Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis

Abstract

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. 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 2007; PubMed Scopus Google Scholar), they a to serological is AIH and other liver such as chronic viral hepatitis E. Dalekos G.N. Autoantibodies and autoantigens in autoimmune hepatitis: in clinical and to of the Autoimmune Dis. 2004; PubMed Scopus Google Scholar). specific an need for the more diagnosis and of AIH. autoimmune hepatitis under the Lanthanide Fluoroscence fibronectin type III domain hepatitis B hepatitis C healthy interleukin-4 receptor metal ion affinity fluorescence analysis of microarray Operating Characteristic. autoimmune hepatitis under the Lanthanide Fluoroscence fibronectin type III domain hepatitis B hepatitis C healthy interleukin-4 receptor metal ion affinity fluorescence analysis of microarray Operating Characteristic. the diagnostic the of AIH autoantigens into the disease Although AIH have identified and is pathogenetic role, and other autoantigens are to be have two the be expressed on the membrane of cells or secreted by cells be to and binding of the autoantibodies to the a or A pathogenetic role in AIH has for autoantibodies specific for or receptor 1 which are both on the membrane E. Dalekos G.N. Autoantibodies and autoantigens in autoimmune hepatitis: in clinical and to of the Autoimmune Dis. 2004; PubMed Scopus Google Scholar). are a as they the of of G. S. H. F. P. J. C. S. 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