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Original article
Annexin A11 is targeted by IgG4 and IgG1 autoantibodies in IgG4-related disease
  1. Lowiek M Hubers1,
  2. Harmjan Vos2,
  3. Alex R Schuurman1,
  4. Robin Erken1,
  5. Ronald P Oude Elferink1,
  6. Boudewijn Burgering2,
  7. Stan F J van de Graaf1,
  8. Ulrich Beuers1
  1. 1 Department of Gastroenterology & Hepatology, Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands
  2. 2 Center for Molecular Medicine, Molecular Cancer Research Section, University Medical Center, Utrecht, The Netherlands
  1. Correspondence to Dr Ulrich Beuers, Department of Gastroenterology & Hepatology, Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; u.h.beuers{at}amc.uva.nl

Abstract

Objective Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan immune-mediated disease that predominantly affects the biliary tract (IgG4-associated cholangitis, IAC) and pancreas (autoimmune pancreatitis, AIP). We recently identified highly expanded IgG4+ B-cell receptor clones in blood and affected tissues of patients with IAC/AIP suggestive of specific (auto)antigenic stimuli involved in initiating and/or maintaining the inflammatory response. This study aimed to identify (auto)antigen(s) that are responsible for the clonal expansion of IgG4+ B cells in IgG4-RD.

Design We screened sera of patients with IAC/AIP (n=50), in comparison to control sera of patients with primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies (n=47), for reactivity against human H69 cholangiocyte lysates on immunoblot. Subsequently, target antigens were immunoprecipitated and analysed by mass spectrometry.

Results Prominent reactivity against a 56 kDa protein was detected in human H69 cholangiocyte lysates exposed to sera of nine patients with IAC/AIP. Affinity purification and mass spectrometry analysis identified annexin A11, a calcium-dependent phospholipid-binding protein. Annexin A11-specific IgG4 and IgG1 antibodies were only detected in serum of patients with IgG4-RD of the biliary tract/pancreas/salivary glands and not in disease mimickers with PSC and pancreatobiliary malignancies. Epitope analysis showed that two annexin A11 epitopes targeted by IgG1 and IgG4 autoantibodies were shared between patients with IAC/AIP and IgG4 antibodies blocked binding of IgG1 antibodies to the shared annexin A11 epitopes.

Conclusion Our data suggest that IgG1-mediated pro-inflammatory autoreactivity against annexin A11 in patients with IgG4-RD may be attenuated by formation of annexin A11-specific IgG4 antibodies supporting an anti-inflammatory role of IgG4 in IgG4-RD.

  • autoimmune biliary disease
  • auto-antibodies
  • pancreatitis
  • antigens

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Significance of this study

What is already known on this subject?

  • Patients with  immunoglobulin G4-related disease (IgG4-RD) of the biliary tract and pancreas are characterised by highly expanded IgG4+ B-cell receptor clones in blood and affected tissues, which disappear on successful corticosteroid treatment.

  • Injection of IgG1 antibodies of patients with autoimmune pancreatitis in mice resulted in pancreatic and salivary gland injuries, but the pathogenic effects of IgG1 were inhibited when IgG4 was simultaneously injected.

What are the new findings?

  • Annexin A11 is a novel autoantigen in IgG4-RD and is targeted by IgG4 and IgG1 antibodies in serum of patients with IgG4-RD of the biliary tract/pancreas/salivary glands, but not in disease mimickers with primary sclerosing cholangitis and pancreatobiliary malignancies.

  • IgG4 antibodies blocked binding of IgG1 to annexin A11 supporting an anti-inflammatory role of IgG4 in IgG4-RD

How might it impact on clinical practice in the foreseeable future?

  • Identification of an IgG4-target autoantigen opens the door to further unravel disease mechanisms underlying IgG4-RD.

Introduction

Imunoglobulin G4-related disease (IgG4-RD) is a recently defined immune-mediated syndrome, which can present with manifestations in a wide range of organs.1–3 The biliary tract (IgG4-associated cholangitis, IAC) and pancreas (autoimmune pancreatitis, AIP) belong to the organs most frequently involved, but the ever-expanding list of localisations includes the hypophysis, orbita, lacrimal and salivary glands, thyroid, lungs and hilum, kidneys, retroperitoneum, aorta, prostate, testis and lymph nodes.4–6 Patients with IgG4-RD share histopathological and serological features and respond remarkably well to corticosteroid treatment. Affected tissues typically show a dense lymphoplasmacytic infiltrate with high numbers of IgG4+ B and plasma cells,7 which contribute to the often elevated IgG4 serum levels,8 a storiform pattern of fibrosis, and obliterative phlebitis.

We recently reported that IgG4+ B cells identified in blood and affected tissue of patients with IAC/AIP originate from a limited number of highly expanded IgG4 class-switched B cell receptor clones.9 10 Mutational analysis disclosed various non-silent mutations in the dominant IgG4+ clones consistent with affinity maturation.9 10 For comparison, control patients with primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies did not exhibit dominant IgG4+ clones in their B cell receptor repertoire. These findings not only made it possible to develop an accurate diagnostic test for this otherwise difficult-to-diagnose disease,10 they also strengthened the hypothesis that specific antigenic stimuli are involved in the antibody response in IgG4-RD.11 To date, however, no disease-specific (auto)antigens have been identified in patients with IgG4-RD.

The role of IgG4 in IgG4-RD remains unclear. IgG4 antibodies possess unique immunological properties, which differ markedly from those of other immunoglobulin subclasses. IgG4 is unable to fix C1q,12 which is necessary for activation of the classical complement cascade, and blocks IgG1-mediated complement activation.13 In addition, IgG4 antibodies mutually exchange half molecules, resulting in bispecific antibodies that cannot crosslink antigen and form large immune complexes.14 As a result, IgG4 is not able to activate low-affinity Fc-gamma receptors on effector cells that only bind IgG-immune complexes.15 In beekeepers, it has been shown that bee venom-specific IgG4 is upregulated on chronic exposure to bee stings. By competitive binding to the allergen IgG4 dampens the pro-inflammatory effect of an IgE-mediated immune response and protects against hypersensitivity reactions.16 17 Similarly, IgG4 block antitumor activity of IgG1 antibodies in melanoma cells.18

Here, we aimed to identify target autoantigens that can be held accountable for the oligoclonal expansion of IgG4+ B cells9 10 and the often highly elevated IgG4 serum levels found in these patients. Identification of autoantigens may broaden our understanding of the pathophysiological mechanisms underlying IgG4-RD and the role of IgG4 in the specific inflammatory response observed in IgG4-RD.

Materials and methods

Study approval

This study was approved by the local medical ethical committee in Amsterdam (MEC 10/007). Participants gave written informed consent prior to inclusion in the study. Serum samples were collected at the Academic Medical Center in Amsterdam between March 2010 and December 2016. Patients with IAC/AIP fulfilled diagnostic Histology, Imaging, Serum IgG4, Organ involvement, Response to therapy (HISORt) criteria.19 20 Patients with PSC were diagnosed according to the 2009 European Association for the Study of the Liver (EASL) Clinical Practice Guidelines on the management of cholestatic liver diseases.21 Diagnosis of pancreatobiliary malignancies was confirmed by histopathology and/or cytology.

Cell cultures

H69 cells, an immortalised non-malignant human intrahepatic cholangiocyte cell line, were kindly provided by Dr Douglas M Jefferson. The medium consisted of 3:1 Dulbecco’s modified Eagle’s Medium (DMEM)/Ham’s F12 medium (Life Technologies/Thermo Fisher Scientific, Waltham, Massachusetts, USA), supplemented with 10% fetal bovine serum (FBS, Gibco/Thermo Fisher Scientific), 180 µM adenine, 865 nM insulin, 62.5 nM transferrin, 2 nM triiodothyronine, 1.1 µM hydrocortisone, 5.5 µM epinephrine and 1.67 µM epidermal growth factor. HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% L-glutamine. All media contained penicillin and streptomycin. Cells were kept at 37ᵒC in a 5% CO2 incubator and passaged twice weekly.

Protein lysates

Cells were cultured on 150 mm dish until confluent. H69 cell monolayers were washed twice with phosphate buffered solution (PBS). H69 cell lysates were prepared using 5.4 mL radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% (w/v) nonyl phenoxypolyethoxylethanol (NP)-40, 0.5% (w/v) sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 mM Tris-HCl, pH 8.0) with addition of protease inhibitor cocktail (Roche, Basel, Switzerland) and 5 mM ethylene glycol tetraacetic acid (EGTA) at 4°C. Insoluble parts were spun down at 12 000 g. Extraction of cytosolic proteins was performed by digitonin permeabilisaton of the cell membrane using 5.4 mL digitonin buffer per 148 cm2 Petri dish (50 µg/mL digitonin in PBS) supplemented with protease inhibitor and 5 mM EGTA. Plates were incubated on orbital shaker at 100 rpm for 20 min at 4°C. The supernatant containing cytosolic proteins was separated. Permeabilised monolayers were immediately washed twice with PBS and incubated with 5.4 mL 0.45% (w/v) NP-40 in PBS with protease inhibitor and 5 mM EGTA for 20 min at 4°C. Monolayers were collected by scraping and spun down at 14 000 rpm for 10 min at 4°C. The supernatant containing membranous fraction was separated. The pellet containing nuclei was washed twice with PBS and lysed in 5.4 mL RIPA buffer with protease inhibitor and 5 mM EGTA.

Primary antibodies

See online supplementary table 2.

Supplementary file 1

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting

Equal amounts of protein (20–30 µg per lane) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane by semidry blotting at 0.06 A per gel for 75 min. Membranes were blocked in 5% non-fat milk in TBST (tris-buffered saline with 0.05% Tween 20) for 1 hour at room temperature or overnight at 4ᵒC. Membranes were cut and incubated with serum diluted in 1% non-fat milk/TBST for 1 hour at room temperature or overnight at 4ᵒC. Sera were diluted to a final IgG4 concentration of 0.04 mg/mL. Membranes were washed three times with TBST for 5 min. Subsequently, membranes were incubated with antihuman IgG4 horseradish peroxidase (HRP)-conjugated antibody or anti-human IgG1 HRP-conjugated antibody in 5% non-fat milk/TBST for 1 hour at room temperature, followed by three washing steps. For immunodetection, the membrane was incubated with home-made enhanced chemiluminescence buffer (100 mM Tris-HCl pH 8.5, 1.25 mM luminol, 0.2 mM p-coumarin and freshly added 3 mM H2O2) for 1 min and detection was performed on an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Chicago, Illinois, USA).

Immunoprecipitation

One hundred microlitres of IgG4 high-affinity beads (CaptureSelect, Thermo Fisher Scientific; binding capacity 6 mg/mL) were washed twice with wash buffer A (PBS with 0.1% NP-40) and incubated with patient serum containing 600 µg of IgG4 for 1 hour at room temperature on a roller bank. After five washes with wash buffer A and transferring beads to a new Eppendorf, IgG4 antibodies were eluted twice for 10 min with 62.5 µL of citrate buffer (pH 3.0). Beads were spun down, after which the supernatant containing IgG4 was transferred to a new Eppendorf and pH was neutralised using 125 µL of 0.5 M borate buffer pH 8.8 to a final pH above 8. Purified IgG4 was added to 250 µL of prewashed tosyl-activated magnetic beads (Dynabeads M-280, Thermo Fisher Scientific). After vortexing the beads, 125 µL of 3.6M ammonium sulfate in 0.1M borate buffer pH 8.8 was added and IgG4 antibodies were covalently bound to the beads during an overnight incubation on a rollerbank at 37ᵒC. Next day, beads were washed with wash buffer B (50 mM Tris pH 7.4, 1 M NaCl, 1% (w/v) NP-40), blocked for 1 hour in 1M Tris pH 7.4, washed again four times in wash buffer B and transferred to a new Eppendorf. Subsequently, 1200 µL of H69 cytosolic cell lysate with addition of 1M NaCl and 1% (w/v) NP-40 was added per 50 µL of beads and incubated overnight at 4ᵒC on a rollerbank. The following day, unbound protein was washed away in three washing steps with wash buffer B and three washes with PBS. Meanwhile, during washing steps, beads were transferred twice to a new Eppendorf. Finally, the supernatant was discarded and beads were frozen for mass spectrometry analysis or bound proteins were eluted from the beads for immunoblotting.

To confirm that the target protein was annexin A11, immunoprecipitation on H69 cytosolic cell lysate was performed using IgG4 CaptureSelect beads coated with IgG4 of patients with IgG4-RD. Precipitated proteins were blotted for annexin A11 using anti-annexin A11 antibody. Again, a pull down with IgG4 of a patient with PSC served as control.

Sample preparation for mass-spectrometry

Precipitated proteins were denatured with 8 M urea in 1 M ammonium bicarbonate (ABC) reduced with tris(2-carboxyethyl)phosphine (TCEP) (10 mM) at room temperature for 30 min after which the cysteines were alkylated with chloroacetamide (40 mM end concentration) for 30 min. After fourfold dilution with ABC, proteins were on-bead digested overnight at room temperature with 150 ng of Trypsin/LysC (Promega, Madison, Wisconsin USA), after which peptides were bound to an in-house-made c18 stage tip, washed with 0.1% formic acid (buffer C) and stored at 4 ˚C until liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis.

LC-MS/MS analysis

After elution from the stage tips, acetonitrile (ACN) was removed from the samples using a SpeedVac and the remaining peptide solution was diluted with buffer C before loading. Peptides were separated on a 30 cm pico-tip column (50 µm ID, New Objective/Ms Wil, Zurich, Switzerland) in-house packed with 3 µm aquapur gold C-18 material (Dr Maisch, Ammerbuch, Germany) using a 140 min or 200 gradient (7% to 80% ACN, 0.1% formic acid), delivered by an easy-nLC 1000 (Thermo Fisher Scientific), and electro-sprayed directly into an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). The latter was set in data-dependent Top speed mode with a cycle time of 1 s, in which the full scan over the 400–1500 mass range was performed at a resolution of 240 000. Most intense ions (intensity threshold of 5000 ions) were isolated by the quadrupole and fragmented with an higher-energy collisional dissociation (HCD) collision energy of 30%. The maximum injection time of the ion trap was set to 35 ms.

Data analysis

Raw files were analysed with the Maxquant software (V.1.5.2.8) with deamidation of glutamine and asparagine as well as oxidation of methionine set as variable modifications, and carbamidomethylation of cysteine set as fixed modification. The Human protein database of Uniprot was searched with both the peptide as well as the protein false discovery rate set to 1%. The intensity-based absolute quantification (IBAQ) and the label-free quantification (LFQ) quantification algorithms were used in combination with the ‘match between runs’ tool (option set at 2 min), all of which are integral parts of the Maxquant software.22–24 Proteins identified with two or more unique peptides were filtered for reverse hits and standard contaminants. LFQ intensities, which are based on the extracted ion current normalised on total peptide abundance, were log2 transformed after which identified proteins were filtered for at least three valid values in at least one sample group. Missing values were replaced by imputation, based on a normal distribution, using a width of 0.3 and a downshift of 1.8, this to simulate a more realistic value for the noise. After a standard t-test, p values were plotted against the difference of the mean log2 transformed LFQ values using the programme R (www.R-project.org). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride) with the dataset identifiers.

Genetic knock-down and overexpression of ANXA11

Lentiviral constructs for shRNA-mediated knock-down of ANXA11 were obtained from MISSION TRC shRNA library (TRCN0000056374, Sigma-Aldrich, Saint Louis, Missouri, USA). Production of Lentivirus was performed as described before.25 H69 cholangiocyte cells were stably transduced and selected with 1 µg/mL puromycin. ANXA11 with V5/HIS-tag was transiently overexpressed in HEK293T cells by topoisomerase I (TOPO) cloning and Gateway cloning (Thermo Fisher Scientific). In short, PCR forward (5’-CACCCATGAGCTACCCTGGCTATCC-3’) and reverse (5’-GTCATTGCCACCACAGATCT-3’) primers covering the full ANXA11 sequence were designed using online resources. For TOPO cloning, a four-base pair sequence (CACC) was added to the forward primer of the 5’ prime end for directional cloning. PCR reactions were performed on H69 cDNA in a BioRad T100 Thermal Cycler. PCR products were analysed by electrophoresis using ethidium bromide-containing agarose gels (1% in TAE buffer). The DNA was gel extracted using a gel extraction kit (Qiagen, Hilden, Germany). Purified PCR products were cloned into TOPO vector (pENTR/D-TOPO) using TOPO cloning kit (Thermo Fisher Scientific) and transformed in chemically competent Escherichia coli bacteria. Subsequently, the construct was ligated into a destination vector with HIS/V5 tag (pcDNA-DEST40) by gateway LR clonase (Thermo Fisher Scientific). Correct cloning was verified by restriction analysis using restriction enzymes (New England Biolabs, Ipswich, Massachusetts). Next, HEK293T cells were transiently transfected with polyethylenimine. Butyrate (5 mM end concentration) was added 24 hours post-transfection. Cells were harvested 48 hours after transfection.

ELISA

To identify the target epitope, 99 linear peptides that cover the full annexin A11 protein (15-aa each, 10-aa overlap) were purchased (Pepscan, Lelystad, The Netherlands). ELISA plates were coated overnight with 50 µL of pooled peptides with a concentration of 50 µg/mL/peptide. Large pools consisted of 24 or 25 different peptides, whereas small pools consisted of six to seven peptides. Plates were washed with PBS and blocked with 3% bovine serum albumin (BSA)/PBS for 2 hours. Plates were washed with PBST (PBS with 0.05% Tween 20) and incubated with sera diluted 1:10 in 2% BSA/PBST for 3 hours, followed by washing and incubation with anti-IgG1 (1:400) or anti IgG4 (1:100) HRP-conjugated antibody in 2% BSA/PBST for 1 hour. Bound antibodies were detected by adding 50 µL of H2O2 activated o-phenylenediamine dihydrchloride (OPD) substrate. The reaction was stopped using 30 µL of 1 M H2SO4. The experiment was performed at room temperature.

Competition assay

In order to show that IgG4 blocked binding of IgG1 of the same patient to the target antigen, a competition ELISA was set up. Briefly, high-binding protein 96-wells ELISA plates (Nunc Microwells plate, Thermo Fisher Scientific) were coated overnight with 50 µL of anti-V5 antibody in PBS (1:400). Plates were washed with PBS and blocked with 2% (w/v) gelatin in PBS. Subsequently, plates were incubated with lysate of HEK293T cells overexpressing annexin A11 labelled with V5-tag (1:6) for 4 hours. HEK293T cell lysate was used as control. Meanwhile, IgG4 antibodies from patients were immunoprecipitated using 40 µL of IgG4 CaptureSelect beads as described above. Proteins were eluted twice with 40 µL 0.1 M glycin pH 3.0 for 10 min and pH was buffered with 80 µL of 1 M Tris pH 7.4 supplemented with 1% (w/v) gelatin. Plates were washed with PBST. Either 0 µL, 10 µL (15 µg IgG4) or 40 µL (60 µg IgG4) of eluted IgG4 was added per well, after which wells were filled to 50 µL with 1% gelatin/PBST. IgG4 from serum of a control patient with cholangiocarcinoma served as control. Plates were placed on a plate shaker. Next day, plates were washed with PBST. Fifty microlitres of serum of IgG4-RD patients diluted in 1% gelatin/PBST (1:50) was added to the wells and incubated for 4 hours. Plates were washed again and incubated with anti-IgG1 HRP-conjugated antibody in 1% gelatin/PBST (1:400) for 2 hours and washed again. Bound antibodies were detected using OPD substrate as described above. The experiment was performed in triplicate and at room temperature.

Statistics

All results are presented as mean±SD. Statistical significance was determined by two-tailed Student’s t-test using SPSS Statistics (V.22, IBM). Statistical significance was considered at p<0.05.

Results

IgG4 and IgG1 autoantibodies from patients with IAC/AIP recognise a human 56 kDa cytosolic protein on immunoblot.

We screened sera of 50 patients with IAC/AIP for reactivity against a lysate of human non-malignant H69 cholangiocytes on immunoblot. This revealed that IgG4 antibodies from sera of multiple patients (n=9) bound to a 56 kDa protein (figure 1A). Clinical characteristics of these patients are shown in table 1. In contrast, none of the sera of control patients with PSC (n=20) or pancreatobiliary malignancies (n=27) demonstrated a band on immunoblot at the same molecular weight, even though we corrected serum dilutions for the differences in IgG4 serum levels between patients. Notably, when we screened the same panel of sera (n=97) for reactivity of IgG1, we found that seven patients with IgG4-reactive antibodies against this 56 kDa protein also exhibited antibodies of the IgG1 subtype against a protein of the same molecular weight (figure 1B). Again, none of the controls showed a similar staining. Secondary mouse antibodies against human IgG4 and IgG1 were specific and did not cross-react with IgG1 and IgG4 protein on immunoblot, respectively (see online supplementary figure 1). Of note, the antibodies remained present in the blood of patients after successful treatment. Next, we found that the target protein was located in the cytosolic fraction of H69 cells (n=4), and not in the membranous or nuclear fraction (see online supplementary figure 2A). Also, screening of sera (n=4) against a human liver lysate, revealed staining of a protein of 56 kDa (see online supplementary figure 2B). This suggests that the protein of interest is present in human organ tissue as well.

Figure 1

Multiple patients with  immunoglobulin G4-related disease (IgG4-RD) have autoreactive antibodies of the IgG4 and IgG1 subclass against a ±56 kDa protein in H69 cholangiocyte cell lysate on immunoblot. (A, B) Sera of patients with IgG4-RD of the biliary tract/pancreas diluted to an end concentration of 0.04 mg IgG4/ml in 1% non-fat milk/TBST were tested for reactivity against H69 cell lysate on immunoblot. Subsequently, bound IgG4 (A) and IgG1 (B) were detected. Lanes were non-continuous and originate from multiple blots. CA, pancreatobiliary carcinoma; IRD, IgG4-RD; kD, kilo Dalton; PSC, primary sclerosing cholangitis.

Table 1

Clinical characteristics of nine patients with IgG4-RD and reactivity against a 56 kDa protein

Identification of annexin A11 as an autoantigen

In order to identify protein autoepitopes in IgG4-RD patients, IgG4 was purified from serum of two patients with IAC/AIP that showed reactivity (IRD2 and IRD4) as well as serum of a control patient (PSC2). Purified IgG4 was covalently bound to beads and used to isolate antigen-containing/epitope-containing proteins from cytosolic fractions of H69 cholangiocyte cell lysates. To test the procedure, precipitated proteins were subjected to SDS-PAGE and probed using serum of the same patient and an HRP-labelled antibody against IgG1 for detection (see online supplementary figure 3). This way the successful precipitation of the target protein was confirmed. Moreover, this proved that both IgG4 and IgG1 antibodies recognised the same 56 kDa protein. Of note, also sera of other patients with autoreactive antibodies could be used for detection on immunoblot suggesting that the target protein was shared between patients (see online supplementary figure 3). Label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the proteins that were enriched in the IgG4-RD patients compared with the PSC2 patient (figure 2A, see online supplementary figure 4, online supplementary table 1). In both IgG4-RD patient cases, we identified the 56 kDa protein annexin A11 as being specifically enriched after immunoprecipitation, leading to the conclusion that this protein is the autoantigen.

Figure 2

Target antigen is annexin A11. (A) Antigen-containing proteins were enriched from H69 cholangiocyte cell lysate by immunoprecipitation using serum  immunoglobulin G4 (IgG4) of a patient with IgG4-associated cholangitis/autoimmune pancreatitis (IAC/AIP) (IRD2) or serum IgG4 of a PSC patient (PSC2) as a control. For each patient four independent pull downs were performed. Enriched samples of patient IRD2 were analysed by label-free quantitative mass-spectrometry, which identified annexin A11 as a target. (B–C) The reactivity of IgG4 (B) and IgG1 (C) from sera of patients with IAC/AIP was tested on immunoblot against the lysate of the H69 cholangiocyte cell line with/without stable knock-down of ANXA11 by ShRNA. This confirmed the identity of the target antigen. (D) Similarly, two patients with IgG4-RD of salivary glands showed reactivity against annexin A11 in H69 cells, but not in the knock-down cell line. Lanes were non-continuous and originate from multiple blots. IRD, IgG4-RD; PSC, primary sclerosing cholangitis.

Genetic knock-down of annexin A11 confirms identity of target antigen

The expression of Annexin A11 was stably knocked down in H69 cholangiocytes using short-hairpin RNA targeting ANXA11. Subsequently, positive sera of patients with IAC/AIP were tested for reactivity against the lysate of aforementioned knock-down cell line on immunoblot. As expected, the band at 56 kDa disappeared, confirming annexin A11 as target antigen of both IgG4 (figure 2B, see online supplementary figure 5A) and IgG1 (figure 2C, see online supplementary figure 5B). Also, the target antigen could be immunoprecipitated from H69 cell lysates using patient sera and blotted with an independent (commercial) antibody against annexin A11 (see online supplementary figure 6). Notably, sera of two patients with IgG4-RD of salivary glands among a total of 11 patients with IgG4-RD without biliary/pancreatic involvement also showed reactivity against annexin A11 (figure 2D), indicating that reactivity against the antigen is not restricted to biliary and pancreatic manifestations of IgG4-RD. Of note, no discernible differences in clinical characteristics could be found between patients with and without antibodies against annexin A11 (table 1).

IgG4 blocks binding of IgG1 to the antigen

In order to determine the epitope(s) of annexin A11 recognised by IgG4 and IgG1, we tested patient sera for reactivity against a peptide library consisting of 99 peptides (15-aa in length with 10-aa overlap) that cover the full range of the protein annexin A11 in an ELISA. We identified two epitopes on the N-terminal domain of the protein, which were shared between patients. Furthermore, both IgG4 and IgG1 antibodies recognised the same epitopes (figure 3A-D), suggesting that they may compete for binding to the antigen. Indeed, IgG4 antibodies were able to block the binding of IgG1 to the antigen in an ELISA (figure 3E). This finding may indicate that IgG4 antibodies are raised in an attempt to dampen an IgG1-mediated immune response through competitive binding.

Figure 3

Epitope mapping reveals two dominant epitopes and binding of  immunoglobulin G1 (IgG1) antibodies to annexin A11 is blocked by IgG4. (A–D) Reactivity of IgG4 (A/C) and IgG1 (B/D) antibodies was tested against different pools of annexin A11 peptides on ELISA. First of all, 99 peptides were pooled in four large pools (A/B). Subsequently, pool 1 was split in four smaller pools with six or seven peptides (C/D). Epitope analysis revealed two dominant epitopes located on peptides 7–12 and 19–25, which were shared among patients and were recognised by both IgG4 and IgG1 antibodies. Data would alternatively be in line with one discontinuous epitope spanning peptides 7–25 yet the quaternary structure of annexin A11 is unknown. (E) Reactivity of IgG1 of patient IRD4 and IRD2 against annexin A11 on ELISA was detected after preincubation of IgG4 of the same patient or control IgG4 of patient CA1 (n=3 per condition). Either 0 µg, 15 µg or 60 µg of IgG4 per well was used. IgG1 binding to the antigen is blocked by IgG4 of the same patient but not by control IgG4. *p<0.05, **p<0.001. IRD, IgG4-RD; CA, pancreatobiliary carcinoma.

Discussion

This study has identified annexin A11 as the first IgG4-target antigen in patients with IgG4-RD, while disease mimics such as PSC or cholangiocarcinoma did not show reactivity against annexin A11. Also, we showed that the epitopes on annexin A11 were shared between patients and that IgG4 blocked binding of IgG1 to the two identified epitopes of the antigen.

The identification of (auto)antigens in IgG4-RD has been addressed in previous studies. However, autoantibodies against suggested autoantigens, such as lactoferrin, carbonic anhydrase, pancreatic secretory trypsin inhibitor and trypsinogen isoforms have not been shown to be of the IgG4 subclass.26–29 Here, we found autoreactive IgG4 and IgG1 antibodies against annexin A11 in serum of 9 out of 50 patients with IAC/AIP. We possibly failed to detect annexin A11 autoantibodies in a larger percentage of the patients, due to the limited sensitivities of the Western blotting and ELISA techniques we used. Confirmation of our findings in independent cohorts is desirable. It could also be that different antigens, yet to be discovered, are relevant for the clonal expansion of IgG4+ B cells in different patients. Still, the by far most prominent 56 kDa band (figure 1) in cholangiocyte lysates exposed to sera of nine of our patients suggests a critical role of annexin A11 in IgG4-RD.

Annexins are a family of 12 calcium-dependent phospholipid-binding proteins. Annexin A11 contains 505 amino acids and migrates on SDS-PAGE at the level of 56 kDa. It shares four highly conserved repeats in the C-terminal domain with other members of the family,30 which are probably accountable for shared functions, including inhibition of phospholipase A2 and binding of anionic phospholipids. Annexin A11 is widely distributed in different tissues,31–33 including pancreatic β-cells where it may have a role in insulin secretion.34 Stainings of annexin A11 in human liver tissue performed by the Human Protein Atlas particularly shows strong expression of annexin A11 in cholangiocytes. In the pancreas tissue, annexin A11 is also highly expressed by duct cells and to a lesser extent in islets of Langerhans.32 33 The expression pattern of annexin A11 fits with the localisation of the inflammation in IAC and AIP, which are characterised by periductal inflammation.35

Why annexin A11 is targeted in IgG4-RD remains to be elucidated. Functional interference of IgG4 antibodies with the antigen, which has been described for instance in MuSK-myasthenia gravis and pemphigus vulgaris, seems unlikely due to the intracellular localisation of annexin A11. Possibly, autoantibodies against annexin A11 are raised when this protein that is otherwise not exposed to the immune system is released on cell injury. This may also explain why autoantibodies against annexin A11 have also been found in other autoimmune diseases, including primary antiphospholipid syndrome, lupus erythematosus and systemic sclerosis.36

We recently identified ‘blue collar work’ with long-term exposure to solvents, diesel or other oil products and industrial toxins as potential risk factors for the development of IgG4-RD of the bile ducts and pancreas.37 This observation might explain the gender distribution of IAC/AIP with a predominantly elderly male patient population.37 It could be speculated that long-term exposure to industrial toxins may trigger an inflammatory process in organs of genetically susceptible individuals which may lead to tissue injury and aberrant expression of intracellular proteins such as annexin A11 which then may contribute to maintaining a chronic autoimmune response.

The role of IgG4 in IgG4-RD is still unclear. We found that IgG4 antibodies blocked binding of IgG1 to the antigen, which could fit with a regulatory function of IgG4 as observed in different inflammatory conditions.16–18 Along the same line, a recent study showed that injection of IgG1 antibodies of patients with AIP in balb/c mice resulted in pancreatic and salivary gland injuries, but the pathogenic effects of IgG1 were inhibited when IgG4 was simultaneously injected.38

In summary, Annexin A11 is a novel autoantigen in IgG4-RD of the biliary tract, pancreas and salivary glands. Identification of this first IgG4-target antigen opens the door to further unravel the pathological mechanisms underlying IgG4-RD.

Acknowledgments

We would like to express our gratitude to Robert M van Es for excellent technical assistance and to the organisations that supported this research project: the German patient organisation ‘Deutsche Morbus Crohn/Colitis ulcerosa Vereinigung’ (DCCV, Sektion PSC), the American patient organisation ‘PSC partners seeking a cure’ and ‘Proteins@Work initiative’ of the Netherlands Proteomics Centre and Netherlands Organisation of Scientific research (NWO). SG is supported by the Netherlands Organisation of Scientific Research (Vidi grant 91713319).

References

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Footnotes

  • SFJG and UB contributed equally.

  • Contributors LH: designing research studies, conducting experiments, analysing data, writing the manuscript. HV: designing research studies, conducting experiments, analysing data, providing reagents, writing the manuscript. AS: conducting experiments, analysing data, writing the manuscript. RE: conducting experiments, analysing data, writing the manuscript. RO: designing research studies, analysing data, providing reagents, writing the manuscript. BB: designing research studies, analysing data, providing reagents, writing the manuscript. SG: designing research studies, analysing data, providing reagents, writing the manuscript. UB: designing research studies, analysing data, providing reagents, writing the manuscript.

  • Competing interests None declared.

  • Ethics approval Medical ethical committee of Academic Medical Center in Amsterdam (MEC 10/007).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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