Proteomic analysis of the autoantibody response following immunization with a single autoantigen

In most autoimmune diseases, the autoantibody response is directed against several antigens of the target organ whose identification is crucial for understanding the physiopathological process. Thus, technologies allowing a characterization of the whole autoantibody pattern of both human and experimental autoimmune diseases are required. Here we have used immunoproteomic analysis of human epidermal extracts to characterize the diversity of the anti-desmosome antibody response induced in normal mice immunized with desmoglein 1, the major autoantigen of pemphigus foliaceus, an autoimmune blistering skin disease. In particular, this analysis enables us to characterize the binding properties of anti-desmosome mAbs derived from these mice and to show that the autoantibody response induced upon immunization with a single autoantigen targets different epidermal autoantigens with a pattern similar to that observed in certain variety of human pemphigus.     

Identification and functional validation of novel autoantigens in equine uveitis

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.     

Autoantigen microarrays for multiplex characterization of autoantibody responses

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.     

Autoantibody profiling for the study and treatment of autoimmune disease

Proteomics technologies enable profiling of autoantibody responses using biological fluids derived from patients with autoimmune disease. They provide a powerful tool to characterize autoreactive B-cell responses in diseases including rheumatoid arthritis, multiple sclerosis, autoimmune diabetes, and systemic lupus erythematosus. Autoantibody profiling may serve purposes including classification of individual patients and subsets of patients based on their 'autoantibody fingerprint', examination of epitope spreading and antibody isotype usage, discovery and characterization of candidate autoantigens, and tailoring antigen-specific therapy. In the coming decades, proteomics technologies will broaden our understanding of the underlying mechanisms of and will further our ability to diagnose, prognosticate and treat autoimmune disease.     

Altered expression level of a systemic nuclear autoantigen determines the fate of immune response to self.

One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self.     

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple α-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.     

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.     

Humanization of autoantigen

Transmissibility of characteristic lesions to experimental animals may help us understand the pathomechanism of human autoimmune disease. Here we show that human autoimmune disease can be reproduced using genetically engineered model mice. Bullous pemphigoid (BP) is the most common serious autoimmune blistering skin disease, with a considerable body of indirect evidence indicating that the underlying autoantigen is collagen XVII (COL17). Passive transfer of human BP autoantibodies into mice does not induce skin lesions, probably because of differences between humans and mice in the amino acid sequence of the COL17 pathogenic epitope. We injected human BP autoantibody into Col17-knockout mice rescued by the human ortholog. This resulted in BP-like skin lesions and a human disease phenotype. Humanization of autoantigens is a new approach to the study of human autoimmune diseases.     

Immune complexes present in the sera of autoimmune mice activate rheumatoid factor B cells

The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF- littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.     

Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis

The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.     

Autoantigen Microarray for High-throughput Autoantibody Profiling in Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components(nucleic acids and associated proteins), cytoplasmic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epitopes. Different isotypes of autoantibodies, including Ig G, Ig M, Ig A, and Ig E, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection.Proteomic microarray as a multiplexed high-throughput screening platform is playing an increasingly-important role in autoantibody diagnostics.In this article,we highlight the use of autoantigen microarrays for autoantibody exploration in SLE.     

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.     

Identification of kinectin as a novel Behçet's disease autoantigen

There has been some evidence that Beh?et's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a lambdaZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sj?gren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.     

Identification of kinectin as a novel Beh?et's disease autoantigen

There has been some evidence that Beh?et's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a λZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sj?gren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.     

Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry

Normal sera contain a large number of naturally occurring autoantibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because of its ability to simultaneous screening for a wide spectrum of different antigens. In previous studies we have shown the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF MS (surface-enhanced laser desorption/ionization-time of flight mass spectrometry) technology for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The microscale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biologically activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n = 12). After washing, the chips were incubated with a complex solution of autoantigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were to be detected by mass spectrometry. Previous studies using monoclonal antibodies have demonstrated that the detection limit is in the attomole level. Furthermore, all sera were analyzed by conventional Western blotting for direct comparison. In the present study, we have shown complex on-chip antibody-antigen reactions. At higher molecular weights (> 30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular mass, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time-consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody distributions for diagnostic purposes and early detection of autoimmune diseases might be possible.     

Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766-775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.     

Retinal Glycoprotein Enrichment by Concanavalin A Enabled Identification of Novel Membrane Autoantigen Synaptotagmin-1 in Equine Recurrent Uveitis

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.