Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica

Abstract

Background: Antibodies targeting membrane proteins play an important role in various autoimmune diseases of the nervous system. So far, assays allowing proper analysis of such autoantibodies are largely missing. A serum autoantibody to aquaporin 4 (AQP4) is associated with neuromyelitis optica (NMO). Although several assays are able to detect this autoantibody, they do not allow determination of the biological activity of anti-AQP4 antibodies.Objective: To develop a bioassay for quantification and characterization of human anti-AQP4 antibodies.Design, Setting, and Participants: We developed a novel bioassay for quantification and characterization of human anti-AQP4 antibodies based on high-level expression of native AQP4 (nAQP4) protein on the surface of human astroglioma cells. The test was validated in 2 independent cohorts of patients with NMO spectrum disease.Results: We detected anti-nAQP4-IgG with a sensitivity of 57.9% and specificity of 100% in patients with NMO spectrum diseases, suggesting that our bioassay is at least as sensitive and specific as the gold-standard NMO-IgG assay. The anti-AQP4 antibodies belonged predominantly to the IgG1 isotype and bound with high affinity to the extracellular domain of nAQP4. Our data suggest that the autoantibody exerts pathological properties because nAQP4-IgGpositive sera induced cell death of nAQP4-expressing cells by antibody-dependent cellular natural killer cell cytotoxic effect and complement activation. Furthermore, nAQP4-IgG titers strongly correlated with in vitro cytotoxic effect. Conclusions:In NMO, this assay may help to unravel the biological function of anti-nAQP4-IgG. Our findings demonstrate the potential of bioassays to characterize biologically relevant antibodies in human autoimmune diseases.

Complement-dependent Cytotoxicity in Neuromyelitis Optica Requires Aquaporin-4 Protein Assembly in Orthogonal Arrays

“…In contrast to CDC, interaction of effector cells with NMO antibody in ADCC does not involve multivalent interaction (38) and so does not require AQP4 clustering in OAPs. It is generally believed that CDC is more important than ADCC in NMO disease pathogenesis (1,39,40); however, direct evidence remains lacking. Involvement of ADCC in NMO would have important implications for NMO therapeutics targeting AQP4 supramolecular assembly.…”

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

“…Mutations in loop A, or loop C, combined with loop E, reduce NMO-IgG binding, suggesting a key role of OAP-specific interactions at the level of extracellular loops (10). It has been reported that the interaction between NMO-IgG and AQP4-OAPs induces pathogenic mechanisms in NMO by complement-dependent cytotoxicity (CDC), and/or by antibody-dependent cellular cytotoxicity (11)(12)(13)(14)(15).…”

Acute inflammatory myelopathies

“…These elegant experiments conclusively proved that the antigen recognized by the NMO-IgG antibody was AQP4. Both the immunohistochemical and the AQP4-transfected HEK cell assays for detecting anti-AQP4 antibody from patient sera were subsequently validated by several groups (Takahashi et al, 2006;Paul et al, 2007;Marignier et al, 2008;Kalluri et al, 2010). These studies definitively prove that the NMO-IgG is an anti-AQP4 antibody.…”