Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan.
Department of Neurology and Strokology, Nagasaki University Hospital, Nagasaki, Japan.
Brain. 2019 Aug 1;142(8):2253-2264. doi: 10.1093/brain/awz168.
Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1β and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.
Lambert-Eaton肌无力综合征 (LEMS) 是一种由自身抗体结合 P/Q 型电压门控钙通道引起的神经肌肉接头自身免疫性疾病。血脑屏障的破坏和小脑颗粒/Purkinje 细胞反应性自身抗体扩散到中枢神经系统对于伴 Lambert-Eaton 肌无力综合征的副肿瘤性小脑变性 (PCD) 的发病机制至关重要。我们最近发现证据表明，视神经脊髓炎患者血浆中的葡萄糖调节蛋白 78 (GRP78) 自身抗体促进 AQP4 自身抗体进入中枢神经系统。在本研究中，我们研究了 PCD-LEMS IgG 中的 GRP78 自身抗体是否会增强穿过血脑屏障的小脑细胞反应性抗体的脑摄取并促进小脑功能障碍。我们首先使用核因子 κB p65 和细胞间黏附分子 1 (ICAM1) 免疫染色的高内涵成像系统评估来自 PCD-LEMS 或 PCD 患者的纯化 IgG 对人脑微血管内皮细胞血脑屏障功能的影响。接下来，我们通过共免疫沉淀和基于活细胞的抗体结合测定鉴定导致 PCD-LEMS IgG 血脑屏障通透性的 GRP78 自身抗体。将脑微血管内皮细胞暴露于来自 PCD-LEMS 患者的 IgG 会诱导核因子 κB p65 核转位、ICAM1 上调、claudin-5 表达减少、通透性增加以及自分泌 IL-1β 和 IL-8 分泌增加；来自 Lambert-Eaton 肌无力综合征患者的 IgG 则没有这些影响。我们在 LEMS-PCD 患者的 IgG 中检测到 GRP78 自身抗体（83.3%，n=18），但在 LEMS 患者中（6.6%，n=15）检测到的较少，在对照组（n=8）中则没有检测到。GRP78 自身抗体的耗竭降低了 LEMS-PCD IgG 对脑微血管内皮细胞的生物学作用。这些发现表明，GRP78 自身抗体的作用超出了视神经脊髓炎的范围，并且它们对 PCD-LEMS 和 LEMS 之间表型差异具有直接影响。
