1 Roudebush Veterans Affairs Medical Center and 2 Departments of Medicine and 3 Pathology, 4 Pediatric Hematology/Oncology, Herman B. Wells Center for Pediatric Research, Riley Hospital for Children; Indiana University School of Medicine, Indianapolis, Indiana 46202; and 5 Scott and White Hospital and The Texas Health Science Center College of Medicine and 6 Central Texas Veterans Health Care System, Temple, Texas 76502
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ABSTRACT |
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Recent studies have detected significant elevations of
interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver
transplantation. In both of these disorders, intrahepatic biliary
epithelial cells (BECs) are the targets of injury. We hypothesized that
BECs may themselves express IL-5 receptors that may modulate key
biliary functions. RNAs coding for IL-5 and -
receptors were
amplified by RT/PCR from a biliary cell line derived from a human
cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5
receptor distribution was detected immunocytochemically on Mz-ChA-1
cells, immortalized murine BEC, bile duct-ligated rat liver, and
isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed
that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated
chloride currents. Additional functional studies showed that IL-5
inhibits secretin-induced bile flow. We conclude that BECs express IL-5
receptors and that IL-5 modulates BEC chloride currents and fluid
secretion. Since IL-5 has previously been associated with cholestatic
liver disease, we speculate that IL-5 may contribute to liver injury
through its effects on biliary secretion.
cholestatic liver disease; chloride channel; patch-clamp recording
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INTRODUCTION |
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THE INTRAHEPATIC BILIARY EPITHELIUM forms the drainage system of the liver and secretes 15-30% of bile (5). Alterations in biliary epithelial cell (BEC) function or damage to this epithelium results in cholestasis, a prominent feature of many liver diseases affecting the intrahepatic biliary epithelium (6, 50). A subset of these disorders, including primary biliary cirrhosis (PBC), primary sclerosing cholangitis, and autoimmune cholangiopathy, are thought to result from immune-mediated bile duct injury (6, 48, 50).
Cytokines released into the microenvironment of the liver contribute to
cholestasis. Earlier studies examining possible mechanisms of
sepsis-induced cholestasis demonstrated that tumor necrosis factor-
and interleukin (IL)-6 inhibit hepatocyte bile transport by inhibition
of sodium-dependent bile acid uptake (25, 65). In
nonsepsis models, the supernatant of lymphocytes from humans with
either alcoholic or viral hepatitis reduced bile flow without any
accompanying morphological changes in the liver when injected into rats
(38). More recent studies of patients with PBC have found
increased levels of serum IL-5 (34) and hepatic parenchyma IL-5 mRNA (39). In parallel, significant increases in the
IL-5 mRNA have been found in liver of patients suffering from acute liver transplant rejection (23). However, the relationship
between these increased IL-5 levels and cholestasis-induced changes in the biliary epithelium have not been established.
In other secretory epithelia, altered regulation of chloride channels has been identified as a key mechanism of impaired secretion (12, 21, 22, 24, 35, 56). One of the better-studied causes for nonobstructive biliary cholestasis is that associated with cystic fibrosis (15, 63). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to defective cAMP-dependent chloride channel transport (49, 51) and may contribute to cholestasis (63). In the liver, CFTR has immunohistochemically and electrophysiologically been identified solely in the biliary epithelia (14, 41). Consequently, efforts are being made to identify how chloride channel function or dysfunction may contribute to other biliary forms of cholestasis.
Previously, IL-5 was referred to as eosinophil recruitment factor on the basis of its characteristic ability to increase migration of eosinophils into an area (59). In allergic forms of asthma, IL-5 is secreted in the airways and recruits eosinophils to this location, promoting the late phase bronchoconstriction characteristic of this disease (2). This effect can be abrogated by treating asthmatic animals with monoclonal antibodies to IL-5 (2). IL-5 is also thought to be pathogenic in Crohn's disease (18), helminthic disease (13), and eosinophilic gastroenteritis (17). Eosinophils and mast cells (46) surrounding the intrahepatic bile ducts is a distinctive feature of early stage PBC and is accompanied by an increase in peripheral eosinophilia (67). More interesting, it was found that those patients who responded best to ursodeoxycholic acid, the only well-established medical treatment for PBC, showed significant reductions in eosinophil counts (67). It is perhaps more than coincidental that cholestatic liver disease has recently been recognized as a complication of hypereosinophilic disorders (30, 64).
Given the propensity of bile duct epithelial cells to be involved in diverse liver diseases in which IL-5 concentration is augmented, we hypothesized that IL-5 receptor (IL-5R) may be present on BECs and may mediate a direct response of the biliary epithelium to IL-5. In other epithelial and nonepithelial cell types, interleukins, including IL-5 (45), have been found to rapidly modulate ion channel or other cellular processes (36, 53-55, 69). Given the established role of chloride transport in BECs, we performed these studies to determine whether IL-5R is expressed on BECs and whether IL-5 alters chloride conductance.
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EXPERIMENTAL PROCEDURES |
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Cell culture and tissue. Mz-ChA-1 cells were maintained in culture at 37°C in 5% CO2 in bicarbonate-containing CMRL-1066 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (684 µM).
Murine bile duct epithelial cells were a generous gift of Dr. Khazal Paradis (47). Cells were grown on Matrigel and maintained in culture at 33°C in 5% CO2 in DMEM-F-12 medium (GIBCO BRL) supplemented with nonessential amino acid solution, glucose, and penicillin/streptomycin. Because BEC hyperplasia is maximal in the first 2 wk (4), liver sections and isolated BECs were obtained from rats 1-2 wk after bile duct ligation. Ligation was performed using previously reported methods (41), and rats were fed a standard chow and maintained in the Animal Care Facilities according to the usual rodent care procedures. All animal procedures were approved by the Roudebush Veterans Affairs Medical Center, IUPUI (Indiana University), or Texas A&M Animal Care Committees as appropriate.RT-PCR.
Total RNA was prepared from Mz-ChA-1 cells by the method of Chomczynski
and Sacchi (11). For IL-5R, the RNA was reverse transcribed using a random primer and subsequently amplified by PCR
(first-strand cDNA synthesis and PCR core kits; Roche Diagnostics, Indianapolis, IN) using primers corresponding to bases 704-720 (5'
primer: CCCTTCACTGCACCTGG) and 1229-1213 (3' primer:
TGGCTCCACTCACTCCA) of the human IL-5R
cDNA sequence
(60). The 525-bp PCR product was isolated, subcloned into
pCR2.1 (Invitrogen, Carlsbad, CA), and sequenced to verify the identity
of the product. IL-5R
was similarly identified using primers
corresponding to bases 215-232 (5' primer: CGCCGGGTGAATGAGGAC) and
839-822 (3' primer: AGCTGGCCACCTCCTTCC) of the human IL-5R
cDNA sequence (28). A 625-bp fragment was isolated,
subcloned, and sequenced for verification. The subcloned fragments were
released by EcoR I digestion, separated on an agarose gel,
and stained with ethidium bromide.
Immunochemistry.
Using polyclonal antibodies specific to either human or rat/mouse
IL-5R (Santa Cruz Biotechnology, Santa Cruz, CA), immunocytochemical analysis was performed on human Mz-ChA-1 cells (33),
immortalized mouse BECs (47), and primary isolated rat
BECs (3). Immunohistochemical studies were performed on
frozen rat liver sections (4 µm), Tissues and cells were air dried
and subsequently fixed with either acetone or methanol for 10 min.
Endogenous peroxidase activity was quenched with 0.9% hydrogen
peroxide. Samples were blocked (1 h at room temperature) in nonimmune
goat serum before incubation with the primary antibodies overnight at
4°C in a humidity chamber. Studies were performed both with the
primary antibody (0.5 µg/ml) or the IL-5R
antibody neutralized
with a 10-fold excess of the peptide antigen fragment of IL-5R
(negative control). Bound antibody was detected by using an ABC Elite
kit and 3,3'-diaminobenzidine as a chromogen according to the
recommended protocols (Vector Laboratories, Burlingame, CA) before
counterstaining with hematoxylin was performed.
Isolation of small and large BECs.
As previously described (7, 8), small and large
cholangiocytes were obtained from normal and 1- to 2-wk bile
duct-ligated (BDL) rats. Following collagenase perfusion, a mixed
nonparenchymal cell fraction was obtained from undissociated liver
tissue by multiple enzymatic digestion (31). The
cholangiocyte-enriched fraction [~50% pure by histochemistry for
-glutamyltranspeptidase (52), a cholangiocyte-specific
marker (7, 8, 31)] was separated into two distinct
subpopulations of small and large cholangiocytes by counterflow
elutriation using a Beckman J6-MI centrifuge equipped with a JE-5.0
rotor (Beckman Instruments, Fullerton, CA). The two subpopulations of
small and large cholangiocytes were further purified by immunoaffinity
purification (7, 8, 31) with the use of a monoclonal
antibody ubiquitously expressed on all intrahepatic cholangiocytes
(31). Immunomagnetic beads were detached from the cells by
enzymatic digestion (31). Cell number and viability were
determined by trypan blue exclusion. Cholangiocyte purity was evaluated
by histochemistry for
-glutamyltranspeptidase. In agreement with
previous studies (7, 8), two pure and distinct
subpopulations of small (~8 µm diameter) and large (~15 µm
diameter) cholangiocytes were obtained from both normal and BDL rats.
Whole cell recording.
Using patch-clamp recording techniques (26), whole cell
currents were measured on Mz-ChA-1 cells ~24 h after passaging cells. Immediately before study, culture medium was replaced with NaCl-rich electrolyte buffer (see below) at room temperature (22-25°C). Cells were viewed through an inverted phase-contrast microscope using
Hoffman optics at a magnification of ×600 (Nikon Diaphot 300). Patch
pipettes were pulled from EN-1 glass capillary tubes (Garner Glass,
Claremont, CA) and had resistances of 3-10 M. Recordings were
made with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA),
and signals were filtered at 1-2 kHz bandwidth using a 4-pole low
pass Butterworth filter. Currents were digitized (1 kHz) for storage on
a Gateway 2000 486/66 computer and analyzed using pCLAMP v. 6.0 software (Axon Instruments).
Animal bile flow studies.
Fischer 344 male rats (150-175 g) were allowed 3-5 days of
acclimatization before undergoing bile duct ligation (61).
After 2 wk to permit hyperplasia of the biliary epithelium, the animals underwent laparotomy with reestablishment of biliary flow for subsequent modulation with saline, secretin (107 M in 1 ml), and various concentrations of IL-5 (1 ml) infused via separate
femoral veins at the rate of 0.1 ml/3 min. Following a 60-min basal
period, infusions were continued for 30 min and bile fractions were
collected in tarred tubes every 10 min. Saline injections were also
administered to replace the volume lost through bile output. Collected
fractions were subsequently weighed, and the volume of bile was
calculated assuming the density of bile to be 1 (61).
Effects on flow were determined by calculating the change in peak flow
following secretin/IL-5 infusion over the last 10 min of the basal
period. Student's t-test was used to determine whether the
effect was significant (P < 0.05).
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RESULTS |
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RT-PCR.
Like many cytokine receptors, IL-5R is a dimer of - and
-subunits
(1). The
-subunit is shared with the IL-3 receptor and
granulocyte-macrophage colony-stimulating receptor (1). In
contrast, the
-subunit is specific to IL-5R (60). The
expression of IL-5R on Mz-ChA-1 cells was initially studied by RT-PCR.
IL-5R
and -
RT/PCR products of 525 and 625 bp, respectively, were
amplified from Mz-ChA-1 cells, subcloned, and subsequently sequenced to confirm their identities (Fig. 1).
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Immunochemistry.
As shown in Fig. 2, A and
B, the expression of the unique IL-5R subunit was
immunocytochemically confirmed in Mz-ChA-1 cells. An immortalized
murine biliary cell line (47) also expresses IL-5R
(Fig. 2C), thereby demonstrating that IL-5R
expression is
not species specific or associated exclusively with biliary malignancy.
To further examine liver IL-5R
expression, we stained both normal
and BDL rat liver. On histological sections from unligated rats (Fig.
3, A and B), there
was no significant biliary cell staining. However, sections from 1- to
2-wk BDL rat liver (Fig. 3, C and D) showed
marked staining that appeared to be heterogeneously distributed.
Smaller ductules (interlobular ducts) stained more densely than larger
intrahepatic (septal) ducts. To better quantitate cell staining, BECs
were isolated from 1- to 2-wk BDL rat livers using the method of Alpini
et al. (7). A high percentage of positively staining cells
from small and large cell preparations were observed (Table
1; Fig. 4,
A and B). In contrast to the histological
sections; however, small and large BECs isolated from unligated animals
also stained positively, albeit at much lower levels (Table 1; Fig. 4,
C and D). In all cases, when the IL-5R
antibody was preabsorbed with the neutralizing peptide, there was no
staining (not shown).
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Whole cell recordings.
Finding IL-5R on BECs raises the possibility that this cell type is
responsive to IL-5. In lymphocytes, IL-5 is thought to modulate cell
growth and differentiation through ion movement (45).
Consequently, we used whole cell recordings of Mz-ChA-1 cells to
determine whether IL-5 inhibited NECA-stimulated chloride currents.
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Animal bile flow.
Because of the well-described effects that secretin has on stimulating
biliary cells to secrete bile, the inhibitory effects of IL-5 (0, 15, 30, 60, and 120 ng/ml) were tested in BDL rats simultaneously treated
with secretin. As shown in Fig. 6,
dose-dependent bile flow inhibition is observed when both secretin and
IL-5 are infused. Compared with 0 ng/ml, the inhibitory effect at both 60 and 120 ng/ml are statistically significant (P < 0.05). The calculated IC50 is ~43 ng/ml.
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DISCUSSION |
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To date, three chloride channels have been reported in BECs: a
high conductance anion channel (40) that is regulated by voltage and GTP-binding proteins (43), an outwardly
rectified calcium-dependent channel (9), and a linear,
cAMP-dependent channel (20). The last type has been
characterized as CFTR (41). Furthermore, CFTR has
previously been detected immunocytochemically on Mz-ChA-1 cells
(9). Since CFTR is the only known cAMP-stimulated chloride
channel on this epithelium, it is a possible target of IL-5 regulation.
Together, these data demonstrate that IL-5 inhibits NECA-stimulated
chloride currents in biliary cells, suggesting a possible link between
IL-5R and CFTR, and that IL-5 inhibits secretin-stimulated bile
flow in a BDL rat model.
Over the past few years, there has been an increasing recognition of
the role of various cytokines in diverse liver ailments, including
cholestasis (10, 44). Cholestatic injury appears to
involve both hepatocytes and biliary cells. A recently proposed model
for cytokine-induced cholestasis supports involvement of IL-1, IL-6,
IL-8, and tumor necrosis factor- (16). Other studies have demonstrated the capacity of an unidentified lymphocyte-derived factor to cause cholestasis in the absence of histological damage (38). IL-5 concentration is increased in both the hepatic
parenchyma (39) and serum (34) of patients
with PBC and acute liver transplant rejection (23),
although its role has not been studied. Our results provide novel
details concerning BEC expression of IL-5R and the effects of IL-5.
IL-5 is secreted by T helper cells (59), and its only
previously reported biological effects have been on hematopoietic cells: B lymphocytes, eosinophils, and IL-5-dependent hematopoietic cell lines (59). In addition, IL-5R was identified on
cells forming the sinus mucosa in humans with allergic and nonallergic rhinitis (66). However, the cell type in the mucosa strip
that expressed the IL-5R was not elucidated. In contrast, using RT-PCR, our study is the first to conclusively demonstrate the expression of
IL-5R on a nonhematopoietic cell type, namely a human
cholangiocarcinoma cell line. Using immunocytochemistry, we identified
IL-5R expression on cells derived from a human cholangiocarcinoma,
mouse immortalized BECs, and isolated rat BECs, and immunohistochemical
staining shows what appears to be enhanced staining of BECs in
BDL rat liver. Bile duct ligation appeared to correlate with enhanced expression both in isolated cells and intact liver, suggesting that the
enhanced staining observed in tissue slices is not simply an
amplification resulting from biliary cell hyperplasia.
Biliary IL-5R expression appears to be regulated by either cell size or location. For example, the expression of the secretin receptor is heterogeneous. Binding of secretin to its receptor results in the activation of CFTR and subsequent secretion of fluid and bicarbonate (41). Secretin receptors are predominantly expressed on medium-to-large intrahepatic BECs (7), whereas CFTR is distributed evenly throughout the liver (68). In contrast, greater expression of IL-5R was observed in the smaller cells associated with smaller bile ducts. Interestingly, it is these smaller ducts and duct cells that appear to be involved in PBC, a disorder associated with increased eosinophils (32) and increased IL-5 (eosinophil recruitment factor) in liver (39). Thus our studies on IL-5R further direct our understanding of the regional distribution and regulation of channels that are broadly distributed throughout the biliary tree.
IL-5, like other cytokines, can regulate transcription
(1). Generally, this requires multiple hours of treatment.
However, a variety of interleukins have also been shown to elicit more immediate effects on ion channels and secretion (Table
3). The inhibition of cAMP-dependent
chloride currents by IL-5 is the first example of such a
cytokine-induced effect in BECs.
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At this point, the timing and connections between increased parenchymal IL-5, the induction of IL-5R expression on BECs, and the functional effects of cAMP-dependent chloride channel inhibition are unknown. Either by a decrease in chloride secretion or an effect on transmembrane potential, inhibition of CFTR may lead to impaired electrolyte and fluid secretion and thus contribute to impaired bile flow and possibly bile acid-induced cytotoxicity. This cytotoxicity toward bile ducts may lead to loss of cell numbers, as seen in ductopenic liver disease (e.g., PBC, rejection). Perhaps at earlier stages, as classically seen in PBC and primary sclerosing cholangitis, IL-5 may promote proliferation either through modulation of transcription factors or inhibition of apoptosis. Although many questions remain unanswered, these studies suggest that the elevated IL-5 concentrations seen in certain cholestatic disorders may be more than coincidental. Future studies will be required to explore the mechanisms of IL-5 regulation of chloride channel function and possible connections between altered ion movement and biliary cell viability.
In summary, IL-5R has been identified on rat BECs as well as mouse and human biliary cell lines. IL-5 inhibits NECA-stimulated biliary cell chloride currents and secretin-stimulated bile flow in BDL rats in a dose-dependent manner. The overall influence of IL-5 on BEC function, including its mechanisms of channel regulation and modulation of bile flow, appears to be a promising area for future investigation.
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ACKNOWLEDGEMENTS |
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We thank Wanda Thurman and Shannon Glaser for their technical assistance and Kathleen Boles for her secretarial support. We appreciate the valuable assistance of Drs. Stephen Hall and Raymond Galinsky for their guidance in determining the optimum time and dosing of IL-5 for the animal studies. Additional thanks to Diana Baxter for her valuable assistance with our illustrations.
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FOOTNOTES |
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Support for this work was provided from separate Veterans Affairs Merit Review Awards (J. M. McGill and G. Alpini), a Glaxo Institute for Digestive Health Award (J. M. McGill), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51080 (A. P. Stansfield), and Scott & White Hospital and Texas A&M University (G. Alpini and G. LeSage).
Address for reprint requests and other correspondence: J. M. McGill, Eli Lilly and Co., Lilly Corporate Center, DC 2133, Indianapolis, IN 46285 (E-mail: jmcgill{at}lilly.com).
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.
Received 2 December 1999; accepted in final form 13 October 2000.
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