van Oostveen Wieke M, Hoekstra Eva M, Levarht E W Nivine, Kotliar Ilana B, Sakmar Thomas P, Toes René E M, de Vries-Bouwstra Jeska K, Heitman Laura H, Fehres Cynthia M
Leiden University Medical Center, Leiden, The Netherlands.
The Rockefeller University and Tri-Institutional PhD Program in Chemical Biology, New York, New York.
Arthritis Rheumatol. 2025 Jul;77(7):901-913. doi: 10.1002/art.43099. Epub 2025 Jan 27.
Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. Although previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II receptor type 1 (AT) and endothelin-1 type A receptor (ETR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays. Our aim was to replicate previous findings and to investigate the functional effects of IgG derived from patients with SSc (SSc IgG) on AT and ETR signaling, the downstream EC response, and the presence of AT-binding autoantibodies in circulation.
Quantitative polymerase chain reaction and cytokine enzyme-linked immunosorbent assay, alongside a real-time cell analyzer, were used to assess receptor-specific functional characteristics of purified SSc IgG (n = 18). Additionally, a novel protein capture assay using solubilized epitope-tagged AT was developed to detect AT-binding autoantibodies in plasma samples from patients with SSc (n = 28) and healthy donors (n = 14).
No evidence for EC activation in an AT- or ETR-dependent manner was revealed. Furthermore, stimulation with SSc IgG did not induce receptor activation or alter G protein-coupled receptor signaling on agonist stimulation in a model with receptor overexpression. Lastly, no AT-binding autoantibodies were detected in plasma samples from patients with SSc when using epitope-tagged solubilized AT.
Overall, our study did not provide evidence to support the presence of AT- or ETR-activating autoantibodies in purified SSc IgG or AT-binding autoantibodies in the circulation of patients with SSc.
系统性硬化症（SSc）是一种罕见但严重的自身免疫性疾病，其特征为免疫失调、纤维化和血管病变。尽管先前的研究强调了存在靶向1型血管紧张素II受体（AT）和A型内皮素-1受体（ETR）的功能性自身抗体，导致自身抗体介导的受体刺激以及随后内皮细胞（ECs）的激活，但目前缺乏对这些自身抗体与其受体之间直接相互作用的全面了解。此外，现有的证实SSc中存在这些自身抗体的数据通常依赖于相似的方法和检测。我们的目的是重复先前的发现，并研究来自SSc患者的IgG（SSc IgG）对AT和ETR信号传导、下游EC反应以及循环中AT结合自身抗体存在情况的功能影响。
使用定量聚合酶链反应和细胞因子酶联免疫吸附测定，以及实时细胞分析仪，评估纯化的SSc IgG（n = 18）的受体特异性功能特征。此外，开发了一种使用可溶性表位标记的AT的新型蛋白质捕获测定法，以检测来自SSc患者（n = 28）和健康供体（n = 14）的血浆样本中的AT结合自身抗体。
未发现以AT或ETR依赖性方式激活EC的证据。此外，在受体过表达模型中，用SSc IgG刺激未诱导受体激活或改变激动剂刺激时的G蛋白偶联受体信号传导。最后，使用表位标记的可溶性AT时，在SSc患者的血浆样本中未检测到AT结合自身抗体。
总体而言，我们的研究没有提供证据支持纯化的SSc IgG中存在AT或ETR激活自身抗体，或SSc患者循环中存在AT结合自身抗体。
