Identification of a target antigen reacting with anti-endothelial cell IgA antibody in Behçet’s disease
Dept. of Medicine/박사
Behçet’s disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease, presenting with various symptoms of vasculitis and/or thrombosis. The purpose of this study was to identify human dermal microvascular endothelial cell (HDMEC) antigen, which binds anti-endothelial cell IgA antibodies in BD.We detected the target protein using western blotting and immunoprecipitation and determined the amino acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/ TOF). We searched for the DNA sequence of the target protein and purified the recombinant target protein by gene cloning. Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by enzyme-linked immunosorbent assay (ELISA). In addition, the sera of BD patients and HC as well as cultured S. sanguis were used for HDMECs stimulation. Subcellular fractions of stimulated HDMECs in each group were extracted and immunoblot analyses for the target protein were performed.The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino acid sequences of hnRNP-A2/B1. Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), four of 30 systemic lupus erythematosus patients (13.3%), eight of 30 rheumatoid arthritis patients (26.7%), nine of 30 Takayasu’s arteritis patients (30%), six of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 in BD were correlated with those against the recombinant streptococcal hsp-65. The hnRNP-A2/B1 was significantly increased in cellular membrane in HDMECs incubated with the sera of BD patients and cultured S. sanguis for 12 hours and 24 hours compared with HDMECs incubated with endothelial cell culture media and the sera of healthy controls. We identified an hnRNP-A2/B1 protein as a target protein of serum anti-endothelial cell IgA antibody in BD patients. We also demonstrated serum IgA reactivity against recombinant human hnRNP-A2/B1 and recombinant streptococcal hsp-65 were correlated.