Abstract
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.
Highlights
Complement-dependent cytotoxicity (CDC) plays a central role in neuromyelitis optica (NMO), in which NMO autoantibodies (NMO-IgG) bind to AQP4 on astrocytes
Our results indicate that in contrast to CDC, Antibody-dependent cell-mediated cytotoxicity (ADCC) was observed in both M1-AQP4-expressing and M23-AQP4-expressing cells, whose efficiency was determined by NMO antibody binding but not by AQP4 OAP formation
Complement-dependent cytotoxicity in NMO depends on the level of AQP4 expression at the cell surface and its supramolecular assembly state, NMO antibody binding, and potentially other factors such as complement regulator proteins
Summary
Complement-dependent cytotoxicity (CDC) plays a central role in neuromyelitis optica (NMO), in which NMO autoantibodies (NMO-IgG) bind to AQP4 on astrocytes. 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. 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. We investigated the role of OAP assembly by AQP4 in NMO-IgG-dependent cell killing by complement and natural killer cells, testing the hypothesis that efficient CDC requires OAP formation by AQP4 but that ADCC does not. We found greatly increased CDC for OAP-assembled AQP4, establishing a second mechanism by which NMO pathology is influenced by AQP4 assembly, independent of NMO-IgG binding
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