In order to elucidate the pathogenic role(s) of autoantibodies in connective tissue disease (CTD), we examined whether autoantibodies against U1-ribonucleoprotein (RNP) and double-stranded (ds) DNA can up-regulate ICAM-1, ELAM-1 and class I and II MHC molecule expression on pulmonary artery endothelial cells (HPAEC). ICAM-1, ELAM-1 and class II MHC molecule expression on HPAEC cultured in the presence of anti-U1-RNP-containing and anti-dsDNA-containing IgG from CTD patients was up-regulated significantly in comparison with that on HPAEC cultured with IgG from normal healthy volunteers. Affinity chromatographic enrichment and depletion of the anti-U1-RNP antibody content of anti-U1-RNP-containing IgG confirmed that the anti-U1-RNP antibody did up-regulate ICAM-1, ELAM-1 and class II MHC molecule expression. The finding that an IgG F(ab')2-purified anti-U1-RNP antibody also up-regulated expression of these molecules may indicate that mechanisms other than Fc receptor-mediated stimulation are involved. These in vitro findings suggest that autoantibodies against U1-RNP and dsDNA play important roles in the immunopathological processes leading to the proliferative pulmonary arterial vasculopathy observed in CTD patients with pulmonary hypertension by up-regulating adhesion and class II MHC molecule expression on endothelial cells.

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SUMMARYIn order to elucidate the immunological properties of anti-U1-ribonucleoprotein (RNP) antibody, one of the autoantibodies detected in patients with connective tissue diseases (CTDs), we tested the endothelial cell-binding by anti-U1-RNP antibodies and epitopes on human pulmonary artery endothelial cells (HPAECs) to which the autoantibody bound. IgG fractions positive for anti-U1-RNP from patients with CTDs bound to the HPAECs. Furthermore, intact and F(ab H ) 2 IgG anti-U1-RNP purified by affinity chromatography also bound to endothelial cells. The binding activity of IgG fractions positive for anti-U1-RNP to the endothelial cells could be effectively absorbed by U1-RNP-Sepharose. An immunoblotting assay of purified IgG anti-U1-RNP antibodies showed that these antibodies could bind to various membrane proteins of NP40-treated HPAECs such as 68, 48, 43,38,33,29,28 and 24 kDa. Some bands, 68, 33, 28 and 24 kDa, seemed to correspond to components of U1-RNP, i.e. 68 kDa, A, B H and C peptides, respectively. We confirmed that the anti-U1-RNP antibody from patients with CTDs can directly recognize a variety of antigens on the endothelial surface of the pulmonary artery, including the components of U1-RNP or other unknown polypeptides. These results suggest that binding to pulmonary artery endothelial cells of this autoantibody may be one of the triggers of endothelial cell inflammation in CTDs.

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