Membrane assembly of aquaporin-4 autoantibodies regulates classical complement activation in neuromyelitis optica
John Soltys, … , Gregory P. Owens, Jeffrey L. Bennett
John Soltys, … , Gregory P. Owens, Jeffrey L. Bennett
Published April 8, 2019
Citation Information: J Clin Invest. 2019. https://doi.org/10.1172/JCI122942.
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Categories: Research Article Inflammation Neuroscience

Membrane assembly of aquaporin-4 autoantibodies regulates classical complement activation in neuromyelitis optica

Abstract

Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG–driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4–specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody–mediated disorders and identifies a potential new therapeutic avenue for treating NMO.

Authors

John Soltys, Yiting Liu, Alanna Ritchie, Scott Wemlinger, Kristin Schaller, Hannah Schumann, Gregory P. Owens, Jeffrey L. Bennett

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Figure 1

AQP4 rAb binding and classical complement activation on M23-AQP4 CHO cells.

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AQP4 rAb binding and classical complement activation on M23-AQP4 CHO cel...
(A) CDC of M23-AQP4 CHO cells induced by AQP4 rAb ON 07-5 no. 58 or isotype control rAb ON 07-5 no. 132 was quantified using an lactate dehydrogenase (LDH) release assay (mean ± SEM; n = 4). Complete or depleted preparations of NHS were used as sources of complement proteins. Recombinant complement C1q (rC1q) was added where indicated. The dashed line indicates 50% lysis. (B) CDC induced by monoclonal AQP4 rAbs with differing epitope specificities measured using an LDH release assay (mean ± SEM; n = 4). The dashed line indicates 50% lysis. (C) The ratio of bound C1q to AQP4 is plotted against the rAb concentration. Solid circles indicate the CDC EC50 for complement activation. (D) The ratio of bound rAb to cell-surface AQP4 (rAb/AQP4) is plotted against the rAb concentration. Solid circles indicate the EC50 for CDC. (E) Lateral and top views of an AQP4 tetramer. Extracellular loops A, C, and E are colored red, green, and blue, respectively. Loop C amino acids His151 and Leu154 are colored purple; membrane-spanning and intracellular amino acids are colored gray. (F and G) Binding of AQP4 rAbs to U87MG cells expressing M23-AQP4 (solid line) or M23-AQP4H151A/L154A is shown as the ratio of bound rAb to cell-surface AQP4 (rAb/AQP4) (mean ± SEM; n = 3). Data were fitted using a single-site total binding model. hC, human complement.