Abstract
IntroductionAntinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Thus, approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). In this work, we aimed to identify new target autoantigens in SSc patients.MethodsUsing a proteomic approach combining two-dimensional electrophoresis and immunoblotting with HEp-2 cell total and enriched nuclear protein extracts as sources of autoantigens, we systematically analysed autoantibodies in SSc patients. Sera from 45 SSc patients were tested in 15 pools from groups of three patients with the same phenotype. A sera pool from 12 healthy individuals was used as a control. Proteins of interest were identified by mass spectrometry and analysed using Pathway Studio software.ResultsWe identified 974 and 832 protein spots in HEp-2 cell total and enriched nuclear protein extracts, respectively. Interestingly, α-enolase was recognised by immunoglobulin G (IgG) from all pools of patients in both extracts. Fourteen and four proteins were recognised by IgG from at least 75% of the 15 pools in total and enriched nuclear protein extracts, respectively, whereas 15 protein spots were specifically recognised by IgG from at least four of the ten pools from patients with unidentified ANAs. The IgG intensity for a number of antigens was higher in sera from patients than in sera from healthy controls. These antigens included triosephosphate isomerase, superoxide dismutase mitochondrial precursor, heterogeneous nuclear ribonucleoprotein L and lamin A/C. In addition, peroxiredoxin 2, cofilin 1 and calreticulin were specifically recognised by sera from phenotypic subsets of patients with unidentified ANAs. Interestingly, several identified target antigens were involved in the transforming growth factor β pathway.ConclusionsWe identified several new target antigens shared among patients with SSc or specific to a given phenotype. The specification of new autoantibodies could help in understanding the pathophysiology of SSc. Moreover, these autoantibodies could represent new diagnostic and/or prognostic markers for SSc.
Highlights
Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc)
Four pools were from patients with identified ANAs, ten pools were from patients with unidentified ANAs, and one pool was from patients without ANAs (Table 1)
Analysis of HEp-2 cell proteomes We found 974 and 832 protein spots stained by silver nitrate in HEp-2 cell total and enriched nuclear protein extracts, respectively (Figures 2B and 2E and Additional file 1)
Summary
Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). A large number of autoantibodies have been identified in the sera of SSc patients. Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients [2]. Other autoantibodies have been found in the sera of SSc patients and include antifibrillarin, antifibrillin 1, anti-Th/To, anti-PM/Scl [3], antifibroblast [4,5,6] and anti-endothelial cell antibodies [7,8,9]. The only specific autoantibodies routinely tested for in SSc patients are ACAs, ATAs and, more recently, anti-RNA polymerase III antibodies
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