Peptide antibiotic human beta-defensin-1 and -2 contribute to antimicrobial defense of the intrahepatic biliary tree

Authors


Abstract

Human beta-defensins (hBDs) are important antimicrobial peptides that contribute to innate immunity at mucosal surfaces. This study was undertaken to investigate the expression of hBD-1 and hBD-2 in intrahepatic biliary epithelial cells in specimens of human liver, and 4 cultured cell lines (2 consisting of biliary epithelial cells and 2 cholangiocarcinoma cells). In addition, hBD-1 and hBD-2 were assayed in specimens of bile. hBD-1 was nonspecifically expressed immunohistochemically in intrahepatic biliary epithelium and hepatocytes in all patients studied, but expression of hBD-2 was restricted to large intrahepatic bile ducts in 8 of 10 patients with extrahepatic biliary obstruction (EBO), 7 of 11 with hepatolithiasis, 1 of 6 with primary biliary cirrhosis (PBC), 1 of 5 with primary sclerosing cholangitis (PSC), 0 of 6 with chronic hepatitis C (CH-C), and 0 of 11 with normal hepatic histology. hBD-2 expression was evident in bile ducts exhibiting active inflammation. Serum C reactive protein levels correlated with biliary epithelial expression of hBD-2. Real-time PCR revealed that in all of 28 specimens of fresh liver, including specimens from patients with hepatolithiasis, PBC, PSC, CH-C and normal hepatic histology, hBD-1 messenger RNA was consistently expressed, whereas hBD-2 messenger RNA was selectively expressed in biliary epithelium of patients with hepatolithiasis. Immunobloting analysis revealed hBD-2 protein in bile in 1 of 3 patients with PSC, 1 of 3 with PBC, and each of 6 with hepatolithiasis; in contrast, hBD-1 was detectable in all bile samples examined. Four cultured biliary epithelial cell lines consistently expressed hBD-1; in contrast these cell lines did not express hBD-2 spontaneously but were induced to express hBD-2 by treatment with Eschericia coli, lipopolysaccharide, interleukin-1β or tumor necrosis factor-α. In conclusion, these findings suggest that in the intrahepatic biliary tree, hBD-2 is expressed in response to local infection and/or active inflammation, whereas hBD-1 may constitute a preexisting component of the biliary antimicrobial defense system. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:925–932.)

Human bile is sterile under normal conditions. However, there have been several reports that bacterial components, such as lipopolysaccharide (LPS), may be detected in normal bile.1–3 Furthermore, in inflammatory biliary diseases, bacteria have been cultured from bile.1, 4–6 The biliary tract drains directly into the duodenum, where the biliary epithelium may potentially be exposed to bacteria and bacterial components. Duodenal microorganisms are believed to be a major source of bacterial infection in several biliary diseases. In particular, enteric bacteria, demonstrable in bile, may be responsible for chronic proliferative cholangitis associated with hepatolithiasis.7, 8 Several defense mechanisms tend to protect the biliary tract from bacterial invasion; these include physical, chemical, and immunological factors.6, 9, 10

The first line of defense against invading pathogens is the innate immune system.11 Key elements of this system are defensins, which are antimicrobial peptides.12 Structurally, defensins are a family of cationic antimicrobial peptides; they are divided into α- and β-subfamilies.11, 12 The β-defensin family is found in the epithelia of several organs; it constitutes an important barrier at mucosal surfaces.11 So far, at least 6 human β-defensins (hBD-1 to -6) have been identified.13 Among them, hBD-1 and hBD-2 are well recognized to mediate salt-sensitive microbicidal activity.14 hBD-1 is extensively expressed in broad epithelia, including that of the gastrointestinal tracts.11, 15–18 hBD-2 is most abundant in inflammed skin and lung; its expression in other epithelial organs is low.19, 20 Interestingly, hBD-2 expression is induced by exposure to bacteria or cytokines, such as tumor necrosis factor (TNF)-α.14

Several studies have demonstrated the importance of the antimicrobial actions of hBD-1 and hBD-2 in several organs, such as the bronchi and gastrointestinal tract.12, 15, 18, 21, 22 So far, the expression of hBD-1 and hBD-2 in the hepatobiliary system and their roles in hepatobiliary pathophysiology have been poorly evaluated.

In this study, we examined immunohistochemically the distribution of hBD-1 and hBD-2 along intrahepatic bile ducts in certain hepatobiliary diseases. Also, using cultured human biliary epithelial cells (hBECs) and intrahepatic cholangiocarcinoma (ICC) cells, we assessed whether hBD-1 and hBD-2 expression in hBECs is influenced by bacterial products and inflammatory cytokines.

Abbreviations:

LPS, lipopolysaccharide; hBD, human beta-defensin; TNF, tumor necrosis factor; hBECs, human biliary epithelial cells; ICC, intrahepatic cholangiocarcinoma; EBO, extrahepatic biliary obstruction; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; CH-C, chronic hepatitis; ISH, in situ hybridization; IgG, immunoglobulin G; mRNA, messenger RNA; RT-PCR, reverse transcription polymerase chain reaction; E. coli, Escherichia coli; IL, interleukin; IFN, interferon; CRP, C-reactive protein; PBS, phosphate-buffered saline.

Patients and Methods

Anatomy of the Intrahepatic Biliary Tree

The intrahepatic biliary tree was classified into large and small bile ducts (Fig. 1).23 The former are macroscopically visible (diameter >300 μm); they have a fibrous wall and are associated with peribiliary glands. They represent the first to the fourth branches of the right and left hepatic ducts. The small bile ducts are recognized under a microscope; they are classified into septal and interlobular bile ducts and bile ductules, based on their size and location within portal tracts.

Figure 1.

Schema of the intrahepatic biliary tree. The intrahepatic biliary tree is classified into large intrahepatic bile ducts, septal bile ducts, interlobular bile ducts, and bile ductules. Large bile ducts are recognizable by the naked eye, and they are consistently associated with peribiliary glands. Septal bile ducts are microscopic; they consist of tall columnar cells, which have oval nuclei at their basal pole, and thick fibrous walls. Interlobular bile ducts are composed of cuboidal cells, in the absence of fibrotic periductal walls; they are accompanied by a small branch of the portal vein and a small branch of the hepatic artery. Bile ductules are the smallest ductal element; they occur in the periphery of portal tracts and are directly associated with hepatocytes.

Patients and Tissue Preparation

All tissue specimens were obtained from our Department of Human Pathology. Consent for research on human subjects was obtained from each patient that had provided a specimen before undertaking experimental procedures. The protocol of this study was approved by the Ethics Committee of Kanazawa University.

Formalin-fixed Specimens.

A total of 49 surgical, autopsy, or explant liver specimens were obtained from 10 patients with extrahepatic biliary obstruction (EBO), 11 with hepatolithiasis, 6 with primary biliary cirrhosis (PBC), 5 with primary sclerosing cholangitis (PSC), 6 with chronic hepatitis C (CH-C), and 11 with normal hepatic histology. Characteristics of these patients are given in Table 1. Explanted livers were obtained at liver transplantation. In patients with hepatolithiasis, stones contained brown pigment, and stone-containing ducts exhibited fibrosis, an inflammatory cell infiltrate, and peribiliary proliferation (chronic proliferative cholangitis).7 Three patients with hepatolithiasis had stones at the bifurcation of common hepatic duct and cholestasis. Liver tissue of 5 of 6 patients with CH-C was obtained from tumor-free parts of liver that had been surgically resected for hepatocellular carcinoma. Specimens of liver tissue that showed no histological abnormality were obtained from tumor-free parts of specimens of liver from 3 patients who had undergone surgical resection of hepatic metastases; these specimens were designated “normal hepatic histology.” Large intrahepatic bile ducts were present in all specimens.

Table 1. Main Clinicolaboratory Features of Hepatobiliary Diseases Studied
DiseaseCase NumberAverage age (yrs)Sex M:FSample S:A:ECRP (mg/dL)ALP (IU/L)γGTP (IU/L)T.bil (mg/dL)
  • NOTE. Laboratory data shown as the mean of each category and parenthesis is range.

  • Abbrevations: EBO, extrahepatic biliary obstruction; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; CVH, chronic viral hepatitis; CRP: C-reactive protein; ALP: alkaline phosphatase; γGTP: γ-glutamyltranspeptitase; T.bil: total bilirubin; M:F, male:female; S:A:E, surgical:autopseid:explanted.

  • *

    Histological stage IV.

  • Except one case (CRP 1.3mg/dL).

Hepatolithiasis11655:611:0:03.5 (0.3–9.6)315 (176–479)125 (39–174)2.0 (0.8–4.5)
EBO10756:42:8:08.8 (2.3–18.3)612 (209–1557)102 (24–260)6.3 (0.7–13.6)
PBC*6520:60:1:53.4 (1.2–5.4)486 (265–898)54 (37–107)18.4 (1.2–32.6)
PSC*5362:30:1:43.0 (0.5–4.8)498 (311–732)66 (58–80)13.7 (1.5–21.6)
CVH6644:25:0:1<0.4249 (182–366)54 (26–83)0.8 (0.3–1.8)
Normal liver11627:43:8:0<0.4233 (154–276)25 (20–33)0.7 (0.2–1.5)

Among the patients with hepatolithiasis, 3 surgically resected gallbladders that did not contain stones were available. In addition, liver specimens were obtained at autopsy of 3 patients (65-86 years old; 2 males and 1 female) who had had fatal extrahepatic sepsis (pulmonary infection in 2, and urinary tract infection in 1).

All of the liver and gallbladder specimens were fixed in neutral formalin and embedded in paraffin. Four-micrometer-thick sections were prepared for routine histology and immunohistochemistry.

Fresh Specimens.

Fresh surgical or explanted liver specimens containing large intrahepatic bile ducts were available from 6 patients with hepatolithiasis, 6 with PBC, 4 with PSC, 6 with CH-C, and 6 with normal hepatic histology. Two of the patients with hepatolithiasis had cholestasis. Fresh liver specimens were cut into several pieces for RNA extraction. The remaining tissue was fixed in 4% paraformaldehyde and embedded in paraffin; 4-μm-thick sections were prepared for in situ hybridization (ISH).

Immunohistochemistry of hBD-1 and hBD-2

Expression of hBD-1 and hBD-2 was assessed immunohistochemically in formalin-fixed sections. Briefly, for assessment of hBD-1, deparaffinized and rehydrated sections were microwaved in 10 mmol/L citrate buffer for 20 minutes; for assessment of hBD-2, similar sections were treated with 0.1 % trypsin for 20 minutes.24 After blocking endogenous peroxidase, sections were incubated with polyclonal antibodies against hBD-1 (goat immunoglobulin G (IgG), 1 μg/mL, a gift from coauthors T.H. and A.W.) or hBD-2 (rabbit IgG, Nagasaki University, Japan24) at 4°C overnight and then incubated with rabbit anti-goat immunoglobulin conjugated to peroxidase-labeled polymer (Simple Staining Kit, Nichirei, Tokyo, Japan) or goat anti-rabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer (Envision, Dako, Tokyo, Japan) at room temperature for 1 hour, respectively. After reacting with benzidine, sections were lightly counterstained with hematoxylin. To generate a negative control, normal rabbit IgG or goat IgG was used instead of the primary antibody.

Detection of Messenger RNA (mRNA) of hBD-1 and hBD-2 in Liver Tissue

To confirm the presence of hBD-1 and hBD-2 mRNA in liver tissue, reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and ISH were undertaken.

RT-PCR and Real-Time PCR.

Total RNA was isolated from fresh liver using the RNeasy Total RNA System (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For RT, 1 μg of total RNA was reverse transcribed with oligo-dT primers and RTase in a total volume of 20 μL; 0.5 μL was used as a template for PCR. Specific primers were designed as follows: hBD-1 forward, 5′-CATGAGAACTTCCTACCTTCTG-3′, and reverse, 5′-GCTCACTTGCAGCACTTGGCC-3′; hBD-2, forward, 5′-CCAGCCATCAGCCATGAGGGT-3′, and reverse, 5′-GGAGCCCTTTCTGAATCCGCA-3′. The predicted product sizes were 210 base pairs for hBD-1 and 255 base pairs for hBD-2. To generate a positive control, the primers for β-actin mRNA (forward, 5′-CAAGAGATGGCCACGGCTGCT-3′; reverse, 5′-TCCTTCTGCATCCTGTCGGCA-3′) were used to amplify a 275 base-pair portion. The PCR profile consisted of an initial denaturation at 94°C for 3 minutes followed by 28 cycles (hBD-1 and hBD-2) or 20 cycles (β-actin): 30-second denaturation at 94°C, 30-second annealing of primers at 62°C, and 60-second extension at 72°C. After PCR, 5-μL aliquots of the products were subjected to 1.5% agarose gel electrophoresis. One of the amplicons was reamplified using the primers containing T7- and Sp6-RNA polymerase promoter to transcribe RNA in vitro. Real-time PCR was undertaken for quantitative analysis using the SYBER Green PCR Master Mix and ABI PRISM 7700 Sequence Detection System (Applied Biosystems Japan, Tokyo, Japan) according to the standard protocol.

ISH.

Using the PCR-amplified products containing T7- and Sp6-RNA polymerase promoter, antisense and sense digoxigenin-labeled RNA probes for hBD-1 and hBD-2 mRNA were generated by in vitro transcription using the Digoxigenin RNA Transcription Kit (Roche Diagnostics, Indianapolis, IN) according to the standard protocol. Deparaffinized sections of paraformaldehyde-fixed specimens were incubated with 10 μg/mL proteinase K for 10 minutes at 37°C and then dehydrated in ethanol and air. The sections were incubated with a hybridization solution containing digoxigenin-labeled probes (1 μg/mL) at 50°C for 16 hours. After washing in a sodium chloride–sodium citrate solution (SSC, final concentration 0.2 × SSC, Sigma, St. Louis, MO), the sections were incubated with alkaline phosphatase-conjugated antidigoxigenin antibody (Roche) for 1 hour. Color development was achieved by adding a prepared substrate solution that contained nitroblue tetrazolium salt (450 μg/mL) and 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt (175 μg/mL) to the slides for 3 hours.

Preparation of Bile and Immunoblot Analysis

Bile samples were obtained from the affected bile ducts of 6 of the patients with hepatolithiasis, and from the gallbladders of 3 of the patients with PBC and 3 of those with PSC.

Protein Extraction.

Bile samples were homogenized in protein lysis solution (Tissue Protein Extraction Reagent, Pierce, Rockford, IL). After centrifugation, the supernatant was transferred to a fresh tube; the concentration of total protein in the supernatant was measured using a Bradford-based assay and the concentration was then adjusted to 1 μg/μL.

Dot Blotting.

Two-microgram protein solution extracted from bile or 3-μL medium of cell culture were directly applied to a nitrocellulose membrane. After blocking in 5% bovine serum albumin, the membrane was first incubated with mouse monoclonal anti-human CD64 (diluted 1:10, Dako) for 30 minutes, to eliminate nonspecific binding to Fc receptors of human immunoglobulin in bile,25 and then probed for 1 hour with polyclonal antibodies against hBD-1 or hBD-2 (goat IgG, 1 μg/mL, Santa Cruz Technologies). After washing, the membranes were incubated with rabbit anti-goat immunoglobulin conjugated to peroxidase-labeled dextran polymer (Nichirei) for 1 hour and then reacted with benzidine.

Cell Cultures

Two human ICC cell lines (CCKS126, 27 and HuCCT128), and 2 hBEC lines (1 isolated from explanted liver of a patient with CH-C, and the other isolated from explanted liver of a patient with PBC) (hBEC1 and hBEC2, respectively) were used. HuCCT1 was purchased from Health Science Research Resources Bank (Osaka, Japan). CCKS1 was established in our laboratory.26, 27 hBEC1 was a gift from Dr. Takashi Kamihira (Kyushu University, Japan).29 hBEC2 was established in our laboratory. CCKS1 and HuCCT1 lines were grown as monolayers in suspension cultures using a standard medium—RPMI 1640 or D-MEM/F-12 (Gibco, Rockville, MD), respectively; the media were supplemented with 10% fetal calf serum and an antibiotic/antimycotic solution (Invitrogen Japan, Tokyo, Japan). hBECs were incubated in a culture medium that contained D-MEM/F-12, Nu-Serum (Becton Dickinson, Bedford, MA), ITS+ (Becton Dickinson), 5 μmol; forskolin (Wako, Osaka, Japan), 12.5 mg/mL bovine pituitary extract (Gibco), 1 μmol dexamethasone (Sigma), 5 μmol triiodo-thyronine (Sigma), 5 mg/mL glucose (Sigma), 25 mmol/L sodium bicarbonate (Sigma), 1 % antibiotic-antimycotic solution, 20 ng/mL of human epidermal growth factor (Gibco), and 10 ng/mL human hepatocyte growth factor (Gibco). A HepG2 cell line (derived from a human hepatocellular carcinoma, RCB1648, RIKEN Cell Bank, Tsukuba, Japan) was used as a control. The cell lines were cultured at 37°C in a water-saturated atmosphere of 95% air and 5% CO2.

Treatment with Escherichia coli (E. coli), LPS, and Cytokines.

The effects of E. coli, LPS, and inflammatory cytokines on the expression of hBDs in cultured cells were determined. A strain of E. coli, ATCC25922 (American Type Culture Collection, Rockville, MD), grown in Luria-Bertani medium (Wako), was collected at the exponential growth phase, and then killed by autoclaving. LPS (derived from E. coli O55:B5, Sigma), and recombinant human cytokines, interleukin (IL)-1β (PeproTech, London, UK), IL-4 (PeproTech), IL-5 (PeproTech), IL-6 (PeproTech), interferon (IFN)-γ (Sigma), and TNF-α(Sigma), were used to stimulate cell lines.

HuCCT1, CCKS1, hBEC1, hBEC2, and HepG2 grown to semiconfluence in culture plates were treated with no stimulants (medium only), LPS (1 μg/mL), or autoclaved E. coli (1 μg/mL) in fresh minimal standard medium for 3 and 12 hours. The cells were then washed 3 times before isolation of their RNA. Expression of mRNA of hBDs in RNA extracts of cultured cells was assessed by RT-PCR and real-time PCR. In addition, these 5 cell lines, grown to semiconfluence in plates, were treated with each of the 6 cytokines at a concentration of 1,000 U/mL in fresh minimal standard medium for 3 hours; the same approach was adopted to assess expression of mRNA of hBDs in cytokine-treated cells.

HuCCT1 and hBEC1 grown to semiconfluence in culture plates were treated with LPS (1 μg/mL) in fresh minimal standard medium for 12 hours; proteins of hBDs were then assessed in culture supernatants as described in Preparation of Bile and Immunoblot Analysis.

RT-PCR and Real-Time PCR for mRNA of hBD-1 and hBD-2.

Total RNA was isolated from cultured cells using the RNeasy Total RNA System (Qiagen) according to the manufacturer's instructions. The methods used for applying RT-PCR and real-time PCR were the same as those applied to fresh liver tissue.

Statistical Analysis

Welch's t test or Mann-Whitney U test was applied when appropriate. A P value of less than .05 was considered to be significant.

Results

Tissue Expression of hBD-1 and hBD-2

Diffuse and granular immunohistochemical patterns of expression of hBD-1 occurred in the cytoplasm of intrahepatic bile duct epithelial cells. In all patients there was no gradient in the intesity of staining at different anatomical sites or in hepatocytes. The staining intensity in hBECs was similar to or weaker than that in hepatocytes (Supplementary Fig. 1). In contrast, expression of hBD-2 was restricted to large intrahepatic bile ducts and peribiliary glands (Supplementary Fig. 1); no expression of hBD-2 was detected in hepatocytes in any liver specimen. Expression of hBD-2 in large bile ducts was found in 8 of 10 patients with EBO, 7 of 11 with hepatolithiasis, 1 of 6 with PBC, 1 of 5 with PSC, 0 of 6 with CH-C, and 0 of 11 with normal hepatic histology; its prevalence in EBO and hepatolithiasis was higher than that in other liver diseases (P < .05). hBD-2 expression was heterogeneous in the intrahepatic biliary tree; it occurred particularly in large bile ducts in which there was an appreciable polymorphonuclear inflammatory infiltrate but not in minimally inflamed portions of bile ducts (Supplementary Fig. 1). hBD-2 was often detectable in the luminal secretion of large intrahepatic bile ducts. Moreover, hBD-1- and hBD-2-positive mononuclear cells were found in inflammed portal tracts; this finding seemed to correlate with mRNA-positive cells demonstrated by ISH (see ISH Study).30

The serum level of C-reactive protein (CRP) was higher in patients with biliary epithelial expression of hBD-2 (7.4 ± 5.1 mg/dL SD) than in those without such expression (1.5 ± 1.7 mg/dL) (Table 1 and Fig. 2). In 3 patients with hepatolithiasis who expressed hBD-1 and hBD-2 in their stone-containing intrahepatic bile ducts, hBD-1 but not hBD-2 was expressed in the lining epithelium of their gallbladders. Furthermore, in autopsy livers from 3 patients with extrahepatic sepsis, biliary epithelial expression of hBD-2 was not detected.

Figure 2.

Mean values and SDs (bars) of serum C-reactive protein (CRP) levels in human beta defensin (hBD)-2-positive and -negative groups. Open circles indicate individual values for all the patients studied using immunohistochemistry. The patients who expressed hBD-2 had higher levels of CRP than those who did not express hBD-2 (*P < .05). Bars are SDs.

Detection of hBD-1 and hBD-2 mRNAs in Tissue

RT-PCR and Real-Time PCR.

Amplification of hBD-1 mRNA occurred in all liver specimens. However, amplification of hBD-2 mRNA occurred in 1 of 4 patients with PSC, 3 of 6 with hepatolithiasis, and in none of the other patients. Real-time PCR revealed that the level of hBD-1 mRNA was not different among the patient groups (Supplementary Fig. 2). However, the level of hBD-2 mRNA was significantly greater in hepatolithiasis (particularly in 3 patients) than in PBC, CH-C, or normal liver (Supplementary Fig. 2). The serum level of CRP was particularly high in the 3 patients with hepatolithiasis who had the highest levels of hBD-2 mRNA; 2 of these patients had choloestasis. There were no other correlations between levels of hBD-2 mRNA and other routine laboratory findings.

ISH Study.

Low levels of hBD-1 mRNA were found in the cytoplasm of large bile ducts and hepatocytes in all specimens; such levels were appreciable in large bile ducts of patients with hepatolithiasis associated with active cholangitis (Fig. 3). hBD-2 mRNA was detectable in large bile ducts in 2 of 6 patients with hepatolithiasis, but it was not detectable in bile ducts of other patients; these ISH-positive patients also exhibited increased levels of hBD-2 mRNA detected by real-time PCR. In addition, mononuclear cells positive for hBD-1 and hBD-2 mRNA were found in periductal zones.30 These specific signals were not detectable when hybridized with the sense probe for these mRNA sequences.

Figure 3.

In situ hybridization staining for human beta defensin (hBD)-1 messenger RNA (mRNA) with antisense (A) and sense (B) probes and for hBD-2 with antisense (C) and sense (D) probes. (A and C) In hepatolithiasis, large intrahepatic bile ducts that exhibit cholangitis and biliary epithelial hyperplasia are positive for both hBD-1 and -2 mRNA. Moreover, several hBD-1 and hBD-2 mRNA-positive mononuclear cells are present in the periductal zone. (B and D) Semiserial sections of (A) and (C), respectively. The specific signals are eliminated when the slides are hybridized with sense probes (B and D).

Immunoblot Detection of hBD-1 and hBD-2 Proteins in Bile.

hBD-1 was detectable to a variable extent in all bile samples; hBD-2 was detected in 1 of 3 bile samples from patients with PBC, 1 of 3 samples from patients with PSC, and all of 6 samples from patients with hepatolithiasis (Fig. 4A). Semiquantitative assessment of the intensity of the immunoreactivity revealed that 3 bile samples from patients with hepatolithiasis exhibited particularly high levels of hBD-2; the average level of hBD-2 protein in patients with hepatolithiasis was higher than that in patients with PBC (<0.05) (Fig. 4B). Three patients with hepatolithiasis and high hBD-2 levels were also positive for hBD-2 mRNA and hBD-2 protein in affected bile ducts and had high serum CRP levels (9.6, 7.0, and 5.3 mg/dL); the serum level of CRP in the remaining 3 patients with hepatolithiasis was not as high (1.2, 2.3, and 4.6 mg/dL).

Figure 4.

Detection of human beta defensin (hBD)-1 and hBD-2 proteins in bile. (A) Dot bolt analysis revealed that hBD-1 is detectable in all bile samples, whereas hBD-2 is detected in 1 of 3 (33%) patients with primary biliary cirrhosis (PBC), 1 of 3 (33%) with primary sclerosing cholangitis (PSC), and each of 6 (100%) with hepatolithiasis. (B) Semiquantitative assessment of the intensity of immunoreactivity, using the National Institutes of Health Image Program, revealed that the average level of hBD-2 protein in hepatolithiasis is higher than that in PBC (**P < .05). Bars are SDs. Open circles indicate individual values. Asterisk is associated with cholestasis.

Expression of hBDs in cultured cells.

Amplification of hBD-1 mRNA occurred in all of the 5 cell lines that were cultured for 3 hours in the absence of a stimulant (medium only). Such expression was not affected by treatment with LPS or E. coli (Supplementary Fig. 3). In contrast, amplification of hBD-2 mRNA was not detected in any of the cell lines in the absence of a stimulant; de novo expression of hBD-2 occurred in all cell lines, except HepG2, after treatment with LPS or E. coli for 3 hrs (Supplementary Fig. 3). Real-time PCR revealed that after treatment of HuCCT1, CCKS1, hBEC1, and hBEC2 with LPS or E. coli, no significant increase of hBD-1 mRNA occurred, but compared to levels in unstimulated cells, there was appreciable upregulation of hBD-2 mRNA (Supplementary Fig. 3). In addition, immunoblotting for hBDs demonstrated hBD-1 protein and LPS-induced hBD-2 protein in supernatants of HuCCT1 and hBEC1 cells that had been cultured for 12 hours (Supplementary Fig. 3).

Amplification of hBD-1 mRNA occurred in 2 ICC cells, 2 hBECs, and HepG2 cells after culture for 3 hours; this expression was not affected by treatment with any cytokines of the stimulants (Supplementary Fig. 4). This finding was also demonstrated using real-time PCR (Supplementary Fig. 4). In contrast, amplification of hBD-2 mRNA occurred in 2 ICC cells and 2 hBECs after treatment with IL-1β or TNF-α for 3 hours, but not after treatment with PBS or any of the other 4 cytokines for 3 hours. hBD-2 amplification did not occur in HepG2 cells after any treatment. Real-time PCR showed that treatment with IL-1β or TNF-α for 3 hours significantly upregulated hBD-2 mRNA—approximately 32-fold and 17-fold, respectively (Supplementary Fig. 4). However, the other 4 cytokines did not affect expression of hBD-2.

Discussion

In this study, we have shown that hBD-1 protein is nonspecifically expressed in hepatocytes and BECs of intrahepatic bile ducts in all of the specimens of liver tissue that we examined. hBD-1 mRNA was also nonspecifically expressed in hepatocytes and BECs. hBD-1 mRNA and protein had a similar distribution within livers. These findings and the consistent detection of hBD-1 in bile in various diseases strongly suggest that hBD-1 plays a general antimicrobial role in the defense of the hepatobiliary system, similar to that documented in other epithelial organs.11, 15–18 Expression of hBD-1 in bile ducts may contribute to the rarity of biliary tract infections. Bile is sterile under physiological conditions. However, the biliary tree is potentially exposed to enteric bacteria.

In contrast, expression of hBD-2 was restricted to large intrahepatic bile ducts and peribiliary glands when it was detectable; hepatocytes did not express hBD-2. hBD-2 expression was particularly frequent in bile ducts exhibiting features of cholangitis in patients with EBO and hepatolithiasis; such expression was infrequent in patients with PBC and PSC and was absent in liver from patients with CH-C and normal liver histology. In EBO and hepatolithiasis, enteric bacteria can usually be cultured from bile,4, 8, 31 suggesting that bacteria-induced cholangitis may contribute to biliary epithelial expression of hBD-2. In patients with biliary epithelial expression of hBD-2, serum levels of CRP are higher than in patients with low expression of hBD-2; high serum CRP levels in this context may reflect active cholangitis involving large intrahepatic bile ducts.

Whether a systemic factor is involved in the expression of hBD-2 in intrahepatic bile ducts is an important issue. Absence of BEC expression of hBD-2 in the liver of patients with extrahepatic sepsis suggests that a systemic factor is unlikely to be involved in BEC expression of hBD-2 in chronic cholangitis. Furthermore, the absence of expression of hBD-2 in the surface epithelium of gallbladders from 3 patients with hepatolithiasis, in whom hBD-2 was expressed in stone-containing intrahepatic bile ducts, suggests that factors involved in hBD-2 expression in such patients are restricted to the affected bile ducts. Three patients with hepatolithiasis, high hBD-2 levels in bile, and high serum CRP levels were positive for hBD-2 mRNA and protein in the liver; 2 exhibited hBD-2 mRNA in bile ducts, suggesting that hBD-2 expressed in BECs of actively inflamed bile ducts might have been secreted into bile. hBD-2 is a secretory peptide antibiotic.11 hBD-1 and hBD-2 were secreted by cultured HuCCT1 and hBEC1 after treatment with LPS. These findings suggest that in cholangitis associated with hepatolithiasis or EBO, biliary epithelial hBD-2 participates in the local defense of biliary tree against bacterial infection. This suggestion is supported by the finding that hBD-2 was predominantly expressed in segments of large bile ducts containing a polymorphonuclear inflammatory infiltrate but not in segments in which inflammation was minimal or absent. hBD-2 may act synergistically with other microbicidal molecules present in bile, such as hBD-1, lactoferrin, and lysozyme.9, 19

There have been several reports, including our own,2, 4, 31, 32 that bacteria and their products, such as LPS, are consistently demonstrable in bile in certain pathological states, particularly in hepatolithiasis and EBO. Accordingly, we examined the influence of local factors, such as bacteria and LPS, on the synthesis and expression of hBD-1 and hBD-2 by cultured human BECs. While spontaneous hBD-1 mRNA expression was consistently detected in the cultured ICCs, hBECs and HepG2 cells, hBD-2 mRNA expression was detected only in cultured ICCs and hBECs after stimulation—e.g., by LPS or E. coli. These findings suggest that enteric bacteria and their products may be local factors involved in the expression of hBD-2 of BECs in situ. We have recently reported that murine BECs possess Toll-like receptors, which recognize pathogen-associated molecular patterns, such as LPS. Thus, BECs may respond directly to bacterial components.31 Interestingly, no expression of hBD-2 mRNA by HepG2 cells was detectable after treatment with LPS, E. coli, or inflammatory cytokines; these cells did consistently express hBD-1 mRNA.

hBD-2 mRNA expression was induced by treatment of 2 ICC and 2 hBEC lines with IL-1β or TNF-α; such expression was not induced by IL-4, IL-5, IL-6, or IFN-γ. However, hBD-1 mRNA was spontaneously expressed by all of the cultured BECs and HepG2 cells. These findings imply that different mechanisms are involved in the synthesis of hBD-1 and hBD-2 in BECs. They also suggest different roles for these peptides in biliary pathophysiology. In addition to BEC lines, hBD-2 is also induced by IL-1β and LPS in gingival keratinocytes and bronchial epithelial cells.16, 22, 33 Moreover, astrocytes and skin keratinocytes have been reported to produce hBD-2 after exposure to TNF-α..20, 34 These findings suggest that hBD-1 is a normal constituent of BECs that plays a role in the antimicrobial defense of bile ducts. In contrast, hBD-2 expression occurs in response to local infection and/or active inflammation; such phenomena are associated with local release of TNF-α and IL-1β. When expressed hBD-2 may also play a role in the antimicrobial defense of bile ducts.

In hepatolithiasis, bacterial infection of the intrahepatic biliary tree, particularly by enteric bacteria, seems to be a common event and is associated with the development of chronic proliferative cholangitis and expression of aberrant mucin.35 Our results have shown that, in this disease, hBD-1 and hBD-2 were expressed on large intrahepatic bile ducts and proliferated peribiliary glands and suggest that they may mediate antimicrobial mechanisms in the biliary tree. However, that hepatolithiasis may become an intractable disease, if surgical or endoscopic treatment is unsuccessful, suggests that expression of hBDs on bile ducts alone is insufficient to completely eliminate bacteria from bile. Both hBD-1 and hBD-2 are salt-sensitive antimicrobial peptides; their actions have been reported to be compromised by salt-dependent inactivation.19, 21 Such inactivation may occur in the bile ducts of patients with cystic fibrosis and may consequently predispose to chronic biliary infection and hepatolithiasis.

In conclusion, hBD-1 appears to be present in BECs under physiological conditions and to constitute a component of the antimicrobial defense of the hepatobiliary system. In active cholangitis due to bacterial infection, as may occur in association with hepatolithiasis, hBD-2 may be induced in large intrahepatic bile ducts, and when induced it may also participate in the antimicrobial defense of the hepatobiliary system. Further analyses of the bactericidal roles of hBD-1 and hBD-2 may provide support for the concept of using hBDs as “peptide antibiotics” to enhance biliary innate immunity in the treatment of infections of the biliary tract.

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