Elsevier

Immunology Letters

Volume 124, Issue 1, 14 May 2009, Pages 35-43
Immunology Letters

Unravelling autoimmune pathogenesis by screening random peptide libraries with human sera

https://doi.org/10.1016/j.imlet.2009.04.001Get rights and content

Abstract

The incidence of autoimmunity is increasing worldwide. The long preclinical period of autoimmune disorders is characterised by an enhanced exposure over time of autoreactive T cells to an increased number of autoantigenic determinants and autoantibodies production. The discovery of novel autoimmune-disease related epitopes is a task that remains extremely challenging in order to establish predictive and preventive strategies of the disease onset. In this Opinion article we highlight the contribution of screening combinatorial random peptide libraries with patients sera in unravelling the etiopathogenesis of autoimmunity.

Introduction

Autoimmunity occurs in up to 3–5% of the population in Western countries [1], [2], [3], [4], [5], [6], [7], [8], [9]. Autoimmune disorders are generally classified according to the organs or tissues that are affected. An autoimmune disease can be recognised for nearly every organ, tissue and even single cell in the body; usually the response to an antigen or a limited series of antigens exclusive of the targeted area is involved. In other autoimmune conditions, such as systemic lupus erythematosus (SLE), no particular cell type seems to be targeted, but the response appears to be directed against widely expressed antigens (e.g. nuclei antigens). Nevertheless these disorders are antigen-specific; furthermore, recognition of widely expressed antigens may produce, unexpectedly, organ-selective clinical symptoms [1].

Patients affected by autoimmune disorders are characterised by the production of several organ- and non-organ specific autoantibodies [1]. These are detectable at diagnosis, but are also present in the latency period which precedes the clinical onset of disease. Clearly this is demonstrated by the natural history of insulin-dependent diabetes mellitus (Type 1 diabetes) [10], a chronic autoimmune disease in which insulin-producing β cells are progressively destroyed by autoreactive T cells. Molecular and cellular mechanisms underline the pathogenesis of autoimmune diseases with the contribution of putative environmental factors and the requisite allele(s) responsible for antigen presentation by antigen-presenting cells for T cell recognition. Although genetic factors have been well dissected [11], few advances have been made in identifying the environmental agents, including putative viruses, that may be causative of disease. In view of the long period that often precedes clinical symptoms in autoimmunity, slow viruses have been suggested as potential candidates [1]. The hypothesis of a superantigen involvement in the pathogenesis of Type 1 diabetes has been pursued as an interesting possibility [12]. Alternatively, autoimmunity may follow the infection with a pathogen that exhibits similarities in some of its epitopes with self-proteins of the host (molecular mimicry) [13]. As a consequence, antibodies produced against the pathogen work indeed as autoantibodies. The long preclinical period may be required for the recognition of a small number of autoantigens by a limited number of autoreactive effector T cells, or may be the result of changes in the number and/or function of regulatory T cells. An alternative explanation is that the chronic state of disease is due to the enhanced challenge over time of autoreactive T cells by an increased number of autoantigenic peptide determinants (epitope spreading) [14]. Proteins to which the immune system is self-tolerant (self-proteins) may evoke autoimmune responses if their expression becomes altered as they undergo post-translational modifications, denaturation, misfolding [14]. Proteins can be physiologically sequestered in organs, but can elicit autoimmune responses when become exposed to the immune system. In the disease progression, secondary events such as environmental factors or metabolic events might contribute to the expression of target cell-associated antigens, increasing their susceptibility to immune destruction. This may occur through the increased expression of co-stimulatory molecules that cause immune activation, enhanced antigen presentation or secretion of inflammatory cytokines.

In some organ-specific autoimmune diseases, directly pathogenetic autoantibodies are described, i.e. those directed against the acetylcholine receptor in myasthenia gravis [15] and the thyroid stimulating hormone receptor antibodies in Graves’ thyrotoxicosis [16]. In other autoimmune disorders, autoantibodies are directed against intracellular autoantigens; therefore, they should not produce disease directly, but represent important serological markers for ongoing damage or overt disease. They often present multiple autoantibodies, indicating subclinical disturbances in other related organs or tissues. In both categories of human autoimmune diseases, many of the target autoantigens have been characterised [17] and are, in most instances, represented by enzymes. The significance of this phenomenon remains at present unknown. In Type 1 diabetes insulin, glutamic acid decarboxylase (GAD) and the insulinoma-associated antigen-2 (IA-2), a member of the tyrosine phosphatase enzyme family have been identified, whereas other potential autoantigens, for example those recognised by cytoplasmic islet cell antibodies (ICA), have not [1], [18].

In the light of the aforegoing, in this Opinion article we emphasise how novel molecular biology approaches propel nowadays the discovery of autoimmune-disease related epitopes, a task that remains extremely challenging in elucidating the pathogenesis of autoimmunity.

Section snippets

Looking for candidate autoantigens

Over the years several approaches have been implemented to identifying candidate autoantigens in organ and non-organ specific autoimmune disorders. For example, tissue preparations from organs target of autoimmunity have been screened in biochemical studies with sera from autoimmune patients by using Western Blotting or immunoprecipitation; the identified proteins were subsequently purified and microsequenced to identify novel autoantigens. Sera have also been used for screening cDNA expression

What are RPLs and their basic and clinical applications?

RPLs consist of short synthetic amino-acid sequences expressed within one of the major phage coat proteins of bacteriophage f1 [24], [25] or chemically synthesised [26], [27], [28], [29], [30], [31]. Smith and co-workers [24], [25] first demonstrated that recombinant DNA technology could be used to display foreign peptides on the surface of filamentous phage. Therefore bacteriophage f1 coat proteins pIII and pVIII [25] started to be employed for the construction of RPLs. There are about five

Searching T1D-related epitopes

Here we discuss the feasibility of screening a RPL with sera from patients with Type 1 diabetes. In initial studies, MoAbs directed against diabetes-related autoantigens were used to screen RPLs. Phagotopes corresponding to known diabetes-related antigens were detected by screening RPLs with anti-GAD MoAbs [104]. Limited homology between the selected phagotopes and the GAD molecule allowed those phagotopes to be mapped to a sequence of the GAD65 isoform. A RPL was also screened with two islet

Searching SLE-related epitopes by RPL screening

We used the thioredoxin RPL system [29] in investigating the etiopathogenesis of a particular pediatric glomerulopathy suggestive of lupus nephritis characterised by the presence of a ‘full-house’ immunofluorescence pattern (full-house nephropathy), but the absence of other clinical and biological evidence of SLE [111]. From clinical experience pediatric patients with ‘full-house nephropathy’ need to be monitored closely because in 10% of them the appearance of autoantibodies and/or clinical

Conclusive remarks and future directions

What we have summarised above suggest that RPL reagents are of great potential and form an unique tool for exploring the immunological specificity in human autoimmunity, thus unravelling the etiopathogenesis of disease. For several autoimmune diseases the technology of RPL screening with human sera has already provided relevant informations in addition to the results obtained with MoAbs. In many investigations peptides were selected that represent the molecular ‘immunofootprint’ characteristic

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