Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Apr 20;287(17):13829-39.
doi: 10.1074/jbc.M112.344325. Epub 2012 Mar 5.

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Puay-Wah Phuan  1 , Julien Ratelade, Andrea Rossi, Lukmanee Tradtrantip, A S Verkman
Affiliations

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Puay-Wah Phuan et al. J Biol Chem. .

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Characterization of M1- and M23-AQP4-expressing CHO-K1 and U87MG cells. A, confocal fluorescence microscopy (top) and TIRFM (bottom) of indicated cells immunostained for AQP4 using a C terminus anti-AQP4 antibody. B, SDS-PAGE (top) and Blue Native-PAGE (BN-PAGE, bottom) of cell homogenates. C, left, surface AQP4 expression quantified in live cells by immunostaining with 50 μg/ml rAb-58 for 1 h at 4 °C. Plasma membranes were stained with wheat germ agglutinin (WGA). Right, data are reported as the ratio of red (surface AQP4) to green (wheat germ agglutinin) fluorescence. (S.E., n = 5, *, p < 0.01).
FIGURE 2.
FIGURE 2.
Cells expressing M1-AQP4 are resistant to NMO-rAb/complement-mediated cytotoxicity. A, LDH cytotoxicity assay showing LDH release from M1- and M23-AQP4-expressing CHO-K1 cells treated for 60 min at 37 °C with rAb-53 or rAb-58 (20 μg/ml) and/or complement (2%). Total LDH content determined by treatment with Triton X-100 (S.E., n = 4, *, p < 0.01). B, dependence of CDC on rAb concentration in CHO-K1 (left) and U87MG (right) cells in the presence of 2% complement (S.E., n = 4). Cells were incubated for 60 min with NMO-rAb and complement at 23 or 37 °C as indicated. Data were fitted to a single-site saturation model. C, dependence of CDC on complement concentration (left) and incubation time (right) in CHO-K1 and U87MG cells in the presence of 2.5 and 5 μg/ml rAb-58, respectively (S.E., n = 4). D, representative fluorescence micrographs showing live/dead (green/red) cell staining after a 60-min incubation with control or NMO rAbs and complement.
FIGURE 3.
FIGURE 3.
Differences in NMO-rAb binding or AQP4 surface expression cannot account for low CDC for M1-AQP4. A, top, representative fluorescence micrographs showing NMO-rAb (green) and AQP4 (red) immunofluorescence at 100 μg/ml rAb-53 and rAb-58 in M1- and M23-AQP4-expressing CHO cells. Bottom, binding curves for rAb-53 (left) and rAb-58 (right) to M1- versus M23-AQP4 (S.E., n = 3) showing green-to-red fluorescence ratios as a function of NMO-rAb concentration. Data were fitted to a single-site saturation model with fitted KD = 44 nm and 2.6 μm (rAb-53) and 83 nm and 202 nm (rAb-58), for M23- and M1-AQP4, respectively. B, cell surface AQP4 expression at 60 min after incubation with 50 μg/ml rAb-58. Data are shown as the percentage of remaining AQP4 at the cell surface at 60 versus 0 min (S.E., n = 5, *, p < 0.01).
FIGURE 4.
FIGURE 4.
Cells expressing M1-AQP4 are resistant to CDC caused by NMO-IgG in human NMO sera. Total IgG was isolated from human NMO (and control) sera. A, dependence of cytotoxicity on total IgG concentration in the presence of 2% complement (S.E., n = 4). M1- and M23-AQP4-expressing CHO-K1 cells were incubated for 60 min with NMO-IgG and complement at 23 or 37 °C. B, representative fluorescence micrographs showing live/dead (green/red) cell staining after a 60-min exposure of cells to complement and control or NMO-IgGs (each 100 μg/ml). C, binding of NMO-IgG (each 100 μg/ml, green) to AQP4 (red) in U87MG cells expressing M1- or M23-AQP4. D, surface AQP4 expression in CHO-K1 cells at 60 min after incubation with 250 μg/ml NMO-IgG from NMO sera. Data are shown as the percentage of remaining AQP4 at the cell surface at 60 versus 0 min (S.E., n = 5, *, p < 0.01).
FIGURE 5.
FIGURE 5.
AQP4 assembly in OAPs is required for CDC. U87MG cells were transiently transfected with M1- or M23-AQP4, G28P-M23-AQP4 (OAP-disrupting mutation), or the indicated mixtures of M1- and M23-AQP4. A, TIRFM of transfected cells immunostained with anti-AQP4 antibody. B, CDC assay on transfected cells (S.E., n = 4, *, p < 0.001). C, fluorescence micrographs of transfected cells stained with 20 μg/ml rAb-58 (green) and anti-AQP4 antibody (red). Values for relative AQP4 cell surface expression are shown, normalized to that of M1-AQP4 (S.E., n = 3).
FIGURE 6.
FIGURE 6.
ADCC does not depend on AQP4 OAP formation. A, live/dead (red/green) staining of M1- and M23-AQP4-expressing CHO-K1 cells incubated with NK cells and 10 or 100 μg/ml control rAb, rAb-53, or rAb-58. B, the percentage of dead cells (red/(red + green)) (S.E., n = 4–6, *, p < 0.01).
FIGURE 7.
FIGURE 7.
Greatly reduced C1q binding to NMO-IgG accounts for resistance of M1-AQP4-expressing cells to CDC. A, left, staining of M1- and M23-AQP4 in CHO-K1 cells with 40 μg/ml NMO rAb-58. Center panels, C1q staining. Cells were incubated with 40 μg/ml rAb-58 (or control rAb) and 120 μg/ml purified recombinant C1q for 30 min, fixed, and stained with FITC-conjugated anti-C1q antibody. AQP4 was stained red with anti-AQP4 antibody. Right, green-to-red fluorescence ratio (S.E., n = 4, *, p < 0.001). B, C9neo immunostaining. Left, cells were incubated with 20 μg/ml rAb-53 or rAb-58 and 2% complement for 30 min, fixed, and stained with anti-C9neo antibody and green fluorescent secondary antibody. AQP4 was stained red. Right, green-to-red fluorescence ratio (S.E., n = 4, *, p < 0.001).
FIGURE 8.
FIGURE 8.
Schematic showing multivalent binding of C1q to Fc regions of NMO-IgG bound to OAP-assembled AQP4. Side view (top) and en-face view (bottom) are shown. Proteins in each view shown on the same size scales as indicated.
See this image and copyright information in PMC

References

    1. Wingerchuk D. M., Lennon V. A., Lucchinetti C. F., Pittock S. J., Weinshenker B. G. (2007) The spectrum of neuromyelitis optica. Lancet Neurol. 6, 805–815 - PubMed
    1. Jarius S., Paul F., Franciotta D., Waters P., Zipp F., Hohlfeld R., Vincent A., Wildemann B. (2008) Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat. Clin. Pract. Neurol. 4, 202–214 - PubMed
    1. Lucchinetti C. F., Mandler R. N., McGavern D., Bruck W., Gleich G., Ransohoff R. M., Trebst C., Weinshenker B., Wingerchuk D., Parisi J. E., Lassmann H. (2002) A role for humoral mechanisms in the pathogenesis of Devic's neuromyelitis optica. Brain 125, 1450–1461 - PMC - PubMed
    1. Misu T., Fujihara K., Kakita A., Konno H., Nakamura M., Watanabe S., Takahashi T., Nakashima I., Takahashi H., Itoyama Y. (2007) Loss of aquaporin-4 in lesions of neuromyelitis optica: distinction from multiple sclerosis. Brain 130, 1224–1234 - PubMed
    1. Lennon V. A., Wingerchuk D. M., Kryzer T. J., Pittock S. J., Lucchinetti C. F., Fujihara K., Nakashima I., Weinshenker B. G. (2004) A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 364, 2106–2112 - PubMed

Publication types

LinkOut - more resources