Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis
2012; Elsevier BV; Volume: 11; Issue: 12 Linguagem: Inglês
10.1074/mcp.m112.018713
ISSN1535-9484
AutoresChiara Zingaretti, Milena Arigò, Angela Cardaci, Monica Moro, Mariacristina Crosti, Antonella Sinisi, Elisa Sugliano, Cristina Cheroni, Francesco Marabita, Renzo Nogarotto, Raoul J. P. Bonnal, Paolo Marcatili, Maurizio Marconi, Anna-Linda Zignego, Paolo Muratori, Pietro Invernizzi, P. Colombatto, Maurizia Brunetto, Ferruccio Bonino, Raffaele De Francesco, Jens Geginat, Massimiliano Pagani, Luigi Muratori, Sergio Abrignani, Mauro Bombaci,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoAutoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH. Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH. Autoantibodies specific for proteins or nonprotein antigens (dsDNA, snRNP, carbohydrates) are often the serological hallmark of autoimmune diseases. Autoantibodies can be simply an epiphenomenon secondary to a chronic inflammatory milieu (1Avrameas S. Ternynck T. Tsonis I.A. Lymberi P. Naturally occurring B-cell autoreactivity: a critical overview.J. Autoimmun. 2007; 29: 213-218Crossref PubMed Scopus (93) Google Scholar), but they can also play a direct pathogenetic role, as antithyroglobulin antibodies do in Hashimoto's thyroiditis (2Pearce E.N. Farwell A.P. Braverman L.E. Thyroiditis.N. Engl. J. Med. 2003; 348: 2646-2655Crossref PubMed Scopus (708) Google Scholar). Autoimmune hepatitis (AIH) 1The abbreviations used are:AIHautoimmune hepatitisAUCarea under the curveDELFIADissociation-enhanced Lanthanide Fluoroscence ImmunoAssayFNIIIfibronectin type III domainHBVhepatitis B virusHCVhepatitis C virusHDhealthy donorIL4Rinterleukin-4 receptorIMACimmobilized metal ion affinity chromatographyMFImean fluorescence intensityPAMpredictive analysis of microarrayROCReceiver Operating Characteristic. 1The abbreviations used are:AIHautoimmune hepatitisAUCarea under the curveDELFIADissociation-enhanced Lanthanide Fluoroscence ImmunoAssayFNIIIfibronectin type III domainHBVhepatitis B virusHCVhepatitis C virusHDhealthy donorIL4Rinterleukin-4 receptorIMACimmobilized metal ion affinity chromatographyMFImean fluorescence intensityPAMpredictive analysis of microarrayROCReceiver Operating Characteristic. is a chronic necro-inflammatory disease of unknown etiology that affects predominantly women with an incidence of 1 to 2 per 100,000 per year and a prevalence of 10 to 20 out of 100,000 (3Czaja A.J. Manns M.P. Advances in the diagnosis, pathogenesis, and management of autoimmune hepatitis.Gastroenterology. 2010; 139: 58-72Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 4Makol A. Watt K.D. Chowdhary V.R. Autoimmune hepatitis: a review of current diagnosis and treatment.Hepat. Res. Treat. 2011; 2011: 390916PubMed Google Scholar). AIH is subdivided into two major types on the basis of autoantibody reactivity (5Invernizzi P. Lleo A. Podda M. Interpreting serological tests in diagnosing autoimmune liver diseases.Semin. Liver Dis. 2007; 27: 161-172Crossref PubMed Scopus (92) Google Scholar). Antibodies to nuclei and/or to smooth muscle characterize type 1 AIH, whereas antibodies to a liver-kidney microsomal constituent define patients with type 2 AIH. Because the detection of these autoantibodies is done by means of immunofluorescence on rodent multi-organ sections (liver, kidney, stomach), there are problems with the standardization and interpretation of the immunostaining patterns (6Bogdanos D.P. Invernizzi P. Mackay I.R. Vergani D. Autoimmune liver serology: current diagnostic and clinical challenges.World J. Gastroenterol. 2008; 14: 3374-3387Crossref PubMed Scopus (163) Google Scholar). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an international committee to define guidelines and develop procedures and reference standards for more reliable testing (7Vergani D. Alvarez F. Bianchi F.B. Cancado E.L. Mackay I.R. Manns M.P. Nishioka M. Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.J. Hepatol. 2004; 41: 677-683Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 8Hennes E.M. Zeniya M. Czaja A.J. Pares A. Dalekos G.N. Krawitt E.L. Bittencourt P.L. Porta G. Boberg K.M. Hofer H. Bianchi F.B. Shibata M. Schramm C. Eisenmann de Torres B. Galle P.R. McFarlane I. Dienes H.P. Lohse A.W. Simplified criteria for the diagnosis of autoimmune hepatitis.Hepatology. 2008; 48: 169-176Crossref PubMed Scopus (1284) Google Scholar). Although ELISA and bead assays with purified or recombinant autoantigens are under development (9Oertelt S. Rieger R. Selmi C. Invernizzi P. Ansari A.A. Coppel R.L. Podda M. Leung P.S. Gershwin M.E. A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA-negative primary biliary cirrhosis.Hepatology. 2007; 45: 659-665Crossref PubMed Scopus (136) Google Scholar), they actually represent a complementary, rather than alternative, approach to traditional immunofluorescence. Moreover, serological overlap is frequently observed between AIH and other non-autoimmune liver diseases such as chronic viral hepatitis (10Zachou K. Rigopoulou E. Dalekos G.N. Autoantibodies and autoantigens in autoimmune hepatitis: important tools in clinical practice and to study pathogenesis of the disease.J. Autoimmune Dis. 2004; 1: 2Crossref PubMed Scopus (121) Google Scholar). Therefore, new, highly specific markers represent an unmet medical need for the more accurate diagnosis and classification of AIH. autoimmune hepatitis area under the curve Dissociation-enhanced Lanthanide Fluoroscence ImmunoAssay fibronectin type III domain hepatitis B virus hepatitis C virus healthy donor interleukin-4 receptor immobilized metal ion affinity chromatography mean fluorescence intensity predictive analysis of microarray Receiver Operating Characteristic. autoimmune hepatitis area under the curve Dissociation-enhanced Lanthanide Fluoroscence ImmunoAssay fibronectin type III domain hepatitis B virus hepatitis C virus healthy donor interleukin-4 receptor immobilized metal ion affinity chromatography mean fluorescence intensity predictive analysis of microarray Receiver Operating Characteristic. Besides the potential diagnostic application, the discovery of novel AIH autoantigens could provide insights into the disease pathogenicity mechanism. Although some AIH target-autoantigens have been identified and characterized, little is known about their pathogenetic role, and other autoantigens are probably still unknown. Autoantibodies, to be considered pathogenetic, must have at least two features: (i) the target-autoantigen should be either expressed on the plasma membrane of target cells or secreted by cells (i.e. should be exposed to autoantibodies), and (ii) binding of the autoantibodies to the target antigen should disturb a cellular function directly or indirectly. A possible pathogenetic role in AIH has been put forward for autoantibodies specific for cytochrome P450 2D6 (CYP2D6) or Asialoglycoprotein receptor 1 (AGPR-1), which are both present on the hepatocyte cell membrane (10Zachou K. Rigopoulou E. Dalekos G.N. Autoantibodies and autoantigens in autoimmune hepatitis: important tools in clinical practice and to study pathogenesis of the disease.J. Autoimmune Dis. 2004; 1: 2Crossref PubMed Scopus (121) Google Scholar). Protein microarrays are a powerful technology, as they allow the simultaneous screening of thousands of analytes (11Song Q. Liu G. Hu S. Zhang Y. Tao Y. Han Y. Zeng H. Huang W. Li F. Chen P. Zhu J. Hu C. Zhang S. Li Y. Zhu H. Wu L. Novel autoimmune hepatitis-specific autoantigens identified using protein microarray technology.J. Proteome Res. 2010; 9: 30-39Crossref PubMed Scopus (62) Google Scholar). In the present study, to identify new autoantigens with potential diagnostic and/or pathogenetic roles in AIH, we printed a microarray with 1626 human proteins whose main feature was being either secreted or membrane associated (i.e. potentially exposed to autoantibody recognition). We used this microarray to screen panels of sera from patients with AIH and identified six new protein antigens that are recognized with high sensitivity and specificity. One of these six autoantigens is the interleukin-4 (IL4) receptor fibronectin type III (FNIII) domain of the IL4 receptor (CD124), and, interestingly, patients' autoantibodies specific for CD124 neutralize IL4 signaling, suggesting a possible pathogenetic role for IL4 neutralization in AIH. Samples used for this study were collected in five different hospitals according to standard operating procedures: (i) IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; (ii) Hepatology Unit, University Hospital, Pisa, Italy; (iii) Sant'Orsola-Malpighi University Hospital, Bologna, Italy; (iv) Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano, Italy; and (v) Center for Systemic Manifestations of Hepatitis Viruses, University of Firenze, Italy. For the discovery phase, 203 sera were used (15 AIH, 78 healthy donor (HD), 110 hepatitis C virus (HCV)), whereas for the validation phase 174 sera were used (50 AIH, 50 HD, 50 HCV, 24 hepatitis B virus (HBV)). The Institutional Review Boards of these hospitals authorized the use of sera for research purposes. Genes whose translated products carry a secretion signal peptide or at least one transmembrane domain were selected, cloned, and expressed in a high throughput system as histidine-tagged products as described elsewhere (12Grifantini R. Pagani M. Pierleoni A. Grandi A. Parri M. Campagnoli S. Pileri P. Cattaneo D. Canidio E. Pontillo A. De Camilli E. Bresciani A. Marinoni F. Pedrazzoli E. Nogarotto R. Abrignani S. Viale G. Sarmientos P. Grandi G. A novel polyclonal antibody library for expression profiling of poorly characterized, membrane and secreted human proteins.J. Proteomics. 2011; 75: 532-547Crossref PubMed Scopus (12) Google Scholar). A total of 1626 polypeptides were cloned and expressed in E. coli. Of these, 1121 were cloned as protein fragments and 505 as full-length proteins (Supplemental Tables S1 and S2). The recombinant proteins were affinity-purified from the bacterial insoluble fraction by means of immobilized metal ion affinity chromatography (IMAC) (GE). Representative SDS-PAGE gels of a panel of purified proteins are shown in Supplemental Fig. S1. Human, viral, or bacterial proteins were used as biological or technical controls in the microarrays (Supplemental Fig. S2). HCV core protein and nonstructural proteins NS3 (from HCV genotype 1), NS3–4a (from HCV genotype 2), and NS5b (from HCV genotype 1); Tetanus toxin; and H1N1 antigen were produced in house by subcloning the corresponding genes in E. coli strain DH5α and expressing them in BL21(DE3). Bovine serum albumin (BSA), human serum albumin, human glutathione-S-transferase, and protein A from Staphylococcus aureus were purchased from Sigma. For DELFIA® experiments, plasmids encoding CYP2D6 and AGPR-1 were purchased from Invitrogen (Ultimate™ Human ORF Clones), subcloned in E. coli strain DH5α, and expressed in BL21(DE3). All the corresponding proteins were purified via affinity chromatography on IMAC resin. Purified recombinant proteins obtained as described above were analyzed via SDS-PAGE (Criterion PAGE system Bio-Rad) followed by Coomassie Blue staining of the gels to assess their integrity and purity (Supplemental Fig. S1). Protein purity was assessed via BioRad ChemiDocTM XRS, with Quantity One® software. Proteins showing purity levels > 70% were used for protein array preparation. To further analyze the quality of the purified proteins, we performed Western blot analysis on the purified proteins with an anti-His monoclonal antibody (mAb). The proteins were resolved on 4% to 12% precast SDS-PAGE gradient Tricine gels under reducing conditions and electroblotted onto nitrocellulose membranes (Bio-Rad) according to the manufacturer's instructions. The membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween 20 (TPBS) for 1 h at room temperature, incubated with α-His mAb (GE Healthcare) diluted 1:1000 in 3% nonfat milk in TPBS for 1 h at room temperature, and washed three times in TPBS. The secondary HRP-conjugated antibody (α-mouse immunoglobulin/HRP) (GE-Healthcare) was diluted 1:1000 in 3% nonfat milk in TPBS and incubated for 1 h at room temperature. The proteins were visualized by means of enhanced chemiluminescence (Super Signal West Pico Chemiluminescence Substrate) (Thermo Scientific) and detected with LAS-3000 (Fujifilm, Wayne, NJ). Protein microarrays were generated by spotting the 1626 affinity-purified recombinant proteins (0.5 mg/ml in 6 m urea) in four replicates on nitrocellulose-coated slides (FAST slides) (GE Healthcare) using Stealth SMP3 spotting pins (TeleChem International, Sunnyvale, CA) and a Microgrid II microarray contact printer (Biorobotics, Veldzigt, The Nederlands), resulting in spots with a diameter of ∼130 μm. As an experimental positive control, a curve of human IgG at 11 different concentrations (from 0.001 to 1 mg/ml) was spotted on the arrays in eight replicates (in 6 m urea) (Supplemental Fig. S3A). Several spots of buffer alone were also printed and used to assess possible nonspecific signals due to cross contamination. A quality control of the spotting procedure was performed on 10% of randomly chosen slides. The percentage of proteins successfully spotted on the slides was assessed by hybridizing the arrays with an α-His mAb followed by an Alexa-647 conjugated α-human IgG secondary antibody and estimating the number of spots with a mean fluorescence intensity (MFI) value significantly above background. A distance matrix was calculated using TIGR Multiexperiment Viewer (version MeV4.7) software (13Saeed A.I. Bhagabati N.K. Braisted J.C. Liang W. Sharov V. Howe E.A. Li J. Thiagarajan M. White J.A. Quackenbush J. TM4 microarray software suite.Methods Enzymol. 2006; 411: 134-193Crossref PubMed Scopus (1421) Google Scholar) to evaluate the system reproducibility (Supplemental Fig. S3B). The spotted microarrays were allowed to remain at room temperature for 1 h before being stored at 4 °C until use. Incubation was automatically performed with a TECAN hybridization station (HS 4800™ Pro) (TECAN, Salzburg, Austria). The microarray slides were prewashed for 3 min in TPBS and saturated with BlockIt™ microarray blocking buffer (Arrayit Corporation, Sunnyvale, CA) for 45 min at 25 °C under mild agitation. After the injection of 105 μl of diluted human serum (1:300 in blocking buffer with 0.1% Tween 20), microarrays were incubated at 25 °C for 45 min with gentle agitation. The microarrays were then washed in TPBS at 25 °C for three cycles of 1 min (wash time) and 30 s (soak time). After that, microarray slides were incubated for 1 h at 25 °C with Alexa-647-conjugated α-human IgG (Invitrogen) (1:800 in blocking buffer) in the dark. The microarrays were then washed at 25 °C two times in TPBS (1 min wash time, 30 s soak time), two times in PBS (1 min wash time, 30 s soak time), and finally one time in milliQ sterile water (15 s). The slides were finally dried at 30 °C under nitrogen for 2 min and scanned using a ScanArray Gx PLUS (PerkinElmer, Shelton, CT). 16-bit images were generated with ScanArrayTM software at a resolution of 10 μm per pixel and analyzed using ImaGene 8.0 software (Biodiscovery Inc., Hawthorne, CA). A 635 nm laser was used to excite the Alexa-647 dye. The fluorescence intensity of each spot was measured, signal-to-local-background ratios were calculated using ImaGene, and spot morphology and deviation from the expected spot position were considered using the default ImaGene settings. For each sample, the background subtracted MFI of replicated spots was determined and subsequently normalized on the basis of the human IgG curve to allow the comparison of data from different experiments (14Bombaci M. Grifantini R. Mora M. Reguzzi V. Petracca R. Meoni E. Balloni S. Zingaretti C. Falugi F. Manetti A.G. Margarit I. Musser J.M. Cardona F. Orefici G. Grandi G. Bensi G. Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.PLoS One. 2009; 4: e6332Crossref PubMed Scopus (51) Google Scholar). Briefly, the MFI values of IgG, spotted at different concentrations (Supplemental Fig. S3A), were fitted by a sigmoid curve, using a maximum likelihood estimator (38Harris J.W. Stocker H. Handbook of Mathematics and Computational Science. Springer-Verlag, New York1998Crossref Google Scholar). The experimental average IgG curve of each slide was adjusted on the reference sigmoid IgG curve, and the background-subtracted MFI values of each protein were normalized accordingly. On the basis of these results, a normalized MFI value of 4.000 (value corresponds to the normalized MFI value of negative controls (BSA, HSA, Hu-GST) plus 2 standard deviations) was chosen as the lowest signal threshold for scoring a protein as positively recognized by human sera. For each protein, a coefficient of variation (CV%) was calculated on four replicate spots for intra-assay reproducibility (14Bombaci M. Grifantini R. Mora M. Reguzzi V. Petracca R. Meoni E. Balloni S. Zingaretti C. Falugi F. Manetti A.G. Margarit I. Musser J.M. Cardona F. Orefici G. Grandi G. Bensi G. Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.PLoS One. 2009; 4: e6332Crossref PubMed Scopus (51) Google Scholar). Each antigen was checked for displaying a CV% correlated to its MFI on the basis of standard IgG curves. If the CV% value was not within the expected range, the antigen was not considered for further analysis. Recognition frequency was defined as the percentage of sera reacting with a particular antigen in protein array with an MFI ≥ 4.000, and it was calculated for each group of sera. TIGR Multiexperiment Viewer (version MeV4.7) software (13Saeed A.I. Bhagabati N.K. Braisted J.C. Liang W. Sharov V. Howe E.A. Li J. Thiagarajan M. White J.A. Quackenbush J. TM4 microarray software suite.Methods Enzymol. 2006; 411: 134-193Crossref PubMed Scopus (1421) Google Scholar) was used to perform an unsupervised bidimensional hierarchical clustering. The identity of selected candidate autoantigens was further confirmed by means of tandem mass spectrometry (MS/MS). Protein spots were excised from the gels, destained with 50 mm ammonium bicarbonate (Fluka Chemie AG, Buchs, Switzerland) in 50% acetonitrile (Mallinckrodt Baker), dehydrated once with pure acetonitrile, and air-dried. Dried spots were digested for 2 h at 37 °C in 12 μl of 0.012 μg/μl sequencing grade modified trypsin (Promega, Madison, WI) in 5 mm ammonium bicarbonate. After digestion, 0.6 μl were loaded on a matrix PAC target (Prespotted AnchorChip 96, set for proteomics) (Bruker Daltonics, Bremen, Germany) and air-dried. Spots were washed with 0.6 μl of a solution of 70% ethanol and 0.1% trifluoroacetic acid. Analysis was performed using an Ultraflex III matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics). Peptides were selected in the mass range of 900–3500 Da. Also, MS/MS spectra were acquired and externally calibrated by using a combination of standards pre-spotted on the target (Bruker Daltonics). MS and MS/MS spectra were analyzed with FlexAnalysis (version 3.0) (Bruker Daltonics). Search parameters were as follows: variable modifications, carbamyl (N-term), oxidation (Met); cleavage by trypsin (cuts C-terminal side of Lys and Arg unless the next residue is Pro); mass tolerance, 300 ppm; missed cleavage, 0; mass values, MH+ monoisotopic. Monoisotopic peaks were annotated with FlexAnalysis default parameters and manually revised. The peptide sequence was determined with Mascot software run on a public database (NCBI nr Homo sapiens, release 20100616, or SwissProt Homo sapiens, release 2010_07, # 536789 sequence entries). The accession number, annotation, Mowse score, percentage of protein coverage, number of unique peptides matched, and number of masses not matched are reported in the Supplemental data. The Dissociation-enhanced Lanthanide Fluoroscence ImmunoAssay (DELFIA)® is a time-resolved fluorescence method that can be used to study antibody binding to solid-phase proteins or peptides. The purified recombinant proteins were used at a concentration of 20 μg/ml (15Frulloni L. Lunardi C. Simone R. Dolcino M. Scattolini C. Falconi M. Benini L. Vantini I. Corrocher R. Puccetti A. Identification of a novel antibody associated with autoimmune pancreatitis.N. Engl. J. Med. 2009; 361: 2135-2142Crossref PubMed Scopus (299) Google Scholar) in 6 m urea to coat DELFIA® plates (PerkinElmer). Plates were then blocked for 1 h at 37 °C with a blocking reagent (PerkinElmer). The serum samples, diluted 1:300 in PBS with 1% BSA (Sigma) and 0.1% Tween 20 (Sigma), were incubated on the plates for 1 h at 37 °C. Plates were then washed five times with washing buffer (PerkinElmer) and incubated for 30 min at room temperature in the dark with europium-labeled α-human IgG serum (1:500 in diluting buffer) (PerkinElmer). After extensive washing, plates were left at room temperature for 10 min and then read on an Infinite F200 PRO instrument (Tecan). Fluorescence intensity values higher than the mean of buffer plus 3 standard deviations were considered as positive. To assess the recognition of native IL4 receptor (IL4R) by human sera, full length IL4R was overexpressed in HeLa cells. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, and 1% penicillin-streptomycin. The human cDNA clone of full length IL4R, transcript variant 1, was purchased from OriGene Technologies (Rockville, MD). HeLa cells were transfected with Lipofectamine 2000 Transfection reagent (Invitrogen) according to the manufacturer's instructions. Briefly, cells were seeded at 5 × 105 cells/well in 24-well dishes and left overnight in medium without penicillin-streptomycin. 1 μg of plasmid DNA and 2 μl of Lipofectamine 2000 reagent were diluted in 100 μl of Opti-MEM (Invitrogen) and pre-incubated for 20 min to allow DNA-Lipofectamine complexes to form; cells were then incubated with Opti-MEM (Invitrogen) containing DNA-Lipofectamine complexes at 37 °C for 16 h. After that, Opti-MEM was replaced with fresh DMEM and, at 24 h post-transfection, cells were assayed for IL4R expression. For surface staining, cells were non-enzymatically detached, harvested, and washed in PBS. To assess IL4R expression on the cell surface, 1 × 105 cells were incubated with PE-conjugated anti-CD124 (Beckman-Coulter, Krefeld, Germany) and with matched mouse Isotype as a control (1:10 in PBS 1% BSA) for 30 min at 37 °C. Non-transfected cells were used as a control. To assess sera reactivity to IL4R expressed on HeLa cells, 1 × 105 transfected cells were incubated with human sera (1:2 in PBS 1% BSA) followed by PE-conjugated anti-Human IgG (1:200 in PBS 1% BSA) for 30 min at 37 °C. Cells stained with secondary antibody alone were used as a control. IL4R staining was analyzed by means of flow cytometry with a FACS Canto II analyzer (BD Biosciences), and data were processed with the program FlowJo (flow cytometry analysis software). To assess the inhibition of IL4-mediated Stat6 phosphorylation by patient sera, peripheral blood mononuclear cells were isolated from healthy donor blood via density gradient centrifugation (Ficoll). CD4-positive T cells were magnetically separated (CD4+T Cell Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Germany), resuspended in RPMI medium (Invitrogen) with 10% fetal calf serum, and seeded in 96 well plates at 1 × 106 cells/ml. Cells were pre-incubated for 1 h at 37 °C with sera of patients or healthy donors (1:2 in RPMI) or with neutralizing anti-IL4R goat polyclonal antibody (100 μg/ml) (R&D, Minneapolis, MN) as a control; cells were then stimulated with IL4 (0.2 ng/ml) (Miltenyi Biotec) for 1 h at 37 °C, fixed for 10 min at 4 °C (BD fixation buffer), washed twice in PBS 1% BSA, and permeabilized (BD Perm Buffer III) for 30 min at 4 °C. Intracellular staining with PE mouse anti-Stat6(Y641)P antibody (BD Biosciences) was performed by incubating cells for 30 min at room temperature in the dark. The percentage of Stat6P positive cells was measured by means of flow cytometry with a FACS Canto II analyzer (Becton Dickinson), and data were processed with the program FlowJo (flow cytometry analysis software). To confirm that the mechanism was mediated by antibodies and not by other factors present in the serum, sera from patients and from healthy donors were depleted from anti-IL4R(FNIII) antibodies and tested via Stat6 phosphorylation assay. To do this, 2 mg of purified protein were blotted on nitrocellulose membrane and then incubated with patients or healthy donor sera. After this incubation, sera were collected and tested as described above. The amount of soluble IL4R was determined using an Abcam sIL4R Elisa kit. Briefly, sera were diluted 1:4 in diluent buffer provided in the kit and incubated in 96 well plates coated with anti-sIL4R. Biotinylated monoclonal antibody specific for sIL4R was added to the wells, and plates were incubated for 1 h at room temperature. After washing, streptavidin-HRP was added to the wells, and plates were incubated for an additional 30 min. 3,3′,5,5′-tetramethylbenzidine substrate was then added, and the optical density at 450 nm was read on an Infinite F200 PRO instrument (Tecan). Results of protein microarray and DELFIA® experiments were analyzed using the two-tailed χ2 test, the Student's t test, the Fisher's exact tests, or the analysis of variance test. The Benjamini-Hochberg correction for multiple testing was used for the analysis of microarray data. Statistical analysis was carried out with the use of TIGR Multiexperiment Viewer or GraphPad software. Predictive analysis of microarray (PAM) was performed using the statistical package PAM 1.51 with the statistical tool R (http://wwwstat.stanford.edu/∼tibs/PAM/index.html) (16Tibshirani R. Hastie T. Narasimhan B. Chu G. Diagnosis of multiple cancer types by shrunken centroids of gene expression.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 6567-6572Crossref PubMed Scopus (2157) Google Scholar). PAM executes a sample classification training routine from expression data via the nearest shrunken centroid procedure to find markers that discriminate best between AIH patients and HDs. Data were log-transformed, mean centered, and standard deviation scaled. After training of the PAM classifier, we performed a 10-fold cross validation in order to check the accuracy of the model and better select the threshold as the one giving the lowest misclassification error. To evaluate the performance of autoantigen combinations in discriminating AIH patients from HDs, logistic regression analysis was performed with R. We created logistic regression models with signals of four autoantigens (IL4R(FNIII), AL137145, C17orf99, APCDD1L) or with signals of two known AIH autoantigens as controls (CYP2D6 and AGPR-1). The probabilities were calculated as follows: p = exp ((Σ(bixi)+ c)/(1+Σ(bixi)+ c)), where p is the probability of each case, i = 1 to n, b is the regression coefficient of a given autoantigen, x is the signal intensity, and c is a constant generated by the model. The ROCR package was used to obtain the Receiver Operating Characteristic (ROC) curves of the models and the area under the curve (AUC)
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