Increased susceptibility of cholangiocytes to tumor necrosis factor-{alpha} cytotoxicity after bile duct ligation

Gianfranco Alpini,1,2,3 Yoshiyuki Ueno,4 Laura Tadlock,5 Shannon S. Glaser,5 Gene LeSage,1 Heather Francis,5 Silvia Taffetani,5 Marco Marzioni,2 Domenico Alvaro,6 and Tushar Patel1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF)-{alpha} plays a critical role in epithelial cell injury. However, the role of TNF-{alpha} in mediating cholangiocyte injury under physiological or pathophysiological conditions is unknown. Thus we assessed the effects of TNF-{alpha} 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-{alpha} alone. However, presensitization by actinomycin D increased apoptosis in cholangiocytes following BDL and was associated with an inhibition of proliferation and secretin-stimulated ductal secretion. Thus TNF-{alpha} mediates cholangiocyte injury and altered ductal secretion following bile duct ligation. These observations suggest that cholestasis may enhance susceptibility to cytokine-mediated cholangiocyte injury.

bile flow; intrahepatic biliary epithelium; proliferation; secretin


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, 6, 7, 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, 33, 34). The increase in cAMP levels leads to the opening of Cl channels (16) and activation of the 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 (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-{alpha} are members of a super family characterized by intracellular domains that mediate death in response to extracellular stimuli. TNF-{alpha} 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-{alpha} are increased following biliary obstruction in mice (12). Furthermore, TNF-{alpha} (in combination with other inflammatory cytokines) inhibits cholangiocyte secretory function in vitro (50). However, the contribution of TNF-{alpha} 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-{alpha} 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-{alpha} modulate cholangiocyte growth or apoptosis in vivo during experimental bile duct ligation? Does TNF-{alpha} 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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {gamma}-glutamyltranspeptidase ({gamma}-GT), N-({gamma}-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-{alpha} was purchased from R&D (Minneapolis, MN). The monoclonal mouse antibodies against the TNF-{alpha} 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 (175–200 g) were purchased from Charles River (Wilmington, MA), maintained in a temperature-controlled environment (20–22°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-{alpha} (50 ng/kg body wt), or actinomycin D (100 µg/kg body wt) + TNF-{alpha} (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 {gamma}-GT (48).

In Vivo Effect of Acute Administration of Actinomycin D, TNF-{alpha}, or Actinomycin D + TNF-{alpha} on Cholangiocyte Apoptosis, Proliferation, and Ductal Functional Activity

We performed studies to determine whether in vivo administration of actinomycin D + TNF-{alpha} 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-{alpha} (50 ng/kg body wt), or actinomycin D (100 µg/kg body wt) + TNF-{alpha} (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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} (0.1–100 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-{alpha} 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-{alpha} 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 {beta}-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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Acute Administration of Actinomycin D + TNF-{alpha} Increases Apoptosis and Inhibits Cholangiocyte Proliferation and Ductal Functional Activity of BDL But Not Normal Rats

Cholangiocyte apoptosis and proliferation. To assess the role of TNF-{alpha}-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-{alpha}, or TNF-{alpha} plus actinomycin D. A single dose of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} 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-{alpha} compared with liver sections from BDL rats treated with NaCl (Fig. 1B). When administered alone, neither actinomycin D nor TNF-{alpha} affected cholangiocyte apoptosis in liver sections from 1-wk BDL rats (Fig. 1B). Although TNF-{alpha} is increased in serum following BDL in experimental animals (12), there may be considerable variation in levels. The temporal discordance between the actual TNF-{alpha} 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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Fig. 1. Effect of a single intraperitoneal injection of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} on cholangiocyte apoptosis (evaluated by TUNEL analysis in liver sections) of normal and 1-wk bile duct-ligated (BDL) rats. A: in normal rat liver sections, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} did not alter cholangiocyte apoptosis (virtually absent) compared with normal rats treated with NaCl. Original magnification, x 625. B: administration of a single dose of actinomycin D + TNF-{alpha} to 1-wk BDL rats induced a significant increase in cholangiocyte apoptosis compared with liver sections from 1-wk BDL rats treated with NaCl. Actinomycin D or TNF-{alpha} alone did not affect cholangiocyte apoptosis, which remained similar to that of BDL rats treated with NaCl. Original magnification, x 625. Data are means ± SE of 3 experiments; ns, not significant. *P < 0.05 vs. NaCl.

 

To begin to understand the intracellular mechanisms by which TNF-{alpha} 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-{alpha} 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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Fig. 2. Measurement of activity and protein expression for caspase-3 in cholangiocytes from 1-wk BDL rats treated with a single injection of NaCl or TNF-{alpha} + 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-{alpha} compared with cholangiocytes isolated from BDL rats treated with NaCl. Data are means ± SE of at least 3 experiments. *P < 0.05 vs. corresponding basal value.

 

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-{alpha}, or actinomycin D + TNF-{alpha} 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-{alpha}-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-{alpha} 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-{alpha} 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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Fig. 3. Immunohistochemistry for proliferating cellular nuclear antigen (PCNA) in liver sections from normal and 1-wk BDL rats treated with a single intraperitoneal injection of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha}. A: there were no PCNA-positive cholangiocytes in liver sections from normal rats. Administration of actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} to normal rats did not alter the number of PCNA-positive cholangiocytes. Original magnification, x1,000. B: administration of actinomycin D + TNF-{alpha} induced a significant decrease in the number of PCNA-positive cholangiocytes in 1-wk BDL rats compared with BDL rats treated with NaCl. Actinomycin D or TNF-{alpha} alone did not affect the number of PCNA-positive cholangiocytes, which remained similar to that of BDL control rats. Original magnification, x1,000. Data are means ± SE of 3 experiments. *P < 0.05 vs. NaCl.

 


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Fig. 4. Immunohistochemistry for cytokeratin 19 (CK-19) in liver sections from normal and 1-wk BDL rats treated with a single intraperitoneal injection of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha}. A: administration of actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} to normal rats did not alter the number of CK-19-positive cholangiocytes. Original magnification, x125. Data are means ± SE of 3 experiments. B: administration of actinomycin D + TNF-{alpha} induced a significant decrease in the number of CK-19-positive cholangiocytes in 1-wk BDL rats compared with BDL rats treated with NaCl. Actinomycin D or TNF-{alpha} alone did not affect the number of CK-positive cholangiocytes, which remained similar to that of BDL control rats. Original magnification, x 125. Data are means ± SE of 3 experiments. *P < 0.05 vs. NaCl.

 


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Fig. 5. Measurement of PCNA protein expression in pure cholangiocytes from normal rats and 1-wk BDL rats treated with a single intraperitoneal injection of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha}. A: administration of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} to normal rats did not alter PCNA protein expression. Immunoblots were quantified by densitometry. Data are means ± SE of 4 experiments. B: a single injection of actinomycin D + TNF-{alpha} decreased PCNA protein expression in purified cholangiocytes compared with cholangiocytes isolated from 1-wk BDL rats treated with a single dose of NaCl. Actinomycin D or TNF-{alpha} alone did not affect PCNA protein expression, which remained similar to that of BDL control rats. Immunoblots were quantified by densitometry. Data are means ± SE of 6–26 experiments. *P < 0.05 vs. BDL + NaCl.

 

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-{alpha} 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-{alpha}, or actinomycin D + TNF-{alpha} (Fig. 6A). Secretin increased cAMP levels of cholangiocytes purified from 1-wk BDL rats treated in vivo with NaCl, actinomycin D, or TNF-{alpha} 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-{alpha} 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-{alpha} (Fig. 6B).



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Fig. 6. Measurement of cAMP levels in pure cholangiocytes from normal rats and 1-wk BDL rats treated with a single intraperitoneal injection of NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha}. A: secretin increased cAMP levels of cholangiocytes from normal rats treated with NaCl, actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha}. Data are means ± SE 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-wk BDL rats treated with NaCl, actinomycin D, or TNF-{alpha}. Basal and secretin-stimulated cAMP levels of cholangiocytes from BDL rats treated with a single intraperitoneal injection of actinomycin D or TNF-{alpha} were similar to those 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-wk BDL rats treated with a single intraperitoneal injection of actinomycin D + TNF-{alpha}. Data are means ± SE of at least 5 experiments. *P < 0.05 vs. corresponding basal value. #P < 0.05 vs. basal cAMP levels of normal cholangiocytes.

 

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-{alpha}, or actinomycin D + TNF-{alpha} 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-{alpha} 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-{alpha} (Table 1).


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Table 1. Effect of actinomycin D, TNF-{alpha}, or actinomycin D + TNF-{alpha} administration on bile flow and bicarbonate concentration and secretion in BDL rats

 

In Vitro Studies in Purified Cholangiocytes from Normal and BDL Rats

Actinomycin D sensitized cholangiocytes from BDL (but not normal) rats to TNF-{alpha}-mediated toxicity. The in vitro cytotoxicity of TNF-{alpha} 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-{alpha} over a wide concentration range (Fig. 7A). Preincubation with actinomycin D did not sensitize normal cholangiocytes to TNF-{alpha} toxicity (Fig. 7A). Similar to normal cholangiocytes, cholangiocytes from BDL rats were resistant to TNF-{alpha}-mediated toxicity (Fig. 7B) However, preincubation with actinomycin D sensitized cholangiocytes from BDL rats to TNF-{alpha}-mediated toxicity (Fig. 7B).



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Fig. 7. Freshly isolated cholangiocytes from normal and BDL rats were treated for 18 h with varying concentrations of recombinant TNF-{alpha} in the presence or absence of 1 µM actinomycin D for 30 min. The number of viable cells was assessed using the MTS assay. A: normal cholangiocytes were resistant to TNF-{alpha} over the concentration range studied. Furthermore, preincubation with actinomycin D did not sensitize normal cholangiocytes to TNF-{alpha} toxicity. Data are means ± SE of at least 3 experiments. B: cholangiocytes from BDL rats were resistant to TNF-{alpha}-mediated toxicity. However, preincubation with actinomycin D sensitized cholangiocytes from BDL rats to TNF-{alpha}-mediated toxicity. *P < 0.05 vs. TNF-{alpha}. Data are means ± SE of at least 3 experiments.

 

Expression of TNF-{alpha} receptor increases in purified cholangiocytes following BDL. Immunoblot analysis shows that following BDL, there was an increase in TNF-{alpha} receptor protein expression in purified cholangiocytes compared with normal cholangiocytes (Fig. 8).



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Fig. 8. Western blot analysis for TNF-R1 receptor in pure cholangiocytes from normal or BDL rats. After BDL, there was an increase in TNF-{alpha} receptor protein expression. *P < 0.05 vs. normal. Data are means ± SE; n = 3 experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this novel study we have shown that a single intraperitoneal injection of actinomycin D + TNF-{alpha} 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 performing in vitro experiments, we found that cholangiocytes from normal and BDL rats were resistant to TNF-{alpha}. Coincubation with actinomycin D sensitized cholangiocytes from BDL (but not normal) rats to TNF-{alpha} 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-{alpha}.

TNF-{alpha} is a multifunctional cytokine that plays a critical role in both hepatic regeneration and injury (26). Cholangiocytes are known to express TNF-{alpha}, and biliary levels of TNF-{alpha} are increased in patients with cholangitis following biliary tract obstruction (47). Cholangiocytes are the primary epithelial source of TNF-{alpha} (35), a key mediator of hepatic regeneration following partial hepatectomy in rats (35). However, there is considerable variability in target cell responses to TNF-{alpha}, and the effects of increased local or circulating TNF-{alpha} on cholangiocyte growth or function have not previously been reported. Our results show that cholangiocytes are sensitized to TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha}, which has been linked with severity of liver damage, in that TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha}. This finding indicates that 1) proliferating cholangiocytes, typical of obstructive cholestasis, are more sensitive to TNF-{alpha}-mediated cell injury, consistent with the increased expression of TNF-{alpha} receptor and basal caspase activities of cholangiocytes from BDL rats; and 2) the increased sensitivity to TNF-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-regulated activation of cholangiocyte apoptosis (24). After cessation of {alpha}-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-{alpha} 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-{alpha} 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-{alpha} has been implicated in many different physiological processes and subserves multiple cell-type or tissue-specific functions (36, 40, 53). Cytotoxicity from TNF-{alpha} 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-{alpha} 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-{alpha} activates both cytoprotective and cytotoxic cellular responses in rat cholangiocytes. In normal cholangiocytes, the former predominates, and cholangiocytes are resistant to TNF-{alpha} 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-{alpha} cytotoxicity as well as cellular oxidative stress in vivo and in vitro and the effect of antioxidants is thus warranted.


    ACKNOWLEDGMENTS
 
This work was supported by a grant from Scott & White Clinic and The Texas A&M University System to T. Patel, G. LeSage, and G. Alpini; Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) 13670488 to Y. Ueno; Ministero dell' Università e della Ricerca Scientifica e Tecnologica Grant (40%) MM06215421/2 progetto nazionale 2000 to D. Alvaro; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-02678 and DK-60637 to T. Patel; NIDDK Grant DK-54208 to G. LeSage; and a Veterans Affairs Merit Award and NIDDK Grant DK-58411 to G. Alpini.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Patel, Division of Gastroenterology, Scott & White Clinic, Texas A&M Univ. System Health Science Center College of Medicine, 2401 South 31st St., Temple, TX 76502 (E-mail: tpatel{at}medicine.tamu.edu).

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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