Multiple target autoantigens on endothelial cells identified in juvenile dermatomyositis using proteomics

Abstract

Objective: Although generally classified within the group of inflammatory myopathies, JDM displays many pathological features of vasculitis. Previous work has shown that AECA are abundant in other forms of vasculitis. We therefore investigated whether such antibodies might also be detected in JDM.

Methods: We screened plasma from children with JDM for the presence of AECA by western blotting and 2D gel electrophoresis (2DE) using proteins extracted from human aortic endothelial cells as the substrate. We performed mass spectrometry to identify candidate antigens from 2DE gels and used ELISA to confirm the presence of specific antibodies.

Results: We identified 22 candidate target autoantigens for AECA probed with JDM plasma. Interestingly, 17 of these 22 target antigens were proteins associated with antigen processing and protein trafficking. ELISA confirmed the presence of antibodies to heat shock cognate 71 kDa protein in JDM plasma, particularly in children with active, untreated disease.

Conclusion: Children with JDM express antibodies to autoantigens in endothelial cells. The clinical and pathological significance of such autoantibodies require further investigation.

Fig. 1

Detection of AECA in JDM (A) Protein extracted from normal human dermal microvascular endothelial cells (HMVEC-D) (a), normal human aortic endothelial cells (HAEC) (b), human aortic adventitial fibroblasts (AoAF) (c), normal human aortic smooth muscle cells (HAoSMC) (d) and human skeletal muscle cells (SkMC) (e) were separated by SDS–PAGE. Proteins reactive to plasma from patients with active JDM (untreated: a) or patients with inactive JDM (off therapy: b) were detected by western blotting (WB). (B) Proteins extracted from HAEC separated by 2D gel electrophoresis (2DE), and antigens detected by WB using plasma from patients with active JDM [untreated (a) or healthy (b) children]. Protein spots in AECA recognized predominantly in JDM compared with control plasma are indicated by arrows and spot ID numbers in (a). (C) Locations of the five protein spots analysed by MS are indicated in a Coomassie Brilliant Blue R-250-stained 2DE gel using HAEC extracts. (D) Results of ELISA assays for anti-HSC70 antibodies. H: healthy children; JD: JDM; JI: JIA; JU: untreated JDM, active disease; JT: treated JDM, active disease; ID: inactive JDM.