P2_163 Analyses of responsible genetic factors for systemic vasculitides using recombinant inbred collagen disease model mice

Abstract

Objectives: Juvenile dermatomyositis (JDM) is a rate systemic autoimmune vasculopathy. The pathogenesis of JDM remains unknown. Among the important challenges concerning JDM is our poor understanding of mechanisms through which blood vessels are identified for immune attack. Anti-endothelial cell antibodies (AECA) have been detected in rheumatic diseases such as vasculitis. Because JDM shares histopathologic features with other forms of vasculitis, we aimed to detect target antigens for AECA and their clinical significance in patients with JDM using proteomics. Methods: To detect antibodies in sera from patients with JDM, proteins extracted from human dermal microvascular endothelial cells, human aortic endothelial cells (HAEC), human aortic adventitial fibroblasts, human aortic smooth muscle cells, human skeletal muscle cells and peripheral blood mononuclear cells were used as antigen sources for SDS-PAGE and immunoblotting. To detect target antigens for AECA, we separated proteins extracted from HAEC by two-dimensional electrophoresis (2DE) and transferred them onto membranes. Autoantigens that were positive only in sera from JDM but not in healthy children sera were detected by western blotting (WB). The detected proteins were identified by mass spectrometry (MS). Bound IgG antibodies to antigens were detected using standard methods. Results: A pool of sera from five patients with JDM had multiple antibodies, in particular, to proteins extracted from HAEC. Furthermore, a 75-kDa target protein of AECA was detected in active patients but not in inactive patients or those in remission. An 88-kDa target protein of AECA was clearly detected in patients who had started treatment with steroids and methotrexate. Five candidate protein spots as JDM-specific proteins were detected in 2DE-WB using proteins extracted from HAEC. One of 34 proteins identified by MS was myosin-9 (MYH9), muscle-related protein. IgG autoantibodies to MYH9 were detected in 31% of the active patients with JDM (n=35), (50% of the untreated JDM patients with active disease), in comparison to 27% of the patients with juvenile idiopathic arthritis (n=15) and 12% (p<0.05) of control children. Conclusions: Antibodies directed toward the proteome of HAEC are present in the sera of patients with JDM. These antibodies could be involved in the pathogenesis of vessel injury in JDM.