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Changes of aquaporin4 antibody-treated primary human astrocytes in culture: a time-lapse microscopic analysis

Background: Neuromyelitis optica (NMO) is characterized by severe optic neuritis and transverse myelitis. Aquaporin-4 (AQP4) antibody is a diagnostic biomarker of NMO. Pathological studies revealed an extensive loss of immunoreactivities to AQP4 and glial fibrillary acidic protein (GFAP), both of which are astrocytic proteins, in the perivascular regions with complement and immunoglobulin depositions in NMO. We also showed a marked increase of GFAP in the cerebrospinal fluids in relapse of NMO. These findings suggest complement-mediated cytotoxicity to astrocytes by AQP4 antibody. A few in-vitro studies with rodent astrocytes or AQP4-transfected HEK293 cells basically supported the theory, but time-lapse microscopic studies of the effects of AQP4 antibody on human astrocytes are lacking. 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, and 3 healthy controls. Confluent 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 changes with a fluorescence microscope, and immunocytochemically analyzed the expression of AQP4, C5b-9, IgG, and annexin V/propidium iodide (AV/PI). GFAP levels in the supernatants were measured by a commercially available ELISA. Result: AQP4 antibody-positive IgG by themselves caused shrinkage of the astrocytic processes from 15 minutes later, but the change was partially reversible by removing the IgG. Following the application of non-heated complements, the cellular AQP4 immunoreactivity was lost, and the cell bodies and the nuclei started to swell soon. In a few hours, many astrocytes lost mobility and adherence property, and floating balloon-like cells and cell debris increased in number as time went by. Remaining adherent cells were often positive for AV/PI. GFAP levels in the supernatants were elevated. AQP4 antibody-negative IgG did not induce the above-described changes. Conclusion: Our results showed that AQP4 antibodies by themselves can cause some morphological changes of human astrocytes, but that with complements the antibodies irreversibly damage the cells in a short time, suggesting AQP4 antibody-mediated astrocytopathy in NMO.

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