Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases. Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases. Autoimmune diseases affect 5% of the world population, and our understanding and treatment of human autoimmune diseases has to be improved (1Davidson A. Diamond B. Autoimmune diseases.N. Engl. J. Med. 2001; 345: 340-350Google Scholar). Often the current diagnostic tools are limited because there is no assay to detect the quality of the patients’ response to drugs. Thus, in addition to improved diagnostics the discrimination between drug responder and drug non-responder before the onset of therapy might be a goal for future strategies in the treatment of autoimmune diseases. A characteristic feature of many autoimmune diseases is the production of autoantibodies (2von Mühlen C.A. Tan E.M. Autoantibodies in the diagnosis of systemic rheumatic diseases.Semin. Arthritis Rheum. 1995; 24: 323-358Google Scholar). Although the pathogenic role for most of the autoantibodies in various autoimmune diseases is not clear, the identification of the autoantigens that are targeted by the autoantibodies during the immune response may present an important tool for diagnosis, classification, and prognosis. Additionally profiling the autoantibody repertoire may help to elucidate the pathophysiology of autoimmunity, enabling novel treatments such as antigen-tolerating therapy (3Robinson W.H. Fontoura P. Lee B.J. de Vegvar H.E. Tom J. Pedotti R. DiGennaro C.D. Mitchell D.J. Fong D. Ho P.P. Ruiz P.J. Maverakis E. Stevens D.B. Bernard C.C. Martin R. Kuchroo V.K. van Noort J.M. Genain C.P. Amor S. Olsson T. Utz P.J. Garren H. Steinman L. Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis.Nat. Biotechnol. 2003; 21: 1033-1039Google Scholar, 4Brocke S. Gijbels K. Allegretta M. Ferber I. Piercy C. Blankenstein T. Martin R. Utz U. Karin N. Mitchell D. Veromaa T. Waisman A. Gaur A. Conlon P. Ling N. Fairchild P.J. Wraith D.C. O’Garra A. Fathman C.G. Steinman L. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.Nature. 1996; 379: 343-346Google Scholar). Protein microarrays have been used for detection and validation of autoantibodies in biological fluids (5Lueking A. Possling A. Huber O. Beveridge A. Horn M. Eickhoff H. Schuchardt J. Lehrach H. Cahill D.J. A non-redundant human protein chip for antibody screening and serum profiling.Mol. Cell. Proteomics. 2003; 2: 1342-1349Google Scholar, 6Robinson W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Google Scholar). Alopecia areata is an autoimmune disease of the hair follicles that affects between 2 and 4% of patients seen in dermatological practice (7Price V.H. Alopecia areata: clinical aspects.J. Investig. Dermatol. 1991; 96: 68SGoogle Scholar) resulting in rapid loss of hair, often featured by nail dystrophy. The loss may reverse completely, become chronic, or progress to loss of all scalp hair (alopecia totalis) and all body hair (alopecia universalis). It is diagnosed by the clinical finding of a patchyhairless area with some dystrophic hairs called “exclamation point” hairs at the border of the patch. It may be confirmed by characteristic histological findings of a dense peri- and intrabulbar inflammatory infiltrate of anagen hair follicles like a bee swarm. The disease is characterized by a frequently relapsing course with good response to systemic immunosuppression (corticosteroids) or topical immunomodulation with diphencyprone (8Freyschmidt-Paul P. Happle R. Hoffmann R. Alopecia areata. Clinical aspects, pathogenesis and rational therapy of a T-cell-induced autoimmune disease.Hautarzt. 2003; 54: 713-722Google Scholar). Progress has been achieved over the past 5–10 years in the characterization of hair follicle antigens targeted by antibodies in alopecia areata (9Tobin D.J. Characterization of hair follicle antigens targeted by the anti-hair follicle immune response.J. Investig. Dermatol. Symp. Proc. 2003; 8: 176-181Google Scholar). Candidate autoantigens that have been identified include the 44/46-kDa hair-specific keratin and trichohyalin. Moreover there is evidence that anti-hair follicle antibodies are modulated during the disease process and can occur before clinically evident hair loss (9Tobin D.J. Characterization of hair follicle antigens targeted by the anti-hair follicle immune response.J. Investig. Dermatol. Symp. Proc. 2003; 8: 176-181Google Scholar). In addition, differentiating anagen keratinocytes are an important structure in the autoimmune etiology of alopecia both in autoimmune endocrine syndrome type I-associated alopecia areata and at least in a subgroup of patients with alopecia areata unrelated to autoimmune polyendocrine syndrome type I (10Hedstrand H. Perheentupa J. Ekwall O. Gustafsson J. Michaelsson G. Husebye E. Rorsman F. Kampe O. Antibodies against hair follicles are associated with alopecia totalis in autoimmune polyendocrine syndrome type I.J. Investig. Dermatol. 1999; 113: 1054-1058Google Scholar). There is evidence for a polygenic inheritance, and diverse candidate genes have so far been identified by case-control and family-based studies: HLA class II genes (11Messenger A.G. McDonagh A.J. Alopecia areata: aetiology and pathogenesis, in Hair and Its Disorders (Camacho, F. M., Randall, V. A., and Price, V. H., eds). Martin Dunitz, London2000: 177-186Google Scholar), interleukin-1 cluster genes (12Tazi-Ahnini R. Cox A. McDonagh A.J. Nicklin M.J. di Giovine F.S. Timms J.M. Messenger A.G. Dimitropoulou P. Duff G.W. Cork M.J. Genetic analysis of the interleukin-1 receptor antagonist and its homologue IL-1L1 in alopecia areata: strong severity association and possible gene interaction.Eur. J. Immunogenet. 2002; 29: 25-30Google Scholar, 13Tarlow J.K. Clay F.E. Cork M.J. Blakemore A.I. McDonagh A.J. Messenger A.G. Duff G.W. Severity of alopecia areata is associated with a polymorphism in the interleukin-1 receptor antagonist gene.J. Investig. Dermatol. 1994; 103: 387-390Google Scholar), and polymorphism in the interferon-induced p78 protein MxA, which is highly expressed in anagen hair bulbs of alopecia areata lesions (14Freyschmidt-Paul P. Happle R. Hoffmann R. Alopecia areata in animal models: insights in pathogenesis and therapy in a T-cell mediated autoimmune disorder.J. Dtsch. Dermatol. Ges. 2004; 4: 260-273Google Scholar). In this study, we combined protein microarray technology with the concept of an arrayed expression library enabling direct connection from the protein product of an individual expression clone to its corresponding cDNA sequence information (for reviews, see Refs. 15Cahill D.J. Nordhoff E. Protein arrays and their role in proteomics.Adv. Biochem. Eng. Biotechnol. 2003; 83: 177-187Google Scholar, 16Lueking A. Konthur Z. Eickhoff H. Büssow K. Lehrach H. Cahill D.J. Protein microarrays—a tool for the post-genomic era.Curr. Genomics. 2001; 2: 151-159Google Scholar, 17Walter G. Büssow K. Cahill D. Lueking A. Lehrach H. Protein arrays for gene expression and molecular interaction screening.Curr. Opin. Microbiol. 2000; 3: 298-302Google Scholar). Using robot technology, a human fetal brain cDNA expression library (hEx1) was picked into microtiter plates, and high density protein arrays were produced on filter membranes followed by in situ expression (18). In an analysis of this of the in the of these on an genes have been in this expression library a high of proteins. this library to be highly for screening protein arrays with body fluids from large of patients with an autoimmune disease the identification of novel autoantigens as well as the diagnosis, and of autoimmune diseases on the of In diagnostic are used to their during our has the to a disease-associated protein chip for diagnosis, we have used and on autoantibodies which present have been successfully used to the autoantibody repertoire W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Google Scholar, G. E. microarray analysis of IgG autoantibody the future response of to U. S. A. 2004; Scholar, The autoantibody repertoire and autoimmune 2004; Scholar). we used alopecia areata as a model for a autoimmune disease and profiled the autoantibody repertoire of alopecia areata identification of putative we the by the identified putative autoantigens on protein microarrays to generate a disease-associated protein chip to and differentiate between these candidate autoantigens. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema were additionally on the disease-associated protein chip. patients with alopecia areata alopecia areata alopecia areata of alopecia areata or alopecia areata alopecia areata data not in an systemic or topical therapy were for in this The patient control of patients with hair and no or of alopecia areata. 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Nordhoff E. C. Lehrach H. G. A human cDNA library for protein expression 2000; Scholar). The were against for J. Z. W. D.J. and a generation of protein Scholar). density protein arrays of the protein expression of the library were obtained from the for and as K. Cahill D. W. D. E. Lehrach H. G. A for protein expression and antibody screening on of an arrayed cDNA Scholar). For serum the were in in for 2 in and with serum patient sera to IgG and in for and with the antibody anti-human or anti-human as in the were for in was followed by with the antibody IgG in the were in for followed by a in and a for in and in in for The were with and the were using a high detection analysis was with protein was expressed in in well microtiter The were from A. Horn M. Eickhoff H. Büssow K. Lehrach H. G. Protein microarrays for gene expression and antibody Biochem. 1999; Scholar) and as K. Nordhoff E. C. Lehrach H. G. 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In the alopecia areata study, we used serum sera from alopecia areata and we identified putative autoantigens on the human protein filter compared with the study, the of identified putative autoantigens is at least of these with were confirmed using both highly sensitive protein microarrays and Western leading to results compared with our that the use of of of serum is a for the screening on the human filter that by using serum putative autoantigens we that the most putative autoantigens be by this For the of a disease-associated protein we that were expressed with to the and confirmed by Western Eight autoantigens were identified by protein chip technology and successfully confirmed by Western blot analysis. 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DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Google Scholar) have that autoantigens such as or were by autoimmune disease and W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Google this we the analysis of patient samples of alopecia areata and other dermatological diseases as a which may to the identification of protein characteristic for the be this an of the disease-associated protein chip. that a protein leading to an detection of about autoantigens may in a discrimination of diseases. A of putative autoantigens can be achieved using for type of expression library such as a or a expression identified putative autoantigens by profiling the autoantibody repertoire of alopecia areata patients with no role which a strong to hair diseases. we the role of some of the identified proteins. to which an important role in the control of hair of was in the and the of hair follicles and in Using in situ was in in the of the hair the of in the autoimmune disease alopecia areata is not The was identified by screening a expression library with autoantibodies from a patient with the skin disease The cDNA a of the this is generated by a from the cDNA Z. C.A. G.J. Characterization of a novel 1996; Scholar, Z. G.J. cDNA of a novel human Scholar). The of this in the pathogenesis of or of other autoimmune diseases is is an and protein of the have a in The by the of the in as to is the identified potential autoantigens are to the autoimmune various putative autoantigens for other autoimmune such as some autoantigens are with known in the For other diseases no was for these autoantigens S. T. K. T. Autoantibodies to protein 2 in systemic autoimmune 2003; Scholar). some of autoantigens with in to the autoimmune disease have been used as of the corresponding disease and immune in 2002; Scholar). In an we have profiled the autoantibody repertoire of an model on a cDNA expression library and identified for the and protein protein as a putative C. D. Lueking A. A. M. J. B. Horn S. Lehrach H. Cahill D.J. protein arrays from a cDNA library for antibody screening and serum Scholar). compared with the human autoantibodies against the human and have been a between to human diseases Lee Lee S. J. B. Lee H. Lee E. of autoantibodies associated with systemic 2002; Scholar). that our strategy to the research of autoimmune diseases to