Biliary epithelial cell antibodies induce expression of toll-like receptors 2 and 3: A mechanism for post–liver transplantation cholangitis?

Authors

  • Xupeng Ge,

    Corresponding author
    1. Division of Transplantation Surgery, Karolinska University Hospital-Huddinge, Karolinska Institute, Stockholm, Sweden
    • Department of Transplantation Surgery, B56, Karolinska University Hospital-Huddinge, S-141 86 Stockholm, Sweden
    Search for more papers by this author
    • Telephone: 46-8-58583988; FAX: 46-8-7743191

  • Mehmet Uzunel,

    1. Division of Clinical Immunology, Karolinska University Hospital-Huddinge, Karolinska Institute, Stockholm, Sweden
    Search for more papers by this author
  • Bo-Göran Ericzon,

    1. Division of Transplantation Surgery, Karolinska University Hospital-Huddinge, Karolinska Institute, Stockholm, Sweden
    Search for more papers by this author
  • Suchitra Sumitran-Holgersson

    1. Division of Transplantation Surgery, Karolinska University Hospital-Huddinge, Karolinska Institute, Stockholm, Sweden
    Search for more papers by this author

  • See Editorial on Page 878

Abstract

Studies to determine the role of preformed antibodies to biliary epithelial cells (BECs) in liver transplant rejections have been initiated. However, the clinical importance of these antibodies in the posttransplantation period still remains to be elucidated. Reactivity to BECs isolated from a normal healthy liver was investigated in sera of 56 patients before and after liver transplantation (LTX) using flow cytometry. Functional capacity of BEC antibodies was determined by the ability to induce expression of Toll-like receptors (TLRs) on BECs. Cytokine and chemokine production induced by BEC antibodies was determined by enzyme-linked immunosorbent assay. In all, 7 patients (13%) had BEC antibodies only pre-LTX, 14 (25%) only after LTX, 18 (32%) both before and after LTX, and 17 (30%) had no detectable antibodies. Presence of preformed BEC antibodies correlated with acute rejections (P < 0.03). Deposition of immunoglobulins in bile ducts was detected in biopsies of patients during rejections. Significantly higher numbers of patients with post-LTX antibodies (9 of 32) developed cholangitis, compared with 0 of 17 without antibodies (P < 0.02). Specificity studies indicated that these antibodies were both non–HLA- and HLA-specific. Normal BECs expressed mRNA but not the proteins for the TLRs. However, treatment with F(ab′)2 fragments of BEC antibodies induced protein expression of TLRs 2 and 3 and significantly high production of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, epithelial neutrophil activating peptide (ENA)-78, and IL-8. In conclusion, BEC antibodies via induction of TLR2 and TLR3 expression, as well as inflammatory cytokine and chemokine production may induce epithelial cell inflammatory responses to bacterial components and contribute to posttransplantation cholangitis. (Liver Transpl 2005;11:911–921.)

Currently, hyperacute rejection is known to be mediated by preformed alloantibodies, and acute rejection is mainly considered to be caused by alloreactive T cells while both humoral and cellular immunity are responsible for chronic rejection.1 The detection and treatment of allograft rejections has been mainly focused on T-cell–mediated processes.2 The presence of antibodies reactive to donor human leukocyte antigen (HLA) and non-HLA antigens frequently found in the sera of recipients undergoing rejection has instigated an interest in the importance of humorally mediated rejections.3 Furthermore, renewed interest in antibody-mediated rejection came partly from Feucht's discovery of using C4d as a marker for the detection of humoral rejection in kidney transplantations,4 and it is believed that the incidence of biopsy-confirmed acute humoral rejections in renal transplant is about 8% to 9%.5 Antibody-mediated rejection is typically unresponsive to conventional antirejection therapy.1 The outcome for acute humoral rejection is worse than for acute cellular rejection and therefore has recently been recognized as a major cause of allograft loss.6–8 Humoral rejection in other organ transplantations such as heart, lung, and liver is also well recognized.9–11

Liver transplantation (LTX) is considered to be the choice of treatment for a wide variety of acute and chronic hepatic disorders. Over the years the 1-year survival rate for recipients of LTX has increased to about 90%.12 These results reflect advances in organ preservation, immunosuppression, surgical techniques, and better management of complications.12, 13 However, infections including cholangitis, septicemia, and pneumonia occur in more than 70% of all transplant recipients within the first year after LTX.14 Liver allograft is known to be resistant to antibody-associated rejection,1 but less resistant to acute rejections.15

In an earlier study, we observed that preformed antibodies to biliary epithelial cells (BECs) are associated with acute rejections of liver allograft, and we discussed the clinical significance of detecting such antibodies prior to liver transplantation.16 As a follow-up, in the present study we monitored the presence of biliary epithelial cell reactive antibodies in the posttransplantation period to evaluate their clinical relevance in liver allograft outcome.

Abbreviations

LTX, liver transplantation; BEC, biliary epithelial cell; RPTECs, renal proximal tubular epithelial cells; TLR, Toll-like receptor; HLA, human leukocyte antigen; ENA-78, epithelial neutrophil activating peptide-78; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α.

Patients and Methods

Approval for the present study was given by the local ethical committee at Huddinge Hospital, Stockholm, Sweden.

Patient Population and Immunosuppression

Fifty-six patients between January 1999 and December 2000 who received a primary orthotopic LTX at Huddinge Hospital from whom sera were available both before and after transplantation were included in this study and in a previous study published by us.16 The median age was 52.0 years (interquartile range, 46.0 to 58.5 years), and 42.9% of the patients were women. The indications of LTX were mainly end-stage chronic liver diseases (Table 1). Recipient-donor selection was based upon ABO blood group compatibility and size matching, but not HLA compatibility. Follow-up time for every patient was 2 years.

Table 1. Indications of Patients for Liver Transplantation (LTx) and the Presence of Biliary Epithelial Cell–Reactive Antibodies
Indications for LTXNo. of patientsBEC antibody–positive patients (%)
Pre-LTX (+)Post-LTX (+)Pre-LTX (+)/Post-LTX (+)
  1. Abbreviations: PSC, primary sclerosing cholangitis; FAP, familial amyloidotic polyneuropathy; PBC, primary biliary cirrhosis; AIH, autoimmune hepatitis.

Hepatitis C cirrhosis113 (27.3)5 (45.5)2 (18.2)
PSC108 (80.0)5 (50.0)5 (50.0)
FAP82 (25.0)3 (37.5)1 (12.5)
PBC64 (66.7)5 (83.3)3 (50.0)
Alcoholic cirrhosis64 (66.7)4 (66.7)3 (50.0)
AIH41 (25.0)2 (50.0)1 (25.0)
Hepatitis B cirrhosis32 (66.7)3 (100.0)2 (66.7)
Others81 (12.5)5 (62.5)1 (12.5)
Total5625 (44.6)32 (57.1)18 (32.1)

One pre-LTX serum sample was taken immediately prior to LTX from each patient. A total of 219 post-LTX sera were collected from all patients during the first 4 weeks after transplantation and at 3 months thereafter, and also during acute rejection episodes and in case of hospitalization. The sera were stored at −20°C.

Immunosuppressive protocol for most of the patients (n = 39) was based on Tacrolimus/FK506 (Prograf, Fujisawa, Munich, Germany), steroids, with or without mycophenolate mofetil (Cellcept, Roche, Stockholm, Sweden). For the other 17 patients Cyclosporine A/CsA (Neoral, Novartis, Basel, Switzerland) based triple immunosuppression included mycophenolate or Imuran (Upjohn, Stockholm, Sweden) was used. Acute rejection episodes were diagnosed by clinical signs and histopathologic confirmation.17 In some rejection episodes, biopsy was contraindicated or not available due to logistic reasons, but all of these patients received standard rejection treatment based on clinical and biochemical data and responded to the treatment. The standard treatment for acute rejection episode in this study was a 1000-mg bolus dose of intravenous methylprednisolone (Solu-Medrol, Pharmacia & Upjohn Co., Stockholm, Sweden) followed by a full recycling of prednisolone (i.e., tapering daily dose from 200 mg to 20 mg). In the case of steroid-resistant rejection, treatment was switched to Tacrolimus/FK506 (Prograf) for cyclosporine patients or treated for 7 to 14 days with OKT3 (Ortho-Biotech, Inc., Raritan, NJ).

The monitoring of bilirary tract after transplantation included ultrasonography, scintigraphy, secondary cholangiogram, endoscopic retrograde cholangiopancreatography, and percutaneous transhepatic cholangiogram. The diagnosis of cholangitis was based on the presence of fever (>38.0°C), right upper quadrant pain, pathological liver function tests, and histological signs of cholangitis in liver biopsy specimen.18 Cytological evidence (granulocytes and/or intracellular bacteria) of infection in the exteriorized bile and isolation of the same microorganism in bile were used as additional criteria in most cases.

Isolation and Culture of BECs

BECs were isolated as previously described.16 Briefly, liver tissue from a normal healthy liver was mechanically disrupted and enzymatically digested. The cell mixture was seeded on tissue-culture flasks coated with 0.2% gelatin until they reached confluence. BECs were purified by immunomagnetic isolation using Dynabeads conjugated with epithelial cell specific antibody, BerEp4 (Dynal, Oslo, Norway) at a concentration of 20 μL immunomagnetic beads per 106 to 107 cells. Mixture of the cells and beads was incubated at 4°C for 30 to 40 minutes with gentle agitation. Cells coupled to the beads were harvested by applying a magnetic particle concentrator (Dynal).

Isolated BECs were cultured under conditions as previously described.19 Characterization indicated that more than 95% of the cells were BECs, which were positive for cytokeratin 19, 7, and a common cytokeratin epitope. Cultured BECs were stimulated with recombinant tumor necrosis factor-alpha and interferon-gamma (20 ng/mL and 200 ng/mL respectively; R&D Systems, Abingdon, England) overnight prior to harvesting for analysis. Control cells renal proximal tubular epithelial cells (RPTECs) were purchased from Clonetics (BioWhittaker, San Diego, CA), and cultivated according to the instructions from the suppliers.

Detection of Antibodies

BEC/RPTEC antibodies were detected using a flow cytometric assay as previously described.16 In short, 5 × 105 BEC/RPTEC were incubated with 50 μL of patient serum for 1 hour at 22°C, then washed three times with phosphate-buffered saline. Ten microliters of mixed 1:4 diluted fluorescein isothiocyanate (FITC) F(ab′)2 fragments of goat anti-human IgG (Fc specific) and IgM antibodies (Jackson Immuno-Research, PA) were added and incubated at 4°C on ice in the dark for 25 minutes. The cells were washed and then analyzed on a Becton Dickinson flow cytometer (FACSorter, Becton Dickinson, San Jose, CA). Fluorescence signals from 10,000 cells were counted and the percentage of FITC-positive cells was recorded. A shift in the mean fluorescence of 20 channels in the test sample as compared to negative control was considered positive.20 All sera giving a positive reaction were further diluted (1:100, 1:500, and 1:1000) in phosphate-buffered saline to determine the titer of the antibodies.

Specificity of BEC Antibodies

Paramagnetic microbeads coated with pooled HLA class I or II antigens (in some cases donor-specific HLA class I– or II–coated microbeads) were used for the removal of HLA antibodies from patient sera.21 The absorbed sera were retested for binding to BECs. RPTECs were used as control target cells to test for tissue specificity.

Purification of IgG Antibodies from Sera of Patients with BEC Antibodies and Preparation of F(ab′)2 Fragments

IgG fractions were isolated from the sera of all patients with cholangitis and BEC antibodies using goat antihuman IgG agarose beads (Sigma, Munich, Germany) according to standard procedure. IgG fractions were also purified from 5 LTX patients without BEC antibodies and rejections.

For the isolation of anti-BEC IgG F(ab′)2 fragments, an ImmunoPure F(ab′)2 preparation kit was used according to the instructions of the manufacturer (PIERCE; Rockford, IL). The purity of antibody and antibody fragment preparations were verified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following silver staining of the gels. The purified IgG F(ab′)2 fragments from patients and controls were concentrated individually and each fraction was used at a concentration of 4 mg/mL for the following experiments.

Reverse Transcriptase-Polymerase Chain Reaction

Since posttransplantation cholangitis are considered to be caused by bacterial infections, we investigated whether the IgG F(ab′)2 fractions from patients with cholangitis could induce the expression of Toll-like receptors (TLRs) on BECs, which may result in development of an innate immune response. We therefore initially tested gene expression for TLRs 1, 2, 3, 4, and 9. For this purpose, RNA was extracted from approximately 1 × 106 untreated BECs, cytokine-treated BECs, and BECs treated with IgG F(ab′)2 fragments from patients with cholangitis according to the instructions of the Qiagen RNA Blood Mini-kit (Qiagen, Hilden, Germany). RNA was eluted with 30 μL of RNAse-free water. The cDNA synthesis was performed in a total volume of 50 μL containing 30 μL RNA, 1× first strand buffer (50 mmol/L Tris-HCl pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2), 105 μg/mL pdN6 (Pharmacia Biosciences, Uppsala, Sweden), 1 mmol/L of each dNTP (Pharmacia Biosciences), 1 mmol/L DTT, 0.48 U/μL RNasin (Promega, Madison, WI) and 4.8 U/μL M-MLV RT (Invitrogen, Paisley, Scotland), at 37°C for 1.5 hours. The reaction was stopped by heating at 68°C for 15 minutes.

PCR: 2.5 μL of cDNA was used in a 25 μL PCR-reaction containing 1× PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% gelatin), 200 mol/L of each dNTP (Applied Biosystems/Roche, Branchburg, NJ), 5% glycerol (Sigma), 100 ng/μL cresol red (Sigma), 0.03 units/μL AmpliTaq polymerase (Applied Biosystems) and 0.5 μmol/L of each primer. Primer sequences were: ABL-F: 5′-CGG CTC TCG GAG GAG ACG ATG A-3′; ABL-R: 5′-CCC AAC CTT TTC GTT GCA CTG T-3′; TLR1-F: 5′-GCA ATG CTG CTG TTC AGC TC- 3′; TLR1-R: 5′-GTC AGA AGT CCA AAG CTC AGA-3′; (389 bp), TLR2-F: 5′-GTG ACC GCA ATG GTA TCT GCA-3′; TLR2-R: 5′-TGT AGA TCT GAA GCA TCA ATC TC-3′; (472 bp), TLR3-F: 5′-GGA GCC AGA ATT GTG CCA GA -3′; TLR3-R: 5′- GAA TTG GCA AAG ATA TCC AGT TC -3′; (321 bp), TLR4-F: 5′-CTC TCC TGC GTG AGA CCA- 3′; TLR4-R: 5′-AGA GGT GGC TTA GGC TCT GA-3′; (262 bp), TLR9-F: 5′-CGG CCT GGT GAA CTG CAA CT-3′; TLR9-R: 5′-GCA GCG CAG GCA CAG TCA T-3′ (305 bp).

PCR conditions were 94°C for 4 minutes followed by 32 PCR amplification cycles. The first 10 cycles were done in a two-segment step at 94°C for 30 seconds and at 61°C for 1 minute. The next 22 cycles were done in a three segment step at 94°C for 15 seconds, 59°C for 50 seconds, and 72°C for 30 seconds. Five microliters of the PCR products were run in a ready-to-use PAGE system (Amersham Biosciences). Nondenaturing, 12.5% polyacrylamide gels were run for 1.5 hours and visualized after an automated silver staining procedure (Amersham Biosciences).

Protein Expression of TLRs on BECs after Pretreatment with BEC Antibodies

We next tested whether BECs expressed the proteins for the various TLRs tested. For this purpose, BECs in the fourth passage were grown to confluence in 6-well culture plates (Falcon, Becton Dickinson). The cells were incubated overnight in the presence of medium alone or stimulated with recombinant tumor necrosis factor-α and interferon-γ at a final volume of 2 mL.

Parallel experiments with cytokine stimulated BECs incubated with IgG F(ab′)2 fractions (4 mg/mL) from patients with posttransplantation cholangitis or control patients at the same final volume were performed. After incubation, the cells were detached by trypsinization, washed and incubated with the following unconjugated monoclonal antibodies anti-TLR1, -TLR2, -TLR3, -TLR4, and -TLR9 (all from eBioscience, Taby, Sweden), followed by labeling with FITC-conjugated secondary goat-antimouse antibodies (Jackson ImmunoResearch Laboratories, Stockholm, Sweden). We tested for surface as well as cytoplasmic expression of the TLRs using 0.5% saponin. Isotype control antibodies were used as negative controls. After incubation on ice at 4°C the cells were washed and analyzed by flow cytometry.

BECs treated in the same manner as above with IgG F(ab′)2 fractions were also double stained by immunofluorescence as previously described.22 Staining with the above-mentioned TLR antibodies was followed by labeling with Texas red–conjugated secondary goat-anti-mouse antibodies and FITC-conjugated CK-19 (DAKO, Glostrup, Denmark).

Cytokine Production by Cultured BECs Treated with BEC Antibodies

Last, we investigated whether BEC antibodies upon binding to BECs could induce production of various inflammatory cytokines and chemokines. For this purpose, purified IgG F(ab′)2 fractions from patients and controls diluted in medium as stated above were added to BECs (1 × 106 cells) and the culture supernatants were collected after 72 hours, sterile filtered and kept frozen at −70°C until assayed. The cytokines and chemokines were measured by standard sandwich ELISA techniques using Quantikine sandwich enzyme immunoassay from R&D Systems. Assays were performed according to the manufacturer's instructions.

Liver Biopsy Staining

Liver biopsies were obtained from 6 patients with rejections and BEC antibodies before and after transplantation, 5 patients with rejections but without BEC antibodies, 5 patients with post-LTX cholangitis, and 2 healthy liver transplant donors. Paraffin-embedded specimens were obtained from the Department of Pathology at Huddinge University hospital. Deparaffinized sections were enzymatically stained. After blocking with normal bovine serum, sections were stained with F(ab′)2 fragments of goat antihuman IgG and IgM antibodies (Jackson ImmunoResearch, West Grove, PA). The immunoperoxidase procedure was carried out using Vectastain Elite ABC kit (ImmunKemi, Stockholm, Sweden) as described by the manufacturers. The DAB- nickel (gives brown/black color staining) or the Vector NovaRed kits were used as color developers. The sections were lastly counterstained with Mayer's hematoxylin (Sigma) and analyzed under a light microscope.

Statistical Analysis

Continuous outcome measures are presented in terms of median and interquartile range, and differences between groups were assessed by Mann-Whitney test. Chi-square test was used to compare categorical data. Fisher's exact test was applied when appropriate. Log-rank test was used to compare patient survival rates between different groups. All statistical analyses were done by using software STATISTICA version 6.0 (StatSoft Scandinavia AB, Uppsala, Sweden), and P values of less than 0.05 were considered to be statistically significant.

Results

Patients

The 2-year patient survival in this selected group of 56 patients was 91.1%. Thirty-five patients (62.5%) developed acute rejection episodes after LTX, in which 31 (88.6%) were confirmed by biopsy result. Acute rejection in 5 of the patients was steroid resistant. There was no difference in acute rejection episodes between FK506-based (60.0%) and cyclosporine A–based (70.6%) immunosuppression regimens (P = not significant [ns]). Nine patients developed cholangitis within 2 years after transplantation (times vary from 20 days to several months after transplantation).

Presence of BEC-Reactive Antibodies in Sera of LTX Patients and its Clinical Correlations

In the present study, 7 patients (13%) had BEC antibodies only before LTX, 14 (25%) only after LTX, 18 (32%) both before and after LTX, and 17 (30%) had no detectable antibodies. There was no significant difference in the distribution of BEC antibodies among different diseases (Table 1).

A higher numbers of patients with BEC antibodies prior to LTX developed acute rejections (20 of 25, 80.0%) compared with 15 (48.4%) of 31 without antibodies (P < 0.03) (Table 2). However, no significant difference was observed in acute rejection episodes between post-LTX BEC antibody–positive and –negative patients (P = ns). Interestingly, 9 (9 of 32, 28.1%) patients with post-LTX BEC antibodies developed cholangitis, while BEC antibodies negative patients did not (P < 0.02, Table 2). In all 9 patients with cholangitis these antibodies were already detected prior to development of cholangitis as well as during the active phase of the clinical episode. Three of the 9 cholangitis patients had a bile duct anastomosis of choledocho-jejunostomy. Choledocho-choledochostomy was performed in the other 6 patients (Table 3). The occurrence of acute rejection episodes and cholangitis is shown in Figure 1 A-B.

Table 2. Correlation Between BEC Antibodies and Acute Rejections and Cholangitis Among Different Patient Groups
 Pre-LTX (+)/Post-LTX (−)Pre-LTX (−)/Post-LTX (+)Pre-LTX (+)/Post-LTX (+)Pre-LTX (−)/Post-LTX (−)
  • *

    6 patients developed both acute rejection and cholangitis after LTx.

No. of patients7141817
Acute rejections (%)7 (100.0)8 (57.1)13 (72.2)*7 (41.2)
Cholangitis (%)0 (0.0)2 (14.3)7 (38.9)*0 (0.0)
Table 3. Some Demographic and Clinical Characteristics of Patients With Post–Liver Transplantation Cholangitis
No.IndicationsAge, yrsSexBEC AntibodiesBile Duct AnastomosisAcute RejectionsExpression of TLR2/TLR3Microbial Infections Causing Cholangitis After LTXGraft Loss
Pre-LTX (titers)Post-LTX (titers)
  • Abbreviations: PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis; Alc, alcoholic cirrhosis; FAP, familial amyloidotic polyneuropathy; HepC, hepatitis C cirrhosis; Acute LF, acute liver failure; CJ, choledocho-jejunostomy; CC, choledocho-choledochostomy.

  • *

    Graft loss is due to severe cholangitis (1), arterial thrombosis (1), arterial pseudoaneurysm (1), and sepsis and renal failure (1).

1PSC30F+ (1:50)+ (>1:500)CJ++/+Enterococcus faecium, Citrobacter species, Candida albicansNo
2PSC36M+ (1:100)+ (>1:100)CJ++/+Enterococcus faecium, Pseudomonas aeruginosaNo
3PSC61M+ (1:100)+ (>1:1000)CJ++/+Candida albicans, Pseudomonas aeroginosaYes*
4PBC58F+ (1:50)+ (>1:500)CC++/−Enterococcus speciesNo
5Alc46M+ (1:50)+ (>1:100)CC++/−Enterococcus faecium, Candida albicans, Saccharomyces cerevisiae, LactobacillusYes*
6Alc60M+ (1:50)+ (>1:100)CC++/−Enterococcus species, Candida albicans, Candida glabrataNo
7FAP30F+ (1:20)+ (>1:500)CC+/−Enterococcus faecium, coagulase-negative staphylococciYes*
8HepC56M+ (>1:1000)CC++/+Not availableYes*
9Acute LF54F+ (>1:500)CC+/−Candida albicans, Stenotrophomonas maltophiliaNo
Figure 1.

Number of acute rejection and cholangitis episodes in LTX patients. (A) Significantly higher numbers of patients with BEC-reactive antibodies present before LTX had acute rejections compared with those without any demonstrable antibodies (P< 0.03). (B) Significantly higher numbers of patients with BEC-reactive antibodies present after liver transplantation had posttransplantation cholangitis compared with those without any demonstrable antibodies (P< 0.02).

We found that lower numbers of patients (5 of 56, 8.9%) had antibodies against RPTECs compared with BECs. The presence of RPTEC antibodies did not correlate with acute rejections, and BEC antibodies from five patients crossreacted with RPTEC.

Specificities of BEC Antibodies

The majority of the immunoglobulins in BEC antibody–positive sera were a mixture of IgG and IgM. The titer of BEC antibodies varied from lower than 1:100 to higher than 1:1000. A higher BEC antibody titer was found in most patients with rejection episodes compared with those without rejection episodes, but the difference was not statistically significant.

We tested whether the antibodies were HLA specific by using absorption assays with HLA class I and class II antigen-coated magnetic beads. Our results demonstrated that in 8 patients reactivity against BECs was caused by HLA antibodies, and 4 (50%) developed cholangitis. In 7 patients reactivity against BECs was partially absorbed by HLA antigen-coated beads, indicating presence of HLA and non-HLA antibodies. In 24 patients no demonstrable HLA antibodies were detected indicating presence of only non-HLA antibodies (Fig. 2).

Figure 2.

Specificities of BEC-reactive antibodies. No correlation of the presence of HLA or non-HLA antibodies reactive with BECs was observed with acute rejections (P= ns) or cholangitis (P= ns). *Some patients had both cholangitis and acute rejections.

Gene and Protein Expression of TLRs on BECs

We first tested whether BECs expressed mRNAs for TLRs 1, 2, 3, 4, and 9 and found that unstimulated and cytokine-stimulated BECs expressed mRNAs for all the TLRs tested. (Fig. 3A). We next tested for the protein expression of the various TLRs on untreated and cytokine-treated BECs and found no constitutive or cytokine-induced surface or intracellular expression of these molecules (Table 4, Fig. 3B). However, treatment of BECs with anti-BEC IgG F(ab′)2 fragments from patients with post-LTX cholangitis induced surface expression of TLR-2 and -3 on cytokine-stimulated but not unstimulated BECs (Table 4, Fig. 3B). No expression of any of the TLRs was observed with IgG F(ab′)2 fractions from control patients (Table 4, Fig. 3B).

Figure 3.

Gene and protein expression of TLRs in BECs. (A) BECs expressed mRNAs for TLRs 1, 2, 3, 4, and 9. ABL was used as housekeeping gene. No obvious difference in amounts of gene expression was observed between unstimulated (lane 1) BECs, cytokine-stimulated (lane 2) BECs, or BECs treated with IgG F(ab′)2 fragments from LTX patients with cholangitis (lanes 3-5). (B) Flow cytometric analysis demonstrated that unstimulated BECs, cytokine-stimulated BECs, and BECs treated with IgG F(ab′)2 fragments from LTX patients without BEC antibodies did not express the proteins for the tested TLRs. However, BECs treated with IgG F(ab′)2 fragments from LTX patients with cholangitis and BEC antibodies showed induced expression of surface proteins for TLRs 2 and 3. (C) Immunofluorescent-positive staining for TLR2 (red, Texas red) and 3 (red, Texas red) on BECs treated with IgG F(ab′)2 fragments from two different liver-transplanted patients with cholangitis. The cells were double stained for cytokeratin 19 (green, FITC). The nucleus is stained blue (DAPI). In some areas the TLRs and CK19 were colocalized (yellow).

Table 4. Expression of Toll-Like Receptors on Biliary Epithelial Cells as Determined by Flow Cytometric Analysis
Antibodies toUnstimulated BECsTNF-alpha & IFN-gamma stimulated BECsBECs + F(ab′)2 Ig fraction from cholangitis patients n = 9BECs + F(ab′)2 Ig fraction from control patients n = 5
TLR1
TLR2++
TLR3++
TLR4
TLR9

BEC- Reactive Antibodies Induced Production of Inflammatory Cytokines and Chemokines

Anti-BEC IgG F(ab′)2 from cholangitis patients induced BECs to produce significantly higher levels of IL-6 and the chemokines IL-8, MCP-1 (monocyte chemoattractant protein-1), MIP-1α (macrophage inflammatory protein-1 alpha), and ENA78 (epithelial neutrophil activating peptide-78) at 72 hours compared with F(ab′)2 from controls (P < 0.005) (Table 5).

Table 5. Cytokine Production by Biliary Epithelial Cells After IgG F(ab′)2 Fraction Treatment
BEC supernatantIL-1β pg/mLIL-6 pg/mLIL-8 pg/mLIL-10 pg/mLIL-12 pg/mLIFN-γ pg/mLTNF-α pg/mLENA-78 pg/mLMIP-1α pg/mLMCP-1 pg/mL
  1. NOTE. Values are shown as median (interquartile range).

Untreated (n = 7)<30<30<30<30<30<30<30<30<30<30
IgG F(ab′)2 (from cholangitis patients) treated (n = 9)<30651 (614–659)10659 (7370–11013)<30<30<30<30292 (150–371)159 (123–348)5002 (4375–9786)
IgG F(ab′)2 (from control patients) treated (n = 5)<30<30<30<30<30<30<30<30<30<30
P valueNS<0.005<0.005NSNSNSNS<0.005<0.005<0.005

In Vivo Deposition of Immunoglobulins on BECs during Rejection Episodes and Cholangitis

No IgG or IgM deposition in bile ducts of normal livers or patients without BEC antibodies was observed (Fig. 4A-C). However, intense binding of IgG and IgM to BECs (black/brown staining) during rejections was observed in livers of patients with BEC antibodies and rejections (Fig. 4D-F). Binding of immunoglobulins (red staining) to BECs was also observed in patients during episodes of cholangitis (Fig. 4G-I).

Figure 4.

Immunohistochemical detection of immunoglobulin deposition in liver biopsy sections. No IgG or IgM deposition in bile ducts of normal livers (A) or patients without BEC antibodies (B-C) was observed. (D-F)However, intense binding of IgG and IgM to BECs (black/brown staining) during rejections was observed in livers of patients with BEC antibodies and rejections. Some positive staining of sinusoidal cells was also observed. Results from three different patients are shown. (G-I), Binding of immunoglobulins (red staining) to BECs was also observed in patients during episodes of cholangitis. Results from three different patients are shown. The sections were counterstained with hematoxylin. Original magnifications ×60 (A-D), and ×40 (E-I).

Discussion

In our previous study we reported that preformed BEC antibodies are associated with acute rejections.12 In the present study we observed that the presence of HLA or non-HLA reactive BEC antibodies in the posttransplantation period was significantly associated with occurrence of cholangitis. Furthermore, binding of these antibodies to BECs was found to induce expression of TLR2 and 3 and production of inflammatory cytokines and chemokines such as IL-6, IL-8, MCP-1, MIP-1α and ENA78, all of which are important in recruitment of inflammatory cells such as neutrophils, monocytes, macrophages, and cytotoxic T cells. The in vivo deposition of immunoglobulins in bile ducts during acute rejections and cholangitis further suggests a role for BEC antibodies in facilitating these processes. Unfortunately, detection of TLRs in bile ducts during episodes of cholangitis could not be demonstrated, since the TLR antibodies were not functional in paraffin embedded biopsy sections. In our earlier study16 we reported that HLA antibodies were significantly associated with cholangitis. In the present study, no difference between the association of either HLA or non-HLA BEC antibodies with cholangitis was observed. The disparity may be explained by the fact that the HLA antibodies are not donor specific. Thus, the exact role of donor-specific HLA antibodies in cholangitis remains to be elucidated.

Cholangitis after transplantation is believed to be caused by ascending bacterial infection often in combination with an obstructed biliary tree. The pathogenic mechanism is unclear, and systemic sepsis may not necessarily occur. Prerequisite conditions are the presence of microorganisms in the bile and increased biliary pressure. Bacteria that commonly cause cholangitis are Escherichia coli, Klebsiella, Enterococcus, Enterobacter, Pseudomonas, and anaerobes.23 Bacterial pathogens are widely recognized as inflammatory stimulants.24, 25 The innate immune system recognizes bacterial pathogens through a family of receptors called TLRs. TLRs provide the elusive link between recognition of invading organisms and development of the innate immune response.

In the present study, we demonstrate that BEC antibodies induce expression of two TLRs on BECs, namely, TLR2 and TLR3. TLR2 responds to a number of bacterial products from both gram-positive and gram-negative organisms, including peptidoglycans, lipoteichoic acid, and mycoplasmal proteins. Fungi are also ligands for TLR2.26 TLR3 has shown to mediate the response to single- and double-stranded viral RNA.24 TLR3 activation leads to an induction of antiviral and proinflammatory cytokines and dendritic cell maturation.27 Bile culture results from the cholangitis patients showed that the majority of microorganisms in the bile during cholangitis episodes were gram-positive bacteria and fungi. In a few cases gram-negative bacteria were also found. This spectrum fits well with the ligands for TLR2. The ligand for TLR3 is double-stranded viral RNA. Since it is rather tedious and difficult to isolate virus from bile, the role of virus in causing posttransplantation cholangitis still needs to be further studied even though some correlations have been reported.28 On the other hand, TLR4 and TLR9 protein expression were not induced on BECs by BEC antibodies in this study. The ligands for TLR4 are gram-negative lipopolysaccharides and TLR9 binds to bacterial DNA.29 The lack of TLR4 and TLR9 expression on BECs in our study might indicate lack of the appropriate stimulating ligands or alternatively a limited role for these rerceptors in post-LTX cholangitis or involvement of mechanisms other than BEC antibodies.

The production of specific cytokines influences local immunity, including neutrophil influx and transmigration across the epithelial cell barrier, which is necessary for bacterial clearance.30 Thus, BEC antibody-mediated enhancement of IL-6, IL-8, ENA-78, MIP-1α and MCP-1 production, as well as TLR2 and TLR3 expression on BECs, suggest that BECs may play an important role in the local immune response, which in turn may be an important determinant of clinical outcome. Since BEC antibodies induce TLR2 and TLR3 expression, it is possible that the induced responsiveness of epithelial cells to bacterial products is the result of enhanced or sustained signaling via newly expressed TLR2 and TLR3. This is supported by the finding that in all 9 patients with cholangitis BEC antibodies were consistently found prior to and persisted long after treatment for cholangitis (as shown by samples taken before, during and after cholangitis episode). The fact that 7 of the 9 patients had recurrent cholangitis (days or months after the first cholangitis episode) also supports the existence of a sustained inflammatory response. In addition, these patients may be at a risk for subsequent tissue scarring or chronic rejections via these sustained inflammatory responses. It is well known that bile duct anastomosis by choledocho-jejunostomy is a risk factor for cholangitis after transplantation.31 However, this anastomosis was performed only in 3 of the 9 cholangitis patients with positive BEC antibodies. Arterial thrombosis, which is another risk factor for cholangitis, occurs only in one patient. Thus, BEC antibodies may be an additional risk factor for development of cholangitis after transplantation. The response to bacterial pathogens via pattern recognition receptors on epithelial cells is important for innate immune defense but their amplified activation may lead to persistent epithelial inflammation.

To our knowledge, this is the first study that demonstrates the ability of antibodies to upregulate TLR2 and TLR3 expression and modulate chemokine secretion in BECs. The induction of TLR expression by antibodies may provide a critical role for antibodies in epithelial cell response to microbial pathogens and activation of inflammatory responses. Not all patients with BEC antibodies in the posttransplantation period had cholangitis. This may be explained by the fact that antibodies are heterogenous with respect to specificities, affinity, titers, and isotypes. In addition, we found some patients with detectable antibodies only in the pre- but not posttransplantation period. These antibodies were found to be significantly associated with acute rejections but not cholangitis. The lack of antibody detection in the posttransplantation serum may not necessarily mean the loss/disappearance of antibodies. The possibility remains that the antibodies may be bound to BECs in the graft during rejections (as demonstrated in the present study) and therefore may not be found in circulation. Furthermore, the specificities of the antibodies associated acute rejections may be different from those facilitating cholangitis.

The results presented here unravel one of the mechanisms by which BEC antibodies induce epithelial cell sensitivity to TLR2 and TLR3 ligands leading to cholangitis. Therefore, BEC antibodies may play a role in the pathogenesis and clinical course of bacterial cholangitis. Even though we here have, presented results using BECs isolated from one liver, we have over the years prepared a panel of primary BEC lines from different healthy liver transplant donors. We are able to confirm the present results with all the primary BEC lines (n = 5) at our laboratory. The results of the present study warrant further investigations, which include larger number of patients to evaluate the role of BEC antibodies in post-LTX cholangitis.

In conclusion, the results suggest that patients who have BEC antibodies in the posttransplantation period may be at a risk for persistent and amplified inflammatory responses to bacterial pathogens. Plasmapheresis and other B-cell–targeting therapies, which have been shown to be effective in blood group ABO-incompatible kidney transplant,32 may be considered for BEC antibody–positive liver transplant patients.

Acknowledgements

The authors wish to thank Silvia Nava for help with immunohistochemical staining and Dr Grzegorz Nowak for the liver biopsy specimens.

Ancillary