OP0224 Anti-AT1R and Anti-ETAR Autoantibodies from Patients with SSC and their Agonistic Effects
Authors: A. Kill, J. Günther, M. O. Becker, D. Dragun, G.-R. Burmester, G. Riemekasten
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Autoantibodies reactive to angiotensin II type 1 receptor (AT<sub>1</sub>R) and endothelin 1 type A receptor (ET<sub>A</sub>R), anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R autoantibodies, are found in patients with Systemic sclerosis (SSc) as a prototypic connective tissue disease with limited therapeutic options. SSc is characterized by three major hallmarks: autoimmunity, vasculopathy and fibrosis. Anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R autoantibodies (Ab) have been demonstrated in association with clinical symptoms of SSc including vascular and fibrotic complications. Therefore, direct agonistic effects of anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab were studied. <h3>Objectives</h3> To analyse anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab-mediated agonistic effects in <i>in vitro</i> and <i>in vivo</i> experimental settings. <h3>Methods</h3> Human microdermal endothelial cells-1 (HMEC-1) were treated with IgG from SSc patients positive for anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab, or with IgG of healthy donors (NC-IgG). To demonstrate angiotensin- and/or endothelin-receptor activation, cells were pre-treated with specific receptor inhibitors, either alone or in combination. Cell activation was measured by IL-8 chemokine expression, adhesion molecule VCAM-1 expression, trans-endothelial neutrophil migration and ROS activation using qRT-PCR, sandwich ELISA, cell culture inserts and fluorescence analysis. <i>In vivo</i> effects were measured by autoantibody transfer into healthy C57Bl/6J mice and analysis of cellular BALF composition. <h3>Results</h3> Upon treatment with anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab positive SSc-IgG, HMEC-1 cells increased IL-8 mRNA levels and IL-8 protein secretion into culture supernatants compared to cells treated with NC-IgG. Levels of IL-8 mRNA and protein were decreased in samples that were pre-treated with specific angiotensin- and endothelin-receptor blockers. Culture supernatants with increased IL-8 protein levels due to SSc-IgG treatment, showed an increased potential to recruit neutrophils through an endothelial cell layer, compared to supernatants of NC-IgG treated samples. Again, specific angiotensin- and endothelin-receptor inhibition showed a reduction in neutrophil recruitment. Culture supernatants conditioned with SSc-IgG increased reactive oxygen species (ROS) production in healthy donor neutrophils compared to supernatants with control treatment. Furthermore, HMEC-1 cells showed also increased mRNA levels of VCAM-1 with significant reduction by receptor inhibition. Moreover, mice that received anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab positive SSc-IgG showed significantly increased neutrophil number in BALF compared to NC-IgG. <h3>Conclusions</h3> Our findings demonstrate the potential of anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab to induce pathogenic effects <i>in vitro</i> and <i>in vivo</i>. Activation of endothelial cells by anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab might reflect in some aspects the situation as seen <i>in vivo</i> and could thereby be an important factor in disease pathogenesis. Furthermore, cell activation by these autoantibodies induced neutrophil recruitment <i>in vitro.</i> Finally, activation of neutrophil recruitment by anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab positive SSc-IgG, demonstrated the potential to directly induce pathogenic effects in healthy mice <i>in vivo</i>. Thus, angiotensin- and endothelin-receptor activation by anti-AT<sub>1</sub>R and anti-ET<sub>A</sub>R Ab could significantly contribute to pathogenesis of SSc. <h3>Disclosure of Interest</h3> None Declared