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

Abstract

Introduction

Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT ₁R) and endothelin-1 type A receptor (ET _AR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT ₁R and anti-ET _AR Abs on initiation of inflammation and fibrosis was analyzed.

Methods

Anti-AT ₁R and anti-ET _AR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT ₁R and ET _AR 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-AT ₁R and anti-ET _AR 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-AT ₁R and anti-ET _AR 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-AT ₁R and anti-ET _AR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT ₁R and anti-ET _AR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.