Autoantibody Profiling of Chinese Patients with Autoimmune Hepatitis

Mar 21, 2008 - Beijing 100850, China, Central Laboratory, Beijing Youan Hospital, Capital University of Medical Sciences,. Beijing 100069, China, Nati...
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Autoantibody Profiling of Chinese Patients with Autoimmune Hepatitis Using Immunoproteomic Analysis Qing Xia,1,† Feng Lu,1,†,|,⊥ Hui-Ping Yan,1,‡ Hong-Xia Wang,§ Xia Feng,‡ Yan Zhao,‡ Bing-Yv Liu,§ Jie Wang,§ Ping Li,§ Yan Xue,§ Mei-Ru Hu,† Lu Qian,† Ning Guo,† Song-Cheng Yang,§ Ming-Yuan Li,| Yuan-Fang Ma,⊥ Bo-An Li,# Xue-Min Zhang,§ and Bei-Fen Shen*,† Department of Molecular Immunology, Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, China, Central Laboratory, Beijing Youan Hospital, Capital University of Medical Sciences, Beijing 100069, China, National Center of Biomedical Analysis, Taiping Road 27, Beijing 100850, China, Department of Cellular & Molecular Immunology, Medical School of Henan University, Kaifeng 475004, China, School of Preclinical and Forensic Medicine, West China Medical Center of Sichuan University, Chengdu 610041, China, and Center of Clinical Laboratory, 302 Hospital, Beijing 100039, China Received December 20, 2007

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P < 0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH. Keywords: autoimmune hepatitis • fumarate hydratase • autoantibody • autoantigen

Introduction Autoimmune hepatitis (AIH) is an unresolving liver inflammation with unknown cause. It responds well to immunosuppressive therapy but has a poor prognosis if untreated. Early and accurate diagnosis is therefore of great importance. However, there are few parameters that can be used to positively predict the presence of AIH. The diagnosis of AIH relies on the exclusion of viral, metabolic, genetic, and toxic etiologies of chronic hepatitis or hepatic injury. The detection * Address correspondence to this author at: Department of Molecular Immunology, Institute of Basic Medical Sciences 27 Taiping Road, Beijing 100850, China. Fax: 8610 68159436 . E-mail: [email protected]. † Institute of Basic Medical Sciences. ‡ Capital University of Medical Sciences. § National Center of Biomedical Analysis. | Medical School of Henan University. ⊥ West China Medical Center of Sichuan University. # 302 Hospital. 1 Lu Feng and Hui-Ping Yan made equal contributions to the work with Qing Xia. 10.1021/pr700861s CCC: $40.75

 2008 American Chemical Society

of autoantibodies is one of the critical components among the diagnostic criteria developed by the International Autoimmune Hepatitis Group.1,2 Over the past 5–10 years, progress has been achieved for the characterization of the autoantigens in AIH. One of the main methods widely employed is serological analysis of recombinant cDNA expression libraries.3 Using this genomic approach as well as other traditionally biochemical methods, multiple autoantigens in AIH have been identified, such as lamin, histone, cyclin A, U1RNP-A, U1RNP-70 kDa, and so on.4 With rapid development of large-scale protein separation and identification technologies, proteomic approaches have gained increasing popularity in screening and identifying autoantigens in autoimmune diseases.5 Compared with genomic approaches, the immunoproteomic analysis used in this study is less laborious and less serum sample consuming. Moreover, it allows experiments to be conducted with autoantibodies and target proteins maintained in natural states, thus improving the detection of antigenicity associated with aberrant postJournal of Proteome Research 2008, 7, 1963–1970 1963 Published on Web 03/21/2008

research articles translational modification of proteins. Indeed, the screening of autoantibodies by two-dimensional immunoblotting analysis using sera from patients with autoimmune diseases, followed by identification of the target proteins with mass spectrometry (MS), has led to the finding of a number of autoantigens, such as aldolase in diabetic retinopathy,6 Hsp70 and sperm outer dense fiber major protein 2 in autoimmune orchitis,7 and fibulin-4 in osteoarthritis.8 Similar attempts had been made to identify autoanitgens in AIH. Utilizing proteomic approach, Ballot et al. analyzed the molecular target of antisoluble liver antigen antibodies (anti-SLA).9 Heterogenous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) has been identified as an autoantigen in type 1 AIH.2 In both studies, rat liver was used as antigen substrate. However, the discrepancy of antigeneity between rodent and primate livers is obvious, and the human liver cell should be a better antigen substrate for the identification of autoantigens in AIH. In the present study, the HepG2 cell line, which was established from a patient with hepatoblastoma, was chosen to screen autoantigens in AIH, since it retains many characteristics of mature human hepatocytes, and was used to identify autoantigen in AIH,10 chronic hepatitis C (CHC),11 as well as other autoimmune diseases previously.12,13 By immunoscreening of a HepG2 cDNA library with an antiliver cytosol type 1-positive serum, Lapierre et al.10 identified formiminotransferase cyclodeaminase as a novel autoantigen in AIH. Using proteins extracted from HepG2 cells, Fukuda et al.11 identified four autoantibodies present in the sera of patients with CHC. By using an immunoproteomic approach, we systematically surveyed the autoantibody repertoire in AIH, identified 13 proteins immunoreactive to the sera of patients with AIH, and subsequently investigated the prevalence of a newly identified autoantibody (anti-FH) in AIH.

Materials and Methods Sera. Sixty-nine sera were collected from patients with AIH. The diagnosis of AIH was made according to the criteria defined by the International Autoimmune Hepatitis Group. Nineteen patients were studied at or close to diagnosis (within 2 months) and 50 during treatment with prednisolone (0.05–2 mg/kg/day), with or without azathioprine (1–2 mg/kg/day): 38 with normal and 12 with high levels of aspartate aminotransferase (AST). Fifty-nine patients were positive for the antinuclear antibody and/or the antismooth muscle antibody, belonging to AIH type 1. Ten patients were positive for the anti-SLA antibody, belonging to AIH type 3. 285 patients with various liver diseases and autoimmune diseases as well as 56 healthy subjects as controls were tested (Table 1). The study was approved by the local institutional ethics committee. Cells and Protein Preparation. The HepG2 cells (ATCC, USA) were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum. Cell lysate was prepared on ice using cool lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris) containing a protease inhibitor cocktail (Roche Diagnostic). The sample was aliquoted and stored at –70 °C until use. 2DE and WB. Proteins were separated by 2DE as described previously and transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham Pharmacia Biotech).14 Protein patterns in gels and PVDF membranes were visualized by Coomassie brilliant blue R250 (CBB R250) staining. The 2D gel images were captured using ImageScanner (Amersham Pharmacia Biotech). Spot detection, quantification, and alignment were performed with the ImageMaster 2D Elite 3.10 software 1964

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Xia et al. a

Table 1. Demographic Data of Patients and Controls study groups

number of patients

sex (F/M)

age range, y (median)

AIH PBC virus-induced liver disease CHB CHC other autoimmune disease SLE RA healthy controls

69 62 128 65 63 95 53 42 56

48/21 46/16 83/45 42/23 41/22 64/31 34/19 30/12 36/20

14–82 (54.3) 31–75 (53.5) 18–73 (51.8) 18–73 (53.0) 21–68 (50.6) 15–73 (43.4) 15–59 (36.1) 40–73 (52.7) 19–85 (51.6)

a AIH, autoimmune hepatitis; F, female; M, male; Y, year; CHB, chronic hepatitis B; CHC, chronic hepatitis C; PBC, primary biliary cirrhosis; RA, rheumatic arthritis; SLE, systemic lupus erythematosus.

(Nonlinear Dynamics, Ltd.). For hybridization with serum, unstained PVDF membranes were incubated with a blocking buffer consisting of Tris-buffered saline, 5% nonfat dry milk, and 0.1% Tween 20 for 2 h, and then with either the sera from patients or with normal control serum at a 1:500–20 000 dilution for 1 h at room temperature. The membranes were incubated with horseradish peroxidase-conjugated antihuman IgG at a 1:10 000 dilution for 30 min at room temperature, washed, and briefly incubated in Enhanced Chemiluminescence (Amersham Pharmacia Biotech). Protein Identification. 2DE gels and PVDF membranes were stained with silver or CBB R250 staining. The protein spots were excised from the 2D gels and in-gel digested as described previously.14 Protein identification was repeated at least twice using spots from different gels. Peptide mass fingerprintings (PMFs) were acquired using a MALDI-TOF REFLEX III instrument (Bruker Daltonics, Bremen, Germany) and used to search through the SWISS-PROT 50.4 and NCBInr 20060731 by the Mascot search engine (http:// www.matrixscience.co.uk). Electrospray ionization MS/MS (ESI-MS/MS) experiments were performed on a Q-TOF2 hybrid quadrupole/TOF MS (Micromass, UK) with a nanoflow Z-spray source. The database search was finished with the Mascot search engine (http:// www.matrixscience.co.uk) using the data processed through MaxEnt3 and MasSeq. FH cDNA Cloning, Expression, and Purification. FH cDNA was generated by RT-PCR from HepG2 cells using the primers 5′-ACTCGGATCCATGGCGACCACGGC-3′and5′-TATCTGCAGCTTCAGATA AGATCAGC-3′. His-tagged FH protein was produced from the pET22b(+)-FH transformed Escherichia coli strain BL21 and then affinity purified from inclusion bodies by detergent extraction and Ni2+-nitrilotriacetic acid affinity chromatography. ELISA. ELISA was performed with a previously published protocol.15 Briefly, the purified recombinant FH (4 µg/well) was coated onto 96-well microtiter plates. Sera were diluted in blocking buffer (1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400). All samples were applied in triplicate. Reaction without the first antibody served as a background control. After washing, plates were incubated with the secondary antibody and quantified using an ELISA plate reader (Anthos labtec, Austria) at 490 nm. Examination of 56 normal control serum samples established the normal range of the assay. The cutoff value of positive ELISA results was defined as an OD exceeding twice the mean of 56 normal controls. The cutoff value was calculated for all dilutions. Statistical Analysis. Differences of prevalences of the antiFH antibodies among the groups of PBC, RA, SLE, CHB, CHC,

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Figure 1. Screening of autoantibodies in AIH. (A) CBB R250 staining of HepG2 cell-derived proteins separated by 2DE. Protein spots (A-S) were recognized by sera from patients with AIH. The gel size was 8 × 7 × 0.1 cm3. After being transferred onto PVDF membranes, proteins were individually reacted with serum samples from 21 patients with AIH and 15 healthy donors diluted at 1/500. (B) Representative 2D-immunoblotting patterns of HepG2 cell proteins reacted with the serum sample from AIH patients (A-C) and from a healthy control (D). (C) Close up section is from the corresponding serum diluted at 1/20 000, and the gradient pH of the immobiline drystrip is pH 4-7 with a size of 20 × 20 × 0.1 cm3.

and normal control (NC) were compared by using the χ2 test. The P values 0.05). Finally, we investigated several parameters in anti-FHpositive and -negative patients with AIH. As for age and gender, anti-FH positive AIH had a preferential age interval of 40–70 years and female predominance (χ2 ) 16.318, P < 0.005). However, with regard to some laboratory parameters, such as alanine aminotransferase (ALT), AST, alkaline phosphatase (ALP), γ-glutamyl transferase (γGT), IgG, IgM, and IgA concentrations, there is no difference with statistical significance observed.

Disccusion In the present study, using immunoproteomic technology, we identified 14 autoantigens in AIH. Among them, five proteins were previously reported to be targets of autoantibodies in AIH, including R-Actin, β-Actin, γ-Actin, R-enolase 1, and hnRNP A2/B1. Notably, eight proteins, including chaperonin containing TCP1, PDI precursor, FH, IMPDH2, phosphoglycerate mutase isozyme B, D-3-phosphoglycerate dehydrogenase, HAD2, and neuropolypeptide h3, have not been described in AIH. Actins are highly conserved proteins that are involved in various types of cell motilities. Polymerization of globular Actin (G-Actin) leads to a structural filament (F-Actin) in the form of a two-stranded helix. F-Actin is a well-known autoantigen in AIH.16,17 A new F-Actin ELISA has been considered as a 1966

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4.57 8.85 9.54 9.54 7.01 7.01 7.01 7.01 6.39 6.67

(19.04) (14.29) (14.29) (14.29)

1/15 1/15 2/15 0/15

(6.67) (6.67) (13.33) (0.00)

(0.00) (0.00) (0.00) (0.00) (26.67) (20.00) (20.00) (20.00) (0.00) (0.00)

useful diagnostic tool with similar specificity and superior sensitivity compared with the current detection of antismooth muscle antibodies by indirect immunofluorescence.16 The binding domain on R-Actin was previously considered as a predominant epitope.18 In our study, all three Actin isoforms, R, β, and γ, were detected in AIH. TCP1, one member of the chaperonin proteins, is essential in the folding of Actin, tubulin, and other cytosolic proteins for forming stable and functionally competent protein conformations.19 TCP1 has a post-translational role in maintaining the native form of Actin and ensuring its correct assembly into microfilaments.20 It is identified as a novel autoantigen of AIH in this study, suggesting that antiactin and anti-TCP1 autoantibodies may be linked to the pathogenesis of the autoimmune process in AIH. Enolase, a ubiquitous glycolytic enzyme, is highly conserved through evolution. It exists as three highly homologous isozymes: R, β, and γ. Ballot et al. identified four isoforms of R-enolase by 1D and 2D immunoblotting analyses using anti-SLA-positive sera against rat liver cytosolic fraction.9 However, anti-Renolase autoantibody has been detected in the sera from various autoimmune diseases, such as SLE, RA, and Hashimoto’s encephalopathy.21 In the present study, the R-enolase was found to be positive to the sera from both AIH patients and normal individuals, suggesting that it might only be one component of targets of the autoantibodies in AIH, but lacks the disease specificity. Interestingly, the patterns of anti-Renolase antibody in sera from AIH patients and normal individuals were different. In our study, the protein spots of healthy volunteers predominantly existed at the highest pI, which was consistent with the data reported by Li. et al.,22 but the spots of AIH were within a lower pI range (Figure 1B). HnRNPs are RNA binding proteins and are involved in the maturation of mRNA precursors and transportation of mRNA to the cytosolic compartment. Protein hnRNP A2/B1 has been described as autoantigens targeted by autoantibodies

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Figure 2. Identification of the spot G recognized by the sera from patients with AIH by MS. (A) Identification of FH by MALDI-TOF MS after trypsin digestion of the spot G. * Indicates the modification of oxidation at the methionine residue; # indicates the modification of carbamidomethyl at the cysteine residue. The protein score is significant (P < 0.05). (B) The peptide at m/z 1490.71 from the PMF for spot G was sequenced by nano-ESI-MS/MS, and the deduced sequence is indicated.

in various autoimmune diseases, such as RA or SLE as well as AIH type 1.23,2 Some other well-characterized autoantigens in AIH, such as asialoglycoprotein receptor,24 formiminotransferase cyclodeaminase,25 cytochrome P450,26 and UGA repressor tRNA-associated protein,27 are absent in our list of autoantigens. The reason may be partly explained by the fact that these antigens are present in trace amounts in HepG2 cells. Some of the missing autoantigens may also arise from technical limitations of the current methods for sample preparation and 2DE, including the loss of acidic and basic proteins that fall outside of the pI

range of the isoelectric focusing step and, importantly, low protein loading levels caused by conductivity effects during the isoelectric focusing, leading to poor coverage of low abundance proteins.28 Among the newly identified autoantigens of AIH, PDI is referred to as an endoplasmic reticulum (ER) stress protein. It regulates processing and folding of membrane and secretary proteins in the ER.29,30 Two critical enzyme families, cytochrome P450 monooxygenases and uridine diphosphate-glucuronosyltransferases harbored in ER, have been identified as the targets of B cell response in autoimmune liver disease.1 Journal of Proteome Research • Vol. 7, No. 5, 2008 1967

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Figure 3. Validation of the antigenicity of FH. (A) 2D immunoblotting analysis with the sera from one AIH patient. The numbers correspond to the proteins identified by MS in Figure 1 and Table 1. Protein spots G, H, and I are identified as FH by MS analysis. (B) 2D immunoblotting analysis of FH with polyclonal anti-FH antibody. (C, D) Enlarged regions of interest. (E) The purified recombinant FH was separated by 12% SDS-PAGE and stained with CBB R250 (lane 1, molecular mass marker; lane 2, purified FH) or transferred on membrane and blotted with serum samples from three patients with AIH that reacted positively to spots G, F, and H in previous 2D immunoblotting analysis (lanes 4-6) as well as the serum samples from three healthy controls (lanes 7-9). Lane 3 represents positive control (polyclonal goat-anti-FH serum).

Figure 4. Level of anti-FH autoantibody in the serum samples determined by ELISA using the recombinant FH as a coating antigen. Sera were diluted at 1:400. All data are expressed as the mean ( the standard error of the mean (s.e.m.). Results were statistically analyzed using an χ2 test.

The finding of anti-PDI antibody reactivity in AIH adds a novel autoantibody to the list of autoantibodies against the antigens of ER in AIH. Previous reports showed that an anti-PDI autoantibody was detected with high frequency in rats treated with various hepatotoxic drugs and in sera from patients with alcoholic liver disease, liver cirrhosis, SLE, and hepatoma.31,32 1968

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Together with our data, it implies that the occurrence of antiPDI antibody may play an immunological role in the progression of hepatic disorders. IMPDH is an established target in immunosuppression following organ transplantation. Mizoribine (MZR), an inhibitor of IMPDH, has been shown to possess immunosuppressive

research articles

Autoantibody Profiling of Chinese Patients with AIH activity in patients who had renal transplantation and patients with RA, lupus nephritis, and primary nephritic syndrome.33 Phosphoglycerate mutase catalyzes the isomerization of 2- and 3-phosphoglycerates and is essential for glucose metabolism in most organisms. D-3-Phosphoglycerate dehydrogenase is a necessary enzyme for de novo L-serine biosynthesis via the phosphorylated pathway.34 The HAD2 protein is involved in the penultimate step of mitochondrial fatty acid oxidation. The biological functions of polypeptide h3 are rarely reported. The above five proteins were not reported to be the targets of autoantibodies in patients with autoimmune diseases. Further analysis with a larger number of samples is needed to elucidate their association with clinical factors. In 2D immunoblotting analysis, 3 of 21 patients with AIH were anti-FH positive, while none of the normal controls were. FH is distributed in both cytosol and the mitochondrial matrix compartment. The mitochondrial enzyme catalyzes the freely reversible hydration of fumarate into malate in the Krebs cycle, whereas the physiological role of the cytosolic isoform is yet unknown.35 Previous studies have led to the realization that some of the major autoantigens in AIH are active enzymes of human hepatic and extrahepatic microsomal xenobiotic metabolism. Therefore, we selected FH as a potential autoantigen and analyzed the occurrences of anti-FH reactivity in AIH and other diseases. The data demonstrate a higher antibody level in AIH than in healthy controls and other disease controls (Figure 4). Notably, the differences of prevalence between AIH and CHC as well as CHB are significant (P < 0.05). The difference between virus-associated autoimmunity and genuine AIH is an important clinical and immunologic issue. Numerous autoantibodies are also associated with CHC (1). An antithyroid antibody was found in CHC with a high prevalence, and an anti-GOR antibody was present in at least 80% of CHC sera.36 Our results suggest that anti-FH might be a possible serological marker to distinguish AIH from viral hepatitis. However, the analysis with a larger number of samples is still needed. In addition, the anti-FH level in AIH is also significantly higher than in PBC. Overlap syndromes between different autoimmune liver diseases are frequent, which not only poses a diagnostic problem but also leads to confusion regarding the appropriate treatment strategy. About 5% of patients with a primary diagnosis of AIH have the signs and symptoms of PBC. On the other hand, 19% of patients with a primary diagnosis of PBC also have signs of AIH. In this report, the occurrence of autoantibody to FH in PBC is 19.35%, which is significantly lower than that in AIH (P < 0.001). Whether anti-FH-positive PBC patients belong to the group of overlap of PBC and AIH still needs more data to elucidate. In summary, our data further demonstrate the serological heterogeneity in AIH and suggest the diversity of mechanisms underlying AIH. Identification of these autoantigens in AIH may permit not only characterizations of the autoimmune responses but also explorations of their pathogenic relevance and the development of more specific diagnostic tests. Further investigations of anti-FH autoantibodies may determine its value for diagnostic purposes and gain insight into the immunologic mechanisms in AIH. Abbreviations Used: 2DE, two-dimensional electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization mass spectrometry; anti-SLA, antisoluble liver antigen; PVDF, polyvinylidene fluoride; CHB, chronic hepatitis B; CHC, chronic hepatitis C; CBB R250, Coomassie brilliant blue R250; AST, aspartate aminotransferase; PMF, peptide mass fingerprinting;

ESI-MS/MS, electrospray ionization MS/MS; hnRNP-A2/B1, heterogeneous nuclear ribonucleoprotein A2/B1; PDI, protein disulfide isomerase; FH, fumarate hydratase; IMPDH2, inosine monophosphate dehydrogenase 2; HAD2, 3-hydroxyacyl-CoA dehydrogenase type 2; PBC, primary biliary cirrhosis; RA, rheumatic arthritis; SLE, systemic lupus erythematosus; WB, Western blot.

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