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.

中性粒细胞胞外陷阱 (NET) 是用抗菌蛋白装饰的胞外染色质网络，由中性粒细胞形成以捕获病原体。NETs 与自身免疫反应的产生有关。在这里，我们研究了类风湿性关节炎 (RA) 血清抗体与 NET 的反应性，并探讨了抗 NET 抗体 (ANETA) 是否具有作为 RA 生物标志物的潜力。为了量化 ANETA，我们使用从受刺激的人类中性粒细胞中分离的 NET 开发了 ELISA，并通过 NET 的免疫荧光染色验证了结果。在 22%–69% 的 RA 血清中检测到 ANETA。RA 血清与源自 RA 患者和健康对照中性粒细胞的 NET 的反应性以及与佛波醇 12-肉豆蔻酸酯 13-乙酸酯或钙离子载体 A23187 诱导的 NET 的反应性未观察到显着差异。ANETA 在新诊断的 RA 患者的基线中已经被检测到，并且在中位随访 7 年的样本中观察到 ANETA 水平升高和降低。通过 ANETA ELISA，我们发现系统性红斑狼疮 (36%)、干燥综合征 (76%) 和硬皮病 (61%) 患者的血清中也存在 ANETA。除了针对 NET 的抗体之外，还使用夹心 ELISA 确定了 RA 血清中 NET 或 NET 片段的存在。在 32% 的血清中检测到 NET 或 NET 片段的水平升高。为了评估 ANETA 作为 RA 生物标志物的效力，我们将 ANETA 阳性与其他临床特征进行了比较。ANETA 的存在在类风湿因子 (RF) 阳性患者中显着更高，但与抗瓜氨酸蛋白抗体 (ACPA) 无关，也没有血清中存在 NET 片段。此外，未观察到与年龄、性别、疾病发作、疾病活动性和炎症标志物的相关性。这些发现表明 ANETA 可能是 RA 和其他自身免疫性疾病的独立生物标志物。