3 experiments.
Statistical analysis was performed by unpaired t-student test.

Figure 1a
Normal +actinomycin D
Normal + NaCl
Normal + actinomycin D + TNF-α
Normal + TNF-α

BDL + NaCl BDL + actinomycin D
BDL + TNF- α
Number of apoptotic cholangiocytes
(per portal tracts)
7
6
5
4
3
2
1
0
Figure 1b
ns vs. control
ns vs. control
BDL + actinomycin D +TNF- α
NaCl Actinomycin D TNF-α Actinomycin D +
TNF-α
*

Caspase 3 activity
6
5
4
3
2
1
0
BDL +
NaCl
Figure 2a
*
BDL +
actinomycin D +
TNF-α

Caspase 3
protein expression x 100
β-actin
200
100
0
BDL +
NaCl
Figure 2b
*
BDL +
actinomycin D +
TNF-α
Caspase 3
32 kD
β-actin
42 kD

Figure 3a
Normal + actinomycin D
Normal + NaCl
Normal + TNF-α Normal + actinomycin D + TNF-α

BDL + NaCl
BDL + TNF-α
Number of PCNA-positive cholangiocytes
(per portal tracts)
20
15
10
5
0
Figure 3b
ns vs. control
BDL + actinomycin D
BDL + actinomycin D +
TNF-α
ns vs. control
NaCl Actinomycin D TNF-α Actinomycin D +
TNF-α
*

Normal + NaCl
Normal + Actinomycin D
Normal + TNF-α Normal + Actinomycin D
+ TNF-α
Number of CK-19 positive cholangiocytes
(per portal tracts)
60
50
40
30
20
10
0
Figure 4a
ns vs. control
ns vs. control
ns vs. control
NaCl Actinomycin D TNF-α Actinomycin D +
TNF-α

BDL + TNF-α
Number of CK-19 positive cholangiocytes
(per portal tracts)
140
120
100
80
60
40
20
0
Figure 4b
BDL BDL + actinomycin D
ns vs. control
BDL + actinomycin D +
TNF-α
ns vs. control
NaCl Actinomycin D TNF-α Actinomycin D +
TNF-α
*

protein expression x 100 (arbitrary units)
PCNA
β- actin
40
30
20
10
0
Normal +
NaCl
Normal
Figure 5a
ns vs.
NaCl
Normal +
actinomycin D
ns vs.
NaCl
Normal +
TNF-α
ns vs.
NaCl
Normal +
actinomycin D +
TNF-α
PCNA
36 kD
β-actin
42 kD

PCNA
protein expression x 100 (arbitrary units)
β-actin
60
50
40
30
20
10
0
BDL +
NaCl
BDL
BDL +
actinomycin D
Figure 5b
ns vs.
NaCl
ns vs.
NaCl
BDL +
TNF-α
*
BDL +
actinomycin D +
TNF-α
PCNA
36 kD
β-actin
42 kD

cAMP levels
(fmol/100,000 cells)
140
120
100
80
60
40
20
0
Normal +
NaCl
*
Figure 6a
Normal +
actinomycin D
* *
Normal +
TNF-α
Basal
Secretin
*
Normal +
actinomycin D +
TNF-α

cAMP levels
(fmol / 100,000 cells)
100
80
60
40
20
0
BDL +
NaCl
*
Figure 6b
BDL +
actinomycin D
*
BDL +
TNF-α
Basal
Secretin
* ns vs.
basal
BDL +
actinomycin D +
TNF-α

Figure 7
TNF-α
Actinomycin D
+ TNF-α
140
TNF-α
Actinomycin D
+ TNF-α
120
120
100
100
80
60
40
Cell Number, % of control
80
60
* *
40
Cell Number, % of control
20
20
0
0 0.1 1 10 100
0
a 0 0.1 1 10 100 b
[TNF-α], ng/ml [TNF-α], ng/ml

Figure 8
8000
*
7000
n =3
6000
5000
4000
3000
2000
TNF-R1 protein expression
(arbitrary units)
1000
0
TNF-R1
55 kD
Normal BDL