Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica

Abstract

BackgroundNeuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA).MethodsPurified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay.ResultAQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes.ConclusionTaken together...

C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder

“…The fluorescence intensity analysis showed that only cells with high deposition of C5b-9 (high signal in ICC analysis) had decreased expression of GFAP (10% of all astrocytes), whereas the rest of astrocytes were characterized by low C5b-9 deposition and normal GFAP expression, comparable to astrocytes incubated with HC or RRMS serum (Figure 3 C, green/red arrows). These findings showed that the majority astrocytes are resistant to AQP4-C hu -induced destruction, probably due to their membrane expression of complement inhibitory protein CD59 ( 8 , 27 ), or sublytic C5b-9 concentration ( 28 ), suggesting that another parallel mechanism of astrocyte destruction in NMOSD inflammation should exist.…”

“…However, only a small subset (~10–11%) of cultured astrocytes was lysed and demonstrated GFAP loss as a result of C5b-9 formation, even though all astrocytes were opsonized by IgG1. This might be a result of membrane expression of complement regulatory proteins on astrocyte surface ( 27 , 28 ) or sublytic concentration of C5b-9 ( 28 ). Postmortem histological analysis revealed that in normal human brain, opposite to other organs, astrocytes are devoid of CD46 (membrane cofactor protein), CD55 (decay accelerating factor), or CD59 (protectin) that inhibit activation of the C hu cascade ( 8 ).…”

Alterations in the Brain Structure and Functional Connectivity in Aquaporin-4 Antibody-Positive Neuromyelitis Optica Spectrum Disorder

“…AQP-4 antibody has been reported to be expressed in the brain (Badaut et al, 2007); it is the most abundant water channel in the brain (Rash et al, 1998) and controls brain water homeostasis (Nielsen et al, 1997). Amassing reports (Misu et al, 2013;Nishiyama et al, 2016;Yick et al, 2018) have shown that AQP-4 can induce complement independent pathologies. Experimental reports have shown that inflammatory sequela follows the binding of NMO-IgG to AQP-4 (Hinson et al, 2012;Pittock et al, 2013).…”

Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor

“…Beside the higher sensitivity to tissue microstructure of the sADC over the ADC, such discrepancy could possibly result from the modified astrocyte phenotype associated with a long-term inhibition of AQP4 expression found in those previous studies [46]. The sub-acute or chronic inhibition of AQP4 activity by AQP4-antibodies or small interfering RNA duplexes alter astrocyte morphology and decrease water permeability [47,48] which could result in an ADC decrease. Also, the effects we observed in baseline conditions should be distinguished from those obtained in conditions of neuronal activation for which AQP4 inhibition by extracellular acidification results in astrocyte swelling, capillary lumen expansion and Virchow-Robin space reduction [49].…”