Department of Medicine and Physiology, University of California, San Francisco, California 94143, USA.
J Biol Chem. 2012 Apr 20;287(17):13829-39. doi: 10.1074/jbc.M112.344325. Epub 2012 Mar 5.
Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.
视神经脊髓炎（NMO）是一种中枢神经系统的炎症性脱髓鞘疾病，其中致病性自身抗体（NMO-IgG）与星形胶质细胞水通道蛋白-4（AQP4）结合导致补体依赖性细胞毒性（CDC）和炎症。我们之前报道了 NMO-IgG 与四聚体中 AQP4 相对于膜内聚集物（正交排列的颗粒，OAPs）的结合亲和力范围很广。我们在这里报告了第二个独立的机制，即 AQP4 组装影响 CDC。利用乳酸脱氢酶释放和活/死细胞细胞毒性测定，我们在不同的细胞系中发现，并且使用不同的单克隆和患者衍生的 NMO-IgG，与表达 M1-AQP4 的细胞相比，CDC 在表达 M1-与 M23-AQP4 的细胞中大大（>100 倍）降低。使用含有 OAP 破坏突变的 M23-AQP4 突变体以及在不同 M1/M23 比值下表达 AQP4 的细胞进行的研究表明，NMO-IgG 依赖性 CDC 需要 AQP4 OAP 组装。相比之下，自然杀伤细胞产生的抗体依赖性细胞介导的细胞毒性不依赖于 AQP4 OAP 组装。C1q 结合和补体攻击复合物（C9neo）的测量结果支持了这样的结论，即 OAP 大大增强的 CDC 是由于 C1q 与 OAP 上聚集的 NMO-IgG 有效、多价结合。我们得出结论，AQP4 在 OAPs 中的组装是 NMO 中 CDC 的必要条件，建立了 OAP 依赖性 NMO 发病机制的新机制。AQP4 OAPs 的破坏可能会大大降低 NMO-IgG 依赖性 CDC 和 NMO 病理学。
