Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis

INTRODUCTION Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT₁R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT₁R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed. METHODS Anti-AT₁R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT₁R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy. RESULTS Anti-AT₁R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs. CONCLUSIONS We conclude that angiotensin and endothelin-receptor activation via anti-AT₁R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT₁R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.