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Aquaporin 4 antibody-mediated cytotoxicity in primary human astrocytes in culture: complement-dependent and -independent cellular changes

Background: Neuromyelitis optica (NMO) is characterized by severe optic neuritis and transverse myelitis. A disease-specific autoantibody against aquaporin (AQP) 4, mainly expressed in astrocytic foot processes, was found in the sera from patients with NMO. In pathological studies in NMO, there is an extensive loss of AQP4 and glial fibrillary acidic protein (GFAP). Previous in vitro studies showed AQP4 antibodies activates complements and they are cytotoxic against astrocytes. However, it is unknown whether AQP4 antibodies alone cause significant alterations on human astrocytes. Objective: To monitor time-dependent changes of primary human astrocytes in culture treated with AQP4 antibody. Methods: Purified IgG (10mg/ml) were obtained from 3 patients with AQP4 antibody-positive NMO, 3 with multiple sclerosis (MS), and 3 healthy controls. Primary human astrocytes were incubated with the IgG (5% volume) for 30 minutes. After removing unbound IgG, the cells were further cultured with rabbit complements (20%) with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes with a fluorescence microscope. We also observed the cytotoxicity by propidium iodide (PI) system and LDH assay. Result: AQP4-IgG alone caused the clustering and then endocytosis of the membranous AQP4. In accordance with these changes, astrocytes remarkably shrank and lost adherence (34.5?7.8%) after 15 minutes ~ 2 hours, but the morphological changes was reversible by removing the IgG. Soon after AQP4-IgG and non-heated complements were added, the cell bodies and the nuclei started to swell. In 60 minutes, many astrocytes lost mobility and adherence property, and floating balloon-like cells and cell debrisincreased in number as time went by. Remaining adherent cells treated with both AQP4-IgG and complements were mostly positive for PI (84.8?4.8 %), which is significantly higher than those with AQP4-IgG or complement alone, or non-treated cells. LDH assays confirmed these results. AQP4 antibodynegative IgG in MS or control did not induce the above-described changes. Conclusion: Our results showed AQP4 antibodies by themselves can cause reversible AQP4 downregulation and loss of adherence of human astrocytes which probably alter the cell function significantly, but by adding complements, the antibodies irreversibly damage the cells in a short time, suggesting a unique AQP4 antibody-mediated astrocytopathy in NMO.

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