Department of Biomolecular Chemistry, Institute of Molecules and Materials (IMM), Radboud University, Nijmegen, the Netherlands.
Department of Rheumatology, Radboud University, Nijmegen, the Netherlands.
J Autoimmun. 2020 Sep;113:102484. doi: 10.1016/j.jaut.2020.102484. Epub 2020 May 23.
Neutrophil extracellular traps (NETs) are networks of extracellular chromatin decorated with antimicrobial proteins, formed by neutrophils to entrap pathogens. NETs have been implicated in the generation of autoimmune reactions. Here, we investigate the reactivity of rheumatoid arthritis (RA) serum antibodies with NETs and explore whether anti-NET antibodies (ANETA) have a potential as biomarker in RA. To quantify ANETA, we developed an ELISA with NETs isolated from stimulated human neutrophils and verified the results by immunofluorescence staining of NETs. ANETA were detected in 22%-69% of RA sera. No significant differences were observed in the reactivity of RA sera with NETs originating from RA patients and healthy control neutrophils, nor with NETs induced by phorbol 12-myristate 13-acetate or the calcium ionophore A23187. ANETA were detected already at baseline in newly diagnosed RA patients and both increased and decreased levels were observed in samples with a median follow-up of 7 years. By ANETA ELISA, we showed that ANETA are also present in sera of patients with systemic lupus erythematosus (36%), Sjögren's syndrome (76%) and scleroderma (61%). In addition to antibodies to NETs, also the presence of NETs or NET fragments in RA sera was determined using a sandwich ELISA. Elevated levels of NETs or NET fragments were detected in 32% of the sera. To assess the potency of ANETA as a biomarker in RA, we compared ANETA positivity with other clinical features. The presence of ANETA was significantly higher in rheumatoid factor (RF)-positive patients, but did not correlate with anti-citrullinated protein antibodies (ACPA), nor with the presence of NET fragments in serum. In addition, no correlation was observed with age, gender, onset of the disease, disease activity and inflammatory markers. These findings suggest that ANETA may be an independent biomarker in RA and possibly also in other autoimmune diseases.
中性粒细胞胞外诱捕网（NETs）是由中性粒细胞形成的一种网络结构，由细胞外染色质和抗微生物蛋白组成，用于捕获病原体。NETs 与自身免疫反应的产生有关。在这里，我们研究了类风湿关节炎（RA）血清抗体与 NETs 的反应性，并探讨了抗 NET 抗体（ANETA）是否有可能成为 RA 的生物标志物。为了定量检测 ANETA，我们开发了一种使用从刺激的人中性粒细胞中分离的 NETs 的 ELISA，并通过 NETs 的免疫荧光染色验证了结果。在 22%-69%的 RA 血清中检测到了 ANETA。RA 患者和健康对照组中性粒细胞来源的 NETs 以及佛波醇 12-肉豆蔻酸 13-乙酸酯或钙离子载体 A23187 诱导的 NETs 与 RA 血清的反应性没有显著差异。在新诊断的 RA 患者中，已经在基线时检测到了 ANETA，并且在中位随访 7 年的样本中观察到了升高和降低的水平。通过 ANETA ELISA，我们还发现 ANETA 也存在于系统性红斑狼疮（36%）、干燥综合征（76%）和硬皮病（61%）患者的血清中。除了针对 NETs 的抗体外，还使用夹心 ELISA 来确定 RA 血清中是否存在 NETs 或 NET 片段。在 32%的血清中检测到 NETs 或 NET 片段的水平升高。为了评估 ANETA 作为 RA 生物标志物的潜力，我们将 ANETA 阳性与其他临床特征进行了比较。在 RF 阳性患者中，ANETA 的存在显著更高，但与抗瓜氨酸蛋白抗体（ACPA）无关，也与血清中 NET 片段的存在无关。此外，与年龄、性别、发病年龄、疾病活动度和炎症标志物均无相关性。这些发现表明，ANETA 可能是 RA 的一个独立生物标志物，也可能是其他自身免疫性疾病的生物标志物。
