■神经精神系统性红斑狼疮(NPSLE)的发病机制尚不清楚,自身抗体的作用仍未确定。
■为了鉴定可能与NPSLE相关的脑反应性自身抗体,对大鼠和人脑进行了免疫荧光(IF)和透射电子显微镜(TEM)。ELISA用于揭示已知循环自身抗体的存在,而Westernblot(WB)用于表征潜在的未知自身抗原。
■我们注册了209个科目,包括SLE患者(n=69),NPSLE(n=36),多发性硬化症(MS,n=22),和82个年龄和性别匹配的健康捐献者(HD)。在几乎整个大鼠大脑中观察到IF的自身抗体反应性(皮质,海马体,和小脑)使用NPSLE和SLE患者的血清,在MS和HD中几乎为阴性。NPSLE的患病率较高(OR2.4;p=0.047),强度,脑反应性自身抗体的滴度高于SLE患者。大多数具有脑反应性自身抗体的患者血清(75%)也对人脑染色。将患者血清与针对神经元(NeuN)或神经胶质标记物的抗体混合的大鼠大脑的双重染色实验显示,自身抗体反应性仅限于含NeuN的神经元。使用TEM,脑反应性自身抗体的靶标位于细胞核中,在较小程度上,在细胞质和线粒体中。鉴于NeuN和脑反应性自身抗体之间的高度共定位,我们假设NeuN是一种可能的自身抗原.然而,使用表达或不表达编码NeuN蛋白(RIBFOX3)的基因的HEK293T细胞裂解物的WB分析显示,携带脑反应性自身抗体的患者血清不识别NeuN相应的条带大小。在NPSLE相关的自身抗体组中(例如,抗NR2,抗P-核糖体蛋白,抗磷脂)通过ELISA测定法进行研究,在那些含有脑反应性自身抗体的血清中只发现抗β2-糖蛋白-I(aβ2GPI)IgG。
■总而言之,SLE和NPSLE患者具有脑反应性自身抗体,但在NPSLE患者中发现的频率和滴度较高。尽管脑反应性自身抗体的许多靶抗原仍未确定,它们可能包括β2GPI。
The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined.
To identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s).
We enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients\' sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients\' sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-β2-glycoprotein-I (aβ2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies.
In conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include β2GPI.