Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts


  • Conflict of interest: Nothing to report.


Fractalkine is a chemokine with both chemoattractant and cell-adhesive functions, and in the intestine it is involved with its receptor CX3CR1 in the chemoattraction and recruitment of intraepithelial lymphocytes. We examined the pathophysiological roles of fractalkine and CX3CR1 in normal and diseased bile ducts. Expression of fractalkine and CX3CR1 were examined in liver tissues from patients with primary biliary cirrhosis (17 cases) and controls (9 cases of primary sclerosing cholangitis, 10 cases of extrahepatic biliary obstruction, 20 cases of chronic viral hepatitis C, and 18 cases of histologically normal livers). Expression of fractalkine in biliary epithelial cells (BECs) in response to cytokine treatments was examined using a human cholangiocarcinoma cell line (HuCC-T1) and human intrahepatic BEC line. The chemotaxis of CX3CR1-expressing monocytes (THP-1) toward fractalkine was assayed using chemotaxis chambers. Fractalkine messenger RNA/protein were expressed on BECs of normal and diseased bile ducts, and their expression was upregulated in injured bile ducts of primary biliary cirrhosis. CX3CR1 was expressed on infiltrating mononuclear cells in portal tracts and on CD3+, CD4+, and CD8+ intraepithelial lymphocytes of injured bile ducts in primary biliary cirrhosis. Fractalkine messenger RNA expression was upregulated in two cultured BECs on treatment with lipopolysaccharide and Th1-cytokines (interleukin 1β, interferon gamma, and tumor necrosis factor α). THP-1 cells showed chemotaxis toward fractalkine secreted by cultured cells. In conclusion, Th1-cytokine predominance and lipopolysaccharide in the microenvironment of injured bile ducts resulting from primary biliary cirrhosis induce the upregulation of fractalkine expression in BECs, followed by the chemoattraction of CX3CR1-expressing mononuclear cells, including CD4+ and CD8+ T cells, and their adhesion to BECs and the accumulation of biliary intraepithelial lymphocytes. (HEPATOLOGY 2005;41:506–516.)

Recent studies showed that a newly identified CX3C-chemokine (CX3CL1), fractalkine, plays an important role in the migration of leukocytes to target sites under physiological as well as pathological conditions.1–5 Unlike other chemokines, fractalkine is expressed as a membrane-bound form on cells and also can be shed as a soluble chemotactic form. The latter is known to be a potent chemoattractant for CD8+ and CD4+ T cells, natural killer cells, and monocytes expressing its receptor (CX3CR1), whereas the former promotes strong adhesion of these leukocytes in an integrin-independent manner.6, 7 Muehlhoefer et al.8 reported fractalkine protein and messenger RNA (mRNA) expression in epithelial cells and endothelial cells in small intestine under normal conditions, and their upregulation during inflammatory bowel disease.8 They also disclosed that fractalkine expressed on the intestinal epithelium may regulate a subpopulation of CD8+ T cells in the epithelial layer (intestinal intraepithelial lymphocytes [IELs]).8

Biliary epithelial cells (BECs) are known as immunologically potent cells, and BECs of inflamed bile ducts actively participate in inflammation by secreting cytokines and expressing immune receptors.9, 10 Chemokines also are expressed on pathological bile ducts.10, 11 For example, Terada et al.12 reported that stromal-derived factor 1 expressed on bile ducts is responsible for the migration and recruitment of lymphoid cells in portal tracts and around bile ducts in primary biliary cirrhosis (PBC) as well as in chronic viral hepatitis (CVH). Although the upregulation of fractalkine and CX3CR1 expression has been reported in hepatic parenchyma and bile ductules of hepatitis C virus–infected livers,13 their expression and immunopathological significance remain unexplored in normal and pathological bile ducts.

Our recent study showed that biliary IELs are present at intrahepatic bile ducts under physiological conditions and mainly belong to CD8+ T cells, as seen in the intestine. Interestingly, numbers of CD4+, CD8+, and CD20+ biliary IELs were increased significantly at injured bile ducts of patients with PBC (K. Isse et al., submitted for publication), and this characterizes chronic nonsuppurative destructive cholangitis.14–19 However, it is not clear how biliary IELs are recruited and regulated in the bile ducts.

In this study, we examined the expression of fractalkine and CX3CR1 in portal tracts, with an emphasis on small bile ducts and recruitment of biliary IELs, using human liver tissue specimens and cultured BECs.


mRNA, messenger RNA; IEL, intraepithelial lymphocyte; BECs, biliary epithelial cells; PBC, primary biliary cirrhosis; CVH, chronic viral hepatitis; CVH-C, hepatitis C virus–related chronic viral hepatitis; PSC, primary sclerosing cholangitis; EBO, extrahepatic biliary obstruction; IgG, immunoglobulin G; RT-PCR, reverse-transcriptase polymerase chain reaction; HIBEC, human intrahepatic biliary epithelial cell; LPS, lipopolysaccharide; IL, interleukin; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; PBS, phosphate-buffered saline; PDC-E2, pyruvate dehydrogenase E2 component.

Materials and Methods

Liver Tissue Specimens and Serum

Wedge-biopsied or surgically resected liver specimens fixed in 10% formalin and embedded in paraffin were obtained from 17 patients with PBC, 9 patients with primary sclerosing cholangitis (PSC), 10 patients with extrahepatic biliary obstruction (EBO) of short duration, 20 patients with hepatitis C virus–related CVH (CVH-C), and 18 patients with histologically normal livers (Table 1). The diagnosis in each case was made based on a combination of clinicolaboratory and histopathological findings. PBC was staged histologically,20 and the grading and staging of CVH-C were carried out according to international criteria.21 Samples obtained by wedged liver biopsies in cases of uncertain diagnosis and nonneoplastic parts obtained from surgically resected livers for liver tumor(s) or for traumatic liver rupture were used. Three-micrometer–thick sections were cut from each paraffin block. Several were processed routinely; the remainder were processed for immunohistochemistry.

Table 1. Liver Tissue Specimens and Sera From the Patients Examined
DiseasesPatient No.Average Age (yr)Sex
  1. Abbreviations: PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; EBO, extrahepatic biliary obstruction; CVH-C, HCV-related chronic viral hepatitis.

Liver tissue specimens    
 PBC (stage 1/2)1761.7215
 Normal liver1859.2810
 PBC (stage 1/2/3/4 = 9/5/4/2)2055.2713
 CVH-C (stage F0/1/2/3 = 2/5/4/5)1666.897
 Normal liver1259.266

Fresh tissue samples from seven livers (two PBC cases, three CVH-C cases, and two normal livers) also were available. They were embedded in optimum cutting temperature compound (Miles Inc., Elkhart, IN) and 5-μm–thick frozen sections were prepared for microdissection and in situ hybridization examination.

Both the formalin-fixed and fresh liver specimens were obtained from the liver disease files of our department.

The intrahepatic biliary tree is classified into intrahepatic large and small bile ducts and bile ductules.15, 22 The large bile ducts are characterized by the presence of accompanying peribiliary glands and roughly correspond to the first to fourth branches of the right and left hepatic ducts. The small bile ducts are classified into septal and interlobular bile ducts according to their size and location within portal tracts. In this study, mainly the small bile ducts were examined.

Serum samples were obtained from 20 PBC patients, 20 EBO patients, including 10 with extrahepatic biliary carcinoma, 16 CVH-C patients, and 12 healthy volunteers (Table 1) and were used for the measurement of soluble fractalkine by an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Informed consent was obtained from all the subjects.

Immunohistochemistry for Fractalkine and CX3CR1

Deparaffinized sections were incubated with rabbit polyclonal antibodies against fractalkine (2 μg/mL; IBL, Fujioka, Japan) or CX3CR1 (5 μg/mL; IBL). The sections then were treated with goat antirabbit immunoglobulins conjugated with peroxidase-labeled dextran polymer (Envision+; DAKO, Tokyo, Japan). After a benzidine reaction, the sections were counterstained lightly with hematoxylin. As negative controls, nonimmunized rabbit IgG was used as primary antibody. Evaluation of immunostaining (positive or negative, and scoring of staining intensity) always was performed blindly and was discussed by two pathologists (K.I. and Y.N.).

Expression of Fractalkine in Bile Ducts.

Seventy-five small bile ducts from normal livers, 50 from EBO livers, 100 from CVH-C livers, 45 from PSC livers, and 85 from PBC livers were chosen for evaluation in each case. In comparison with the intensity in endothelial cells (strongly positive) and parenchymal hepatocytes (negative), fractalkine expression in bile ducts was evaluated as either absent/faint (±), slightly positive (+), or strongly positive (++; Fig. 1A-B).

Figure 1.

Immunohistochemistry for fractalkine and CX3CR1 in the liver tissue. (A) Normal liver. Fractalkine is expressed in endothelial cells of small vessels (arrowhead), whereas small bile duct is faintly positive (arrow). Hepatocytes are negative for fractalkine. (B) Primary biliary cirrhosis. Fractalkine is evidently expressed in pathological bile ducts (arrow). Small vessels in portal tract also express fractalkine (arrowhead). (C) Normal liver. CX3CR1 expression is faint in the normal small bile duct (arrow) and weak in the smooth muscle of hepatic artery (arrowhead). (D) Primary biliary cirrhosis. CX3CR1 is evidently expressed in injured bile ducts in the membranous and cytoplasmic parts (arrow). CX3CR1-positive mononuclear cells are accumulated around the damaged bile ducts and portal area. (A-D) Visualized by benzidine reaction and counterstained by hematoxylin (original magnification, ×200).

CX3CR1-Positive Mononuclear Cells.

With the National Institutes of Health (Bethesda, MD) image program, CX3CR1-positive mononuclear cells were counted in portal tracts excluding the portal vein, bile ducts, and arteries, and their number per area (mm2) was calculated.

Expression of CX3CR1 in Bile Ducts.

The immunohistochemical expression of CX3CR1 in small bile ducts was classified as negative (no or faint staining) or positive (Fig. 1C-D). Furthermore, to confirm the intracellular distribution of CX3CR1, alkaline phosphatase-labeled Envision (DAKO) with Fast Red (Vector Laboratories, Burlingame, CA) was used and the samples were viewed under a confocal laser scanning microscope (Zeiss LSM5 Pascal; Carl Zeiss Japan, Tokyo, Japan).

Double Immunostaining of CX3CR1/CD3, CD4, or CD8.

To characterize infiltrating lymphocytes, double-immunostaining for CX3CR1 and either CD3, CD4, or CD8 was performed in normal and diseased livers. Sections were incubated with polyclonal rabbit anti-CX3CR1 antibody and monoclonal mouse anti-CD3 antibody (clone PS1, neat; Nichirei, Tokyo, Japan), anti-CD4 antibody (clone 1F6, neat; Nichirei), or anti-CD8 antibody (clone C8/44B, ×200; DAKO). After treatment with Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 594 goat antimouse IgG (10 μg/mL; Molecular Probes, Eugene, OR), fluorescence was observed under a fluorescent microscope.

In Situ Hybridization Study

Polymerase chain reaction (PCR)-amplified products containing the human fractalkine mRNA sequence and T7-RNA and Sp6-RNA polymerase promoters were obtained. Then, single-stranded RNA probes complementary (antisense) and anticomplementary (sense probe) to the corresponding gene transcripts were obtained using in vitro transcription according to the standard protocol of the digoxigenin RNA Transcription Kit (Roche, Indianapolis, IN). Paraformaldehyde-fixed frozen sections (from one normal and two PBC livers) were incubated with hybridization solution (DAKO) mixed with digoxigenin-labeled fractalkine antisense and sense probes at 50°C for 16 hours. Slides were washed finally in 0.2× sodium chloride–sodium citrate. After blocking reagent (Roche) was applied, the sections were incubated with alkaline phosphatase–conjugated antidigoxigenin antibody (Roche) for 1 hour. Color development was performed with a nitroblue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate toluidine salt solution (Roche) for 3 to 6 hours in a dark room.

Microdissection and Reverse-Transcriptase PCR for Fractalkine mRNA

Frozen sections from one CVH-C and four PBC livers in which bile ducts were identifiable were fixed gently in 75% ethanol and then stained with hematoxylin–eosin. An Arcturus laser capture microscope (Laser Capture Microdissection System PixCell II; Arcturus, Mountain View, CA) was used to microdissect BECs. Briefly, intrahepatic bile ducts were identified, and approximately 500 BECs from several bile ducts in each case were microdissected and captured on a microcentrifuge cap. Total RNA was extracted from these microdissected materials using a PicoPure RNA Isolation kit (Arcturus).

For reverse-transcriptase PCR (RT-PCR), total RNA, RTase (ReverTra Ace; Toyobo, Tokyo, Japan), and random primers were used. The nucleotide sequences for fractalkine, CX3CR1, and CD45 were analyzed and primers were generated. RT-PCR for CD45 (leukocyte common antigen) was performed to check for possible contamination by leukocytes of the microdissected bile duct samples. PCR conditions were denaturation at 94°C for 1 minute, annealing for 1 minute, and elongation at 72°C for 2 minutes. The primer sequences and annealing temperature are shown in Table 2.

Table 2. Primers for Human Fractalkine, CX3CR1, and CD45 mRNA
GenePrimer SequencesPCR ProductAnnealing
  1. Abbreviation: bp, base pairs.

FractalkineForward 5′-GATGGCTCCGATATCTCTG-3′262 bp60°C
CD45Forward 5′-CAAAGGAACAGGCTGAAGGT-3′220 bp55°C
β-actinForward 5′-CAAGAGATGGCCACGGCTGCT-3′275 bp60°C

Cell Culture

One human intrahepatic cholangiocarcinoma cell line, HuCC-T123 (JCRB0425; Health Science Research Resources Bank, Osaka, Japan), and one nonneoplastic human intrahepatic BEC cell line (HIBEC), primary culture, originating from an explanted liver with hepatitis C virus–related cirrhosis24 were used. These cells grew on culture dishes with standard medium, RPMI 1640 medium (Gibco, Rockville, MD) with 10% fetal bovine serum for HuCC-T1, and DMEM/F12 (Gibco) with 10% fetal calf serum for HIBEC. They were cultured at 37°C in 95% air and 5% CO2.

Three-Dimensional Culture of HuCC-T1 Within Collagen Gel.

As described in a previous study,25 HuCC-T1 cells were embedded in collagen gel (Nitta Gelatin, Osaka, Japan) and cultured. Three days later they were fixed in formalin and embedded in paraffin. The expression of fractalkine and CX3CR1 was examined immunohistochemically (see Immunohistochemistry for Fractalkine and CX3CR1).

Treatment of Cultured Cells With Lipopolysaccharide and Inflammatory Cytokines

Lipopolysaccharide (LPS) and interleukin (IL)-1β, IL-4, IL-6, interferon gamma (IFN-γ), and tumor necrosis factor α (TNF-α) were purchased from PeproTech (Rocky Hill, NJ). Both HuCC-T1 and HIBEC possess receptors for LPS (Toll-like receptor 4) and for these cytokines (K. Harada et al., submitted for publication). When cultured cells reached a semiconfluent state, the medium was replaced with fresh medium and LPS 1 μg/mL or either of the cytokines at 1,000 U/mL was added for a 3-hour culture.

RT-PCR for Fractalkine mRNA Expression

Total RNA was isolated from cultured HuCC-T1 and HIBEC cells with the RNeasy Total RNA System (Qiagen, Hilden, Germany) following the manufacturer's directions and was used for RT-PCR. For quantitative analysis, the real-time PCR assay was performed with by SYBER Green PCR Master Mix and an ABI PRISM 7700 Sequence Detection System (both from Applied Biosystems, Tokyo, Japan). The primers for fractalkine (forward, 5′-CTGCTGCCCTAACTCGAAAT-3′; reverse, 5′-AGGACCACAGACTCGTCCAT-3′) and glyceraldehyde 3-phosphate dehydrogenase (forward, 5′-GGCCTCCAAGGAGTAAGACC-3′; reverse, 5′-AGGGGTCTACATGGCAACTG-3′) were set according to the instruction. Data were assessed as the relative fold compared with unstimulated cells (phosphate-buffered saline [PBS]).

Detection of Soluble and Cell-Bound Fractalkine in HuCC-T1 Cells

HuCC-T1 was cultured for 3 days after stimulation with PBS or either LPS, IL-1β, IFN-γ, or TNF-α. Soluble fractalkine in the supernatant and cell-bound fractalkine in the cell lysate from HuCC-T1 were detected by enzyme-linked immunosorbent assay and Western blotting, respectively. Western blotting was performed with the same antibody used in the immunohistochemistry.

In Vitro Chemotaxis Assay of Fractalkine

The chemoattractant activity of fractalkine secreted by the HuCC-T1 cells stimulated with LPS was assessed in 24-well plates assembled with Chemotaxicell (8-μm pore size; Kurabou, Osaka, Japan)26 and THP-127 (RCB1189; RIKEN, Tokyo, Japan), which are monocytes expressing CX3CR1 and showing efficient chemotaxis and adherence in a fractalkine-dependent manner.28, 29 Briefly, HuCC-T1 was cultured with LPS (1 μg/mL) for 3 days. As described in Results, soluble fractalkine was detectable by enzyme-linked immunosorbent assay in the supernatant of subconfluent HuCC-T1 after LPS treatment. After assembly of the Chemotaxicell, a THP-1 suspension (2 × 105 cells/100 μL) was seeded and incubated for 90 minutes. The migrated THP-1 cells were counted in 10 randomly chosen high-power fields (×400) under a microscope, according to Vitale et al.30 To evaluate further the chemotactic processes, THP-1 cells were preincubated with human recombinant fractalkine (1 μg/mL; Chemicon, Temecula, CA) comprising only the chemokine domain,31 which is expected to bind CX3CR1 expressed on THP-1 cells or sufficient anti-CX3CR1 antibody (10 μg/mL; IBL) in the culture for 15 minutes, which is expected to block CX3CR1 on THP-1. Normal rabbit IgG (10 μg/mL; Santa Cruz Biotechnology, Santa Cruz, CA) was used as a negative control.

Statistical Analysis

Data were analyzed using the Kruskal-Wallis test and Spearman's correlation coefficient. P values less than .05 were considered statistically significant.


Fractalkine Expression in Liver Tissue


In normal livers, small bile ducts were negative or slightly positive for fractalkine (Fig. 1A). Some infiltrating mononuclear cells, endothelial cells of small vessels, smooth muscle of arteries, and periportal hepatocytes were negative or faintly positive. In EBO, CVH-C, and PSC livers, small bile ducts and proliferated bile ductules were negative or slightly positive for fractalkine. In PBC livers, expression of fractalkine in BECs was rather strong in approximately two thirds of small bile ducts, particularly injured bile ducts (Fig. 1B), whereas the remaining one third were negative or slightly positive. Endothelial cells around injured bile ducts also were positive.

RT-PCR for Fractalkine mRNA in Microdissected Samples.

Fractalkine mRNA was detected by RT-PCR in microdissected samples of small bile ducts of CVH-C and PBC in which CD45 amplification was not evident (Fig. 2), suggesting that fractalkine mRNA was present in BECs.

Figure 2.

Fractalkine and CX3CR1 messenger RNA (mRNA) in microdissected small bile ducts by Laser Capture Microdissection System PixCell II. By reverse-transcriptase polymerase chain reaction, fractalkine and CX3CR1 mRNA were amplified at the predicted size (263 bp and 296 bp, respectively) from RNA samples of microdissected bile ducts in chronic viral hepatitis C (CVH-C) and primary biliary cirrhosis (PBC). β-actin (275 bp) was used as an internal control. CD45 (leukocyte common antigen, 220 bp) is not amplified. RT+, addition of reverse transcriptase for reverse transcription; RT−, addition of distilled water for reverse transcription.

In Situ Hybridization for Fractalkine mRNA.

Signals for fractalkine mRNA in small bile ducts were weak in normal liver and strong in PBC liver (Fig. 3A). Fractalkine mRNA also was detectable in smooth muscle, endothelial cells, and several mononuclear cells, including fibroblasts in portal tracts and fibrous septa. The distribution of cells positive for fractalkine mRNA was similar to that of expression of fractalkine protein. No signals were detected in these cells using sense probes as a negative control (Fig. 3B).

Figure 3.

(A) In situ hybridization shows fractalkine messenger RNA in the cytoplasm of bile duct (arrow) of primary biliary cirrhosis. Positive signals also are detected around the bile duct, corresponding to infiltrating mononuclear cells and endothelium of small vessels. (B) These signals disappear in the bile duct (arrow) and also in the periductal nonbiliary cells when the slide is hybridized with a sense probe. Panels A and B are serial sections from the same area of primary biliary cirrhosis (original magnification, ×200).

Serum Level of Soluble Fractalkine

As shown in Fig. 4A, the soluble fractalkine level was significantly elevated in the sera of PBC patients compared with healthy volunteers, CVH-C patients, or EBO patients. The serum level was higher in CVH-C and EBO patients than in healthy volunteers.

Figure 4.

(A) Serum level of soluble fractalkine measured by enzyme-linked immunosorbent assay is higher in primary biliary cirrhosis (PBC; 3,736 ± 838 pg/mL, mean ± SE) than in healthy subjects (32 ± 6.8 pg/mL), chronic viral hepatitis C (CVH-C; 1,622 ± 417 pg/mL), and extrahepatic biliary obstruction (EBO; 1,910 ± 425 pg/mL; *P < .05, Kruskal-Wallis test). Serum value of soluble fractalkine is higher in EBO and CVH-C livers than in those of healthy volunteers (*P < .05, Kruskal-Wallis test). Numbers of patients with each disease examined are shown in parentheses. (B) Density of CX3CR1-positive mononuclear cells in an area of the portal tract excluding the portal vein, bile ducts, and arteries. The number of CX3CR1-expressing mononuclear cells in the portal tract is 1.6 ± 1.5/mm2 (mean ± SE) in normal liver (NL), 2.8 ± 1.0/mm2 in livers with extrahepatic biliary obstruction (EBO), 7.0 ± 2.5/mm2 in livers with chronic viral hepatitis C (CVH-C), 3.3 ± 1.6/mm2 in livers with primary sclerosing cholangitis (PSC), and 21.5 ± 4.2/mm2 in livers with primary biliary cirrhosis (PBC). CX3CR1-positive cell numbers are increased in PBC compared with other diseases (*P < .01, Kruskal-Wallis test).

CX3CR1 Expression in Liver Tissue


Although CX3CR1-positive mononuclear cells were very few in portal tracts of normal livers and infiltrated variably in portal tracts and fibrous septa in EBO, CVH-C, and PSC livers, they were significantly increased in PBC livers (P < .05; Fig. 4B). CX3CR1 also was expressed in BEC (Fig. 1C-D) and in arterial and venous branches of portal tracts. Small bile ducts expressing CX3CR1 were more frequent in PBC livers (77% of 85 bile ducts) than in the others, except for PSC livers (24% of 75 bile ducts in normal livers, 44% of 50 bile ducts in EBO livers, 31% of 45 bile ducts in CVH-C livers, and 51% of 45 bile ducts in PSC livers; P < .05, Kruskal-Wallis test).

Confocal laser microscopy showed that CX3CR1 was expressed in the membranous parts (basolateral and luminal) of bile ducts and also in their perinuclear cytoplasm (Fig. 5A). In addition, endothelial cells of small arteries and fibroblast-like spindle cells32 in portal tracts were positive for CX3CR1 (Fig. 5B).

Figure 5.

Immunofluorescent staining for CX3CR1 in the liver of primary biliary cirrhosis using the Envision-AP method with Fast Red (Envision+; DAKO, Tokyo, Japan) and viewed under a confocal laser microscope. (A) In one small bile duct, membranous staining of CX3CR1 (arrows) and perinuclear cytoplasmic staining are evident (original magnification, ×400). (B) Endothelium of the hepatic artery (arrowhead) and that of portal veins is strongly positive for CX3CR1. Such staining is not evident in the smooth muscles of the artery (original magnification, ×630).

RT-PCR for CX3CR1 mRNA in Microdissected Samples.

CX3CR1 mRNA was detected by RT-PCR in the microdissected samples of small bile ducts of CVH-C and PBC livers in which CD45 amplification was not evident (Fig. 2), suggesting that CX3CR1 mRNA was present in BECs.

Double Immunostaining of CX3CR1 and CD3, CD4, or CD8.

CD3+ T cells (Fig. 6A, red) mainly were present in portal tracts and fibrous septa. In PBC livers, CX3CR1 was shown evidently in the cytoplasm and occasionally in the membranous parts, whereas in other diseases, CX3CR1 was slightly or faintly positive on the membranous parts or in the cytoplasm of bile ducts (Fig. 6B, green). Most biliary IEL of injured bile ducts and of lymphoid cells around these bile ducts in PBC livers were positive for both CX3CR1 and CD3 (Fig. 6C, yellow), although some mononuclear cells in portal tracts were positive for CD3 alone (Fig. 6C, red). In normal and other diseased livers, a few biliary IELs detectable in small bile ducts and lymphoid cells in portal tracts infrequently were positive for both CX3CR1 and CD3.

Figure 6.

Double immunostaining of CX3CR1 and CD3, CD4, or CD8 in the liver of primary biliary cirrhosis. CX3CR1 was visualized by Alexa Fluor 488 (green fluorescence) and CD3, CD4, or CD8 was visualized by Alexa Fluor 594 (red fluorescence). Panels A, B, and C are from the same area of the same section, and panels D and E are also from the same area of the same section. All panels are viewed by fluorescence microscope. *Lumen of the bile duct. (A) Many CD3+ T cells (red) are seen around the bile ducts and also within the biliary epithelia (original magnification, ×200). (B) CX3CR1+ mononuclear cells (green) are seen around the bile duct. Biliary epithelial cells of this bile duct also show membranous and supranuclear cytoplasmic staining of CX3CR1. (original magnification, ×200). (C) Lymphocytes positive for CD3+ and CX3CR1 (yellow) are seen within the biliary epithelia (arrows) and also around the bile ducts. In addition, red mononuclear cells expressing CD8 alone are also seen (original magnification, ×200). (D) CD8+ T cells (red) are found within the biliary epithelia and also around the bile ducts (original magnification, ×400). (E) Lymphocytes positive for CD8+ and CX3CR1 (yellow) are seen within the biliary epithelia (arrows) and also around the bile ducts (original magnification, ×400).

CD4+ and CD8+ T cells were seen within biliary epithelia (Fig. 6D, red), and most of them also expressed CX3CR1 (Fig. 6E, yellow). CD4+ and CD8+ T cells expressing CX3CR1 also were found around the injured bile ducts of PBC patients.

Expression and Secretion of Fractalkine and CX3CR1 in Cultured BECs

Expression of Fractalkine mRNA on Cytokine Treatment.

Quantitative analysis using real-time PCR revealed that fractalkine mRNA was weakly expressed in both cultured HuCC-T1 cells and HIBECs without any stimulants (PBS), whereas treatment with LPS, IL-1β, IFN-γ, and TNF-α induced an upregulation of the expression in these cultured cells, especially LPS and IFN-γ in HuCC-T1 and IFN-γ in HIBECs (P < .05; Fig. 7). Stimulation with IL-4 or IL-6 did not result in such upregulation in these cultured cells, suggesting that Th1 cytokines and LPS upregulate fractalkine expression in cultured BECs.

Figure 7.

Expression of fractalkine messenger RNA (mRNA) induced by cytokines in cultured cells assessed by real-time polymerase chain reaction. Cultured HuCC-Tl cells and human intrahepatic biliary epithelial cells (HIBECs) were treated with either lipopolysaccharide (LPS) at 1 μg/mL, or interleukin (IL)-1β, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor α (TNF-α) at 1,000 U/mL for 3 hours. The increase in fractalkine levels on treatment with LPS, IL-1β, IFN-γ, or TNF-α are 11.7-fold, 8.9-fold, 11.1-fold, and 5.3-fold in HuCC-T1 cells, and 37.5-fold, 12.5-fold, 39.6-fold, and 17.9-fold in HIBECs, respectively, compared with each nonstimulant. Compared with control (phosphate-buffered saline [PBS]), the relative fold increase in the expression of fractalkine mRNA is higher for LPS and IFN-γ in HuCC-T1 cells and for INF-γ in HIBEC cells (*P < .05, Kruskal-Wallis test).

Fractalkine Protein in Culture Supernatants and in Cell Lysate of HuCC-T1 Detected by Western Blotting.

Supernatants of HuCC-T1 cultured for 3 days contained soluble fractalkine, and its level was increased by treatment with IL-1β (126.9 ± 39.6 pg/mL), LPS (39.8 ± 19.9 pg/mL), IFN-γ (54.0 ± 10.8 pg/mL), or TNF-α (16.1 ± 2.5 pg/mL) when compared with no stimulant (PBS; 10.4 ± 7.6 pg/mL). The increase caused by IL-1β treatment was significant (P < .05). Fractalkine protein was detected in cell lysate of HuCC-T1 cultured for 3 days with no stimulant and was increased after stimulation with LPS, IL-1β, IFN-γ, and TNF-α by Western blotting.

Fractalkine and CX3CR1 Expression in HuCC-T1 Cells Cultured Within Collagen Gel.

HuCC-T1 cells cultured within collagen gels for 3 days showed cystic or branching growth.26 Fractalkine and CX3CR1 were detected immunohistochemically diffusely in the cytoplasm of cultured cells and also in their lumen, suggesting that these cultured cells synthesized and secreted fractalkine and CX3CR1.

Chemotactic Effects of Fractalkine to THP-1 Cells

The number of THP-1 cells that transmigrated in the chemotaxicell chamber after LPS treatment (29.4 ± 1.5 cells/high-power field) was larger than that in the negative control (PBS; 13.9 ± 0.6 cells; P < .01; Fig. 8), suggesting that the fractalkine synthesized and secreted by HuCC-T1 cells treated with LPS chemoattracted THP-1. The number of LPS-induced transmigrated THP-1 cells was significantly reduced on preincubation with recombinant fractalkine (1 μg/mL; 20.8 ± 0.8 cells) or anti-CX3CR1 antibody (10 μg/mL; 12.6 ± 2.3 cells), confirming that increased migration of THP-1 was dependent on fractalkine derived from HuCC-T1 cells and also was dependent on CX3CR1 expressed on THP-1.

Figure 8.

Chemotactic effects of fractalkine (secreted by HuCC-T1) on THP-1 cells. Number of THP-1 cells transmigrated in chemotaxicell chambers after lipopolysaccharide (LPS) treatment (1 μg/mL; 29.4 ± 1.5 cells/high-power field [HPF]) are larger than those without (13.9 ± 0.6 cells/HPF; *P < .01, Kruskal-Wallis test). Preincubation with anti-CX3CR1 antibody (10 μg/mL) or recombinant fractalkine (1 μg/mL) significantly reduced LPS-induced THP-1 transmigration (20.8 ± 0.8 cells/HPF and 12.6 ± 2.3 cells/HPF, respectively; **P < .05, Kruskal-Wallis test). However, normal rabbit immunoglobulin G (IgG; 10 μg/mL) did not affect LPS-induced THP-1 migration. Ab, antibody; FRK, fractalkine.


In this study, fractalkine mRNA and protein were detectable in BECs of small bile ducts and also in endothelial cells of portal tracts of normal and diseased livers. Although the expression was weak in normal bile ducts, it was increased in injured bile ducts of PBC.

In normal intestine, fractalkine is expressed in epithelial and endothelial cells8, 33 and is involved in chemoattraction and adhesion of CX3CR1-expressing mononuclear cells into the propria,8, 33 and interestingly, in recruitment of lymphocytes, including CD8+ T cells into the intestinal epithelium.1, 8, 33, 34 The fractalkine–CX3CR1 system plays an important role in the mucosal immune response in inflammatory bowel disease.1, 8, 33, 34 This study showed that CX3CR1-positive mononuclear cells infiltrated into portal tracts, and their number increased in PBC. Furthermore, most biliary IELs in injured bile ducts of PBC livers were positive for CX3CR1, suggesting that increased expression of fractalkine in BECs is responsible for chemoattraction of CX3CR1-positive lymphocytes into portal tracts and into biliary epithelia. Fractalkine expression was not strong in BECs of bile ducts in CVH-C and EBO livers, compatible with the finding that biliary IELs expressing CX3CR1 were few in these livers.

We investigated the regulation of fractalkine expression in two cultured BECs. The upregulation or induction of fractalkine expression in cultured endothelial or smooth muscle cells in response to inflammatory stimuli has been reported.8, 31, 35–38 In nonstimulated conditions, cultured HuCC-T1 and HIBECs expressed fractalkine mRNA negligibly. Interestingly, treatment with LPS, TNF-α (Th1 and Th2 types), IL-1β (Th1-type), or IFN-γ (Th1-type) upregulated fractalkine mRNA expression significantly in these cultured cells. In contrast, Th2 cytokines (IL-4 and IL-6) induced no such upregulation. Furthermore, HuCC-T1 cells cultured for 3 days after treatment with LPS, INF-γ, TNF-α, and especially IL-1β increased secretion of soluble fractalkine in culture supernatant. These findings suggest that the Th1 predominance around in vivo small bile ducts may lead to increased expression of fractalkine in BECs, followed by periductal chemoattraction of T cells and recruitment of biliary IELs in injured bile ducts of PBC livers. LPS in bile39 to which BECs are facing also may contribute to this scenario. Th1-dominant cytokine milieu around bile ducts is known to be important for the development of cholangiopathy in PBC.40, 41

Brand et al.33 disclosed that fractalkine was able to induce expression of fractalkine via CX3CR1 on intestinal epithelial cells in an autocrine manner.33 This study showed the expression of mRNA and protein of CX3CR1 and fractalkine in the bile ducts, and that the expression was upregulated in injured bile ducts of PBC. Furthermore, it was found that HuCC-T1 cells cultured within collagen gel produced and secreted both fractalkine and CX3CR1, raising the possibility that their coexpression leads to an autocrine/paracrine effect on fractalkine-induced fractalkine expression.29, 33

Recent studies suggest potential pathophysiologic roles for fractalkine in glomerulonephritis and intestinal inflammatory diseases.29, 33, 35–38, 42–44 This study showed that expression of fractalkine and CX3CR1 was upregulated in injured bile ducts of PBC, CX3CR1-expressing mononuclear cells were dense around bile ducts and within the biliary epithelium, and cultured BEC expressed cell-bound fractalkine and secreted soluble fractalkine. So, we examined the chemotactic activity of mononuclear cells expressing CX3CR1, toward a fractalkine gradient, by using chemotaxicell microchemotaxis chambers and HuCC-T1 and THP-1 (monocytes expressing CX3CR128, 31). Transmigration of THP-1 toward cultured HuCC-T1 cells treated with LPS was increased, indicating that the fractalkine gradient difference might have chemoattracted THP-1. This fractalkine-dependent chemotaxis of THP-1 cells was confirmed by preincubation of THP-1 with either recombinant fractalkine or anti-CX3CR1 antibody, indicating that fractalkine secreted by HuCC-T1 is functional and that the binding of CX3CR1 by its ligand fractalkine inhibited the chemotactic transmigration activity of THP-1. These results suggest that fractalkine expressed on and secreted from BECs of small bile ducts, particularly injured ones in PBC livers, plays an important role in the migration and adhesion of CX3CR1-expressing mononuclear cells in portal tracts and into biliary epithelia.

T-cell–mediated cytotoxicity toward BECs of small bile ducts reportedly is important in PBC livers, and granzyme B- and Fas ligand-expressing T cells infiltrating around bile ducts in PBC are known to be effecter cells to induce biliary epithelial apoptosis.45 This study showed that CD3+, CD4+, and CD8+ T cells were present within epithelial layer as well as around injured bile ducts, and most of these T cells also were positive for CX3CR1. Interestingly, most of these injured bile ducts showed upregulation of fractalkine. It seems likely, therefore, that T-cell migration into biliary epithelium was mediated via the fractalkine–CX3CR1 system, and these T cells eventually could damage the bile ducts. PBC is characterized by a serological occurrence of antimitochondrial antibodies that recognize 2-oxo-acid dehydrogenase complex, particularly pyruvate dehydrogenase E2 component (PDC-E2).46 Furthermore, autoreactive T cells against PDC-E2 are also detectable in the serum and liver in PBC.47 CD4+ T cells, especially CD4+ and CD28 T cells recognizing PDC-E2 and CD8+ T cells recognizing PDC-E2, were increased in number in the peripheral blood and liver tissue of PBC livers,24, 47 and these autoreactive T cells may be cytotoxic and responsible for immune-mediated BEC damage. This finding and the circumstantial evidences given above support that idea that fractalkine and CX3CR1 expression are relevant to duct injury in PBC livers via autoreactive and cytotoxic T-cell migration. Further study to demonstrate that CD4+ and/or CD8+ biliary IELs are reactive against PDC-E2 is needed.

Elevated serum soluble fractalkine levels of PBC may emphasize the systemic inflammatory component of PBC and seem to support the above-mentioned scenario regarding fractalkine and bile duct lesions. Eventually, secreted and cell-adherent fractalkine on injured bile ducts may be responsible for chemotaxis of mononuclear cells expressing CX3CR1 and in recruitment of biliary IELs. Similar data on the increased serum level of fractalkine and chemotactic migration of CD4+ lymphoid cells into target tissues were reported in allergic asthma and rhinitis.43 This study also raises the possibility that immunoneutralization of CX3CR1 attenuates development and persistence of chronic cholangitis of PBC, as is speculated to be the case in other organs.31, 48, 49

In conclusion, this study showed that fractalkine expression was upregulated in injured bile ducts in PBC. Upregulation of fractalkine was induced by LPS and Th1-cytokines in cultured BECs, suggesting that in PBC livers, the Th1-cytokine predominance around the bile duct and possibly LPS in bile upregulate fractalkine expression in bile ducts. Secreted soluble fractalkine may be involved in the chemoattraction of mononuclear cells expressing CX3CR1 around these pathological bile ducts and in the recruitment of biliary IELs.