Interleukin-5 inhibition of biliary cell chloride currents and bile flow

James M. McGill1,2, Margaret S. Yen2, Oscar W. Cummings3, Gianfranco Alpini5,6, Gene LeSage5, Karen E. Pollok4, Barbara Miller1, Steven K. Engle2, and Ann P. Stansfield2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-5alpha and -beta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-5Ralpha , 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-5Ralpha 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-5Rbeta was similarly identified using primers corresponding to bases 215-232 (5' primer: CGCCGGGTGAATGAGGAC) and 839-822 (3' primer: AGCTGGCCACCTCCTTCC) of the human IL-5Rbeta 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-5Ralpha (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-5Ralpha antibody neutralized with a 10-fold excess of the peptide antigen fragment of IL-5Ralpha (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 gamma -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 gamma -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.

Cells were prepared for immunohistochemistry by cytospinning (100 g for 5 min) smears of small and large cholangiocytes from normal or BDL rats. Following purification, cytospun smears of small and large cholangiocytes were fixed in cold methanol for 10 min, air dried, rinsed in 1× PBS, pH 7.4, and stored at -70°C before continuing with the quenching of endogenous peroxidase activity (see Immunochemistry).

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 MOmega . 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).

Following formation of the whole cell configuration, cells were unperturbed for a 2-min basal period. During this period, cells were clamped at -40 mV and depolarized to +60 mV. Current-voltage relationships were determined by measuring the current generated from -120 mV to +120 mV at 20 mV incremental 400-ms sweeps from a holding potential of -40 mV. 5'-N-ethylcarboxamidoadenosine (NECA, 200 µM; RBI, Natick, MA) was subsequently applied to the extracellular bathing medium. NECA is a selective A1/A2 adenosine receptor agonist that modulates intracellular cAMP levels (19) and has previously been shown to activate cAMP-dependent chloride currents in this cell type (42). Peak currents were recorded after 90-120 s. Following stimulation with NECA, IL-5 (Sigma, St. Louis, MO) was applied to the extracellular bathing medium at either 0.11 or 0.33 ng/ml, the half-maximal dose for lymphocyte activation in cell culture conditions (62).

The standard NaCl-rich extracellular buffer contained (in mM): 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 4.8 glucose, and 10 HEPES/NaOH, (pH ~7.30) with a total chloride of 150 mM. The standard KCl-rich pipette solution contained (in mM): 130 KCl, 10 NaCl, 2 MgCl2, and 10 HEPES/KOH, (pH ~7.20), with free calcium adjusted to ~150 nM (0.5 CaCl2, 1 EGTA) and a total chloride of 145 mM.

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 (10-7 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).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RT-PCR. Like many cytokine receptors, IL-5R is a dimer of alpha - and beta -subunits (1). The beta -subunit is shared with the IL-3 receptor and granulocyte-macrophage colony-stimulating receptor (1). In contrast, the alpha -subunit is specific to IL-5R (60). The expression of IL-5R on Mz-ChA-1 cells was initially studied by RT-PCR. IL-5Ralpha and -beta 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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Fig. 1.   Interleukin (IL)-5 receptor (R) subunits alpha  and beta  are both expressed in Mz-ChA-1 cells. Specific IL-5Ralpha - and beta -specific fragments were amplified by RT-PCR from total RNA. The cDNA products were subsequently subcloned into pCR2.1 plasmid and sequenced to verify their identities. Plasmids were digested with EcoR I and separated on a 1% agarose gel. std; Molecular weight standard.

Immunochemistry. As shown in Fig. 2, A and B, the expression of the unique IL-5Ralpha subunit was immunocytochemically confirmed in Mz-ChA-1 cells. An immortalized murine biliary cell line (47) also expresses IL-5Ralpha (Fig. 2C), thereby demonstrating that IL-5Ralpha expression is not species specific or associated exclusively with biliary malignancy. To further examine liver IL-5Ralpha 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-5Ralpha antibody was preabsorbed with the neutralizing peptide, there was no staining (not shown).


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Fig. 2.   Immunohistochemical detection of IL-5Ralpha on bile duct epithelial cell (BDC) lines. Mz-ChA-1 cells were incubated with IL-5Ralpha antibody preabsorbed with antigenic peptide (A, negative control) or incubated with the antibody alone (B). Immortalized murine BDCs were incubated with IL-5Ralpha antibody (C). Detection of bound antibody is outlined in EXPERIMENTAL PROCEDURES.



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Fig. 3.   Detection of IL-5Ralpha on normal and bile duct-ligated (BDL) rat liver sections. Frozen sections from normal (A and B) or 1- to 2-wk BDL rat liver (C and D) were fixed in methanol and stained for IL-5Ralpha as described in EXPERIMENTAL PROCEDURES. Photos of the bile duct epithelium were taken at ×13 (A and C) and ×66 (B and D) magnification. Control slides (antibody preabsorbed with antigenic peptide) were negative (data not shown).


                              
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Table 1.   Isolated small and large BECs staining positively for IL-5Ralpha from bile duct unligated and ligated rats after 1 wk



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Fig. 4.   Detection of IL-5Ralpha on isolated large and small biliary epithelial cells (BECs) from normal and BDL liver. Small or large BECs from 1- to 2-wk BDL or normal rat livers were isolated as described in EXPERIMENTAL PROCEDURES. Expression of IL-5Ralpha on these cells was examined using immunohistochemical approaches. Cytokine expression was detected in small, 1- to 2-wk BDL (A), large, 1- to 2-wk BDL (B), small, normal (C), and large, normal (D) BECs to various degrees. The small spherical structures are the residual magnetic beads.

Whole cell recordings. Finding IL-5Ralpha 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.

Similarly to T84 colonocytes (58), NECA (an A1/A2 receptor agonist) increases chloride secretion in both primary isolated rat BECs and Mz-ChA-1 cells (42). Both primary and cholangiocarcinoma cells exhibit a reduced response to the A1-selective agonist R(-)-PIA, indicating that the adenosine receptor subtype associated with chloride secretion is of the A2 category. A2 receptors activate cAMP signal transduction pathways (19). In Mz-ChA-1 cells, as well as human immortalized H69 biliary cells, cAMP-dependent whole cell currents consistent with CFTR have been observed by St-Pierre et al. (57). Because of the reported effects of NECA on this cell line and the absence of demonstrated secretin receptors on Mz-ChA-1 cells, NECA was chosen as the pharmacological stimulus. Cells stimulated with NECA (200 µM) showed a characteristic 900% increase in currents at 100 mV (Fig. 5; Table 2). Subsequent addition of IL-5 (0.33 ng/ml) to NECA-stimulated cells resulted in near complete inhibition of chloride currents (n = 9). IL-5 alone had no effect on whole cell currents.


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Fig. 5.   IL-5 inhibits cAMP-dependent chloride currents in BDCs. 5'-N-ethylcarboxamidoadenosine (NECA, 200 µM) was applied to the extracellular bathing medium, which resulted in an ~9-fold increase in linear conductance (B) compared with baseline currents (A). This response is characteristic of the cAMP-dependent current in BECs (42). When exposed to a partial dose of IL-5 (0.11 ng/ml; C), there was an ~50% decrease in currents. When exposed to additional IL-5 (0.33 ng/ml; D), there was a near-complete inhibition of current. The linear current-voltage (I-V) plots for the NECA-activated and NECA-activated, IL-5-inhibited currents are shown (E). These linear current traces are characteristic of cAMP-dependent chloride currents in BECs (41).


                              
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Table 2.   BEC whole cell currents resulting from serial treatment with NECA and IL-5

Although IL-5 can bind to IL-5Ralpha alone, it does so with low affinity [dissociation constant (Kd) 20 nM; Ref. 37], whereas the IL-5Ralpha beta dimer binds IL-5 with much higher affinity (Kd 0.15 nM; Ref. 37). The low dose of IL-5 used in these experiments is consistent with the activation of the alpha beta -dimer and is within the EC50 of 0.1-0.5 ng/ml previously demonstrated in hematopoietic cells (62).

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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Fig. 6.   IL-5 inhibits secretin-stimulated bile flow in BDL rats. Two weeks after bile duct ligation, bile flow was reestablished and 10-min fractions of bile were collected from 0 to 300 min. Following a 60-min basal period, secretin (10-7 M) and IL-5 [0 (saline), 15, 30, 60, and 120 ng/ml] were infused over 30 min into separate femoral veins. The mean change in peak flow (± SE) compared with the last 20 min of basal was measured and reported in ml · kg-1 · min-1. Both 60 and 120 ng/ml IL-5 resulted in statistically significant inhibition of bile flow compared with saline infusion (P < 0.05). The approximate IC50 of this dose response effect was 43 ng/ml.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-5Ralpha beta 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-alpha (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-5Ralpha 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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Table 3.   Examples of interleukin binding to receptor cells resulting in rapid (seconds to minutes) cellular responses

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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