NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement

doi:10.1016/j.jns.2013.05.035

Journal of the Neurological Sciences

Volume 331, Issues 1–2, 15 August 2013, Pages 136-144

https://doi.org/10.1016/j.jns.2013.05.035 Get rights and content

Abstract

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for neuromyelitis optica (NMO). However, the molecular mechanism of NMO still remains unclear.

The purpose of this study was to identify the possible humoral mechanisms responsible for the occurrence of astrocytic damage. Human primary astrocytes (AST) were immortalized by retroviral vectors harboring temperature-sensitive SV40 T antigen gene and AQP4 cDNA (M23), designated as hAST-AQP4. The effects of NMO sera on the content and localization of AQP4, including cytotoxicity and astrocytic morphology, were evaluated. In addition, this study examined whether the amount and localization of AQP4 protein in astrocytes were influenced by direct contact with the immortalized human brain microvascular endothelial cell line, TY09. NMO sera alone induced cytotoxicity and addition of complement had a more harmful effect on hAST-AQP4. NMO sera also decreased AQP4 mRNA and protein. NMO sera alone up-regulated TNFα and IL-6 in astrocytes and co-incubation with anti-TNFα and anti-IL-6 neutralizing antibodies blocked both the cytotoxicity and reduction of AQP4 in astrocytes. In the experiment using the in vitro BBB models, AQP4 protein mainly localized at the astrocytic membrane after co-culture with TY09, in contact with TY09. The future elucidation of factors that up-regulate AQP4 in astrocytes presumably released by blood brain barrier forming endothelial cells and that block the production of inflammatory cytokines may therefore lead to the development of a novel therapeutic strategy.

Introduction

Neuromyelitis optica (NMO) is an inflammatory disorder of the central nervous system that affects mainly the optic nerves and the spinal cord [1]. A highly disease-specific autoantibody named NMO-IgG wasdiscovered in NMO [2] and its target antigen was identified as aquaporin-4 (AQP4) [3]. AQP4 exists as two different heterotetramers, M-1 and M-23 AQP4, which result from usage of different start codons [4], [5], [6]. Although AQP4 antibodies have been reported to alter the subcellular localization of AQP4 proteins or down-regulate AQP4 protein [7], [8], [9], [10], Ratelade et al. demonstrated that NMO-IgG brings little internalization to in vivo mouse brain and mouse primary astrocytes [11]. These studies used HEK 293 cells transfected with M1-AQP4 [7], [8] or rodent astrocytes [8], [9], [10], [11]. On the other hand, in vitro studies using human astrocytes, particularly cells dominantly expressing the M23 isoform, are rare. In addition, although there is only one report in which a model of co-culture of human astrocytes and endothelial cells was used in NMO research [12], no studies performing quantification of AQP4 using Western blotting (WB) with co-culture of conditionally-immortalized human brain microvascular endothelial cells (HBMECs) [13] and human astrocytic cell lines have been reported.

This study established a human astrocytic cell line expressing dominantly M23 protein. This line was used to determine whether NMO sera influence the amount of AQP4 and inflammatory cytokines. In addition, the effects of NMO sera on the morphology and viability of astrocytes and the subcellular localization of AQP4 were investigated. The influence of co-culture with HBMECs on the expression level and localization pattern of AQP4 protein in astrocytes was also evaluated using both immunofluorescence and WB. The results suggest a novel underlying mechanism which can provide a better understanding of the pathophysiology of NMO.

Section snippets

Antibodies and sera

Polyclonal rabbit anti-AQP4 (H 80), polyclonal rabbit anti-GFAP, polyclonal goat anti-excitatory amino acid transporter 2 (EAAT2), and polyclonal mouse anti-β-actin were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). The polyclonal anti-interleukin (IL)-6 and anti-TNFα were purchased from R&D systems (Minneapolis, Minnesota, USA). FITC-conjugated secondary antibodies were obtained from Invitrogen (CA, USA). HRP-conjugated secondary antibodies were purchased from Merck Millipore

Establishment of hAST-AQP4 cell lines

Human primary astrocytes and a conditionally-immortalized human astrocytic cell line, designated “hAST”, expressed AQP4 mRNA (Fig. 1A), but the immunocytochemical and WB analyses did not confirm AQP4 at the protein level (Fig. 1B, D). hAST was further transfected with retrovirus harboring AQP4 cDNA to establish a human astrocytic cell line expressing plenty amounts of AQP4 protein which is useful for the in vitro study of NMO. The obtained cell lines named hAST-AQP4 expressed plenty of AQP4

Discussion

AQP4 is present in two major isoforms, a longer (M1) isoform with translation initiation at Met-1 and a shorter (M23) isoform with translation initiation at Met-23 [4]. M23 AQP4 assembles in membranes as regular square arrays called orthogonal arrays of particles (OAPs) [6], [25]. M23 involves specific intermolecular interactions, while M1, which does not make up OAPs on its own, has residues upstream of M23 that block the N-terminus association [26]. Importantly, primary human astrocyte and

Conflict of interest

There is no conflict of interest.

Acknowledgments

This study was supported in part by JSPS KAKENHI Grant Number 23790994, and also by the Health and Labour Sciences Research Grant on Intractable Diseases (Neuroimmunological Diseases) from the Ministry of Health, Labour and Welfare of Japan.

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Dynamics of AQP4 upon exposure to seropositive patient serum before and after Rituximab therapy in Neuromyelitis Optica: A cell-based study
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Neuromyelitis Optica (NMO) is an autoimmune inflammatory disease that affects the optic nerves and spinal cord. The autoantibody is generated against the abundant water channel protein of the brain, Aquaporin 4 (AQP4). Of the two isoforms of AQP4, the shorter one (M23) often exists as a supramolecular assembly known as an orthogonal array of particles (OAPs). There have been debates about the fate of these AQP4 clusters upon binding to the antibody, the exact mechanism of its turnover, and the proteins associated with the process. Recently several clinical cases of NMO were reported delineating the effect of Rituximab (RTX) therapy. Extending these reports at the cell signaling level, we developed a glioma based cellular model that mimicked antibody binding and helped us track the subsequent events including a variation of AQP4 levels, alterations in cellular morphology, and the changes in downstream signaling cascades. Our results revealed the extent of perturbations in the signaling pathways related to stress involving ERK, JNK, and AKT1 together with markers for cell death. We could also decipher the possible routes of degradation of AQP4, post-exposure to antibody. We further investigated the effect of autoantibody on AQP4 transcriptional level and involvement of FOXO3a and miRNA-145 in the regulation of transcription. This study highlights the differential outcome at the cellular level when treated with the serum of the same patient pre and post RTX therapy and for the first time mechanistically describes the effect of RTX.
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Cell cultures. TY-10 cells, brain microvascular endothelial cells, and hAST, astrocytic cells, are adult human immortalized cell lines, transfected with plasmid expressing temperature sensitive Simian virus-40 large T-antigen (ts-SV40-LT) and the catalytic subunit of human telomerase, as previously described [11,12]. Both cell lines were developed at Yamaguchi University (Japan), in the labs of Dr. Sano and Kanda.
Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood–brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 μM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.
Affinity-matured ‘aquaporumab’ anti-aquaporin-4 antibody for therapy of seropositive neuromyelitis optica spectrum disorders
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Aquaporumab antibody therapy is unlikely to have significant toxicity based on the observation that NMO patients can have circulating AQP4-IgG autoantibodies for many years without clinical disease (Jarius and Wildemann, 2010; Jarius et al., 2014), and, except for a few case reports of NMO-related myositis (Guo et al., 2014; Malik et al., 2014), peripheral disease in AQP4-expressing tissues such as kidney, lung and stomach has not been seen. We note, however, that some cell culture studies report actions of AQP4-IgG on cytokine release that are independent of complement and cellular cytotoxicity mechanisms (Haruki et al., 2013; Howe et al., 2014), as well as AQP4-IgG cellular internalization (Hinson et al., 2012), though we have not seen AQP4-IgG internalization to toxicity in polarized astrocytes in vivo (Ratelade et al., 2011; Rossi et al., 2012). In conclusion, our study advances the development of an aquaporumab blocking antibody strategy for potential therapy of AQP4-IgG seropositive neuromyelitis optica spectrum disorder.
Pathogenesis in seropositive neuromyelitis optica spectrum disorders (herein called NMO) involves binding of IgG1 autoantibodies to aquaporin-4 (AQP4) on astrocytes in the central nervous system, which initiates complement and cellular injury. We previously developed an antibody blocking approach for potential therapy of NMO in which an engineered, monoclonal, anti-AQP4 antibody lacking cytotoxicity effector functions (called aquaporumab) blocked binding of NMO autoantibodies to astrocyte AQP4 (Tradtrantip et al. Ann. Neurol. 71, 314–322, 2012). Here, a high-affinity aquaporumab, which was generated by affinity maturation using saturation mutagenesis, was shown to block cellular injury caused by NMO patient sera. Anti-AQP4 antibody rAb-53, a fully human antibody with effector function neutralizing Fc mutations L234A/L235A and affinity-enhancing Fab mutations Y50R/S56R, called AQmab^AM, bound to AQP4 in cell cultures with K_d ~ 18 ng/ml (~0.12 nM), ~8-fold greater affinity than the original antibody. AQmab^AM, but without L234A/L235A Fc mutations, produced complement-dependent cytotoxicity (CDC) with EC₅₀ ~ 82 ng/ml. AQmab^AM prevented CDC produced by sera from eight NMO patients with IC₅₀ ranging from 40 to 80 ng/ml, and similarly prevented antibody-dependent cellular cytotoxicity (ADCC). Mechanistic studies demonstrated that AQmab^AM blocked binding of serum NMO autoantibodies to AQP4. AQmab^AM offers a targeted, non-immunosuppressive approach for therapy of seropositive NMO. Autoantibody blocking may be a useful therapeutic strategy for other autoimmune diseases as well.
Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica
2016, Biochemistry and Biophysics Reports
Citation Excerpt :
In addition, pure AQP4 loss without astrocyte damage has been reported in neuropathological studies of NMO [9–11]. Astrocyte lysis may also be mediated by either complement-dependent cytotoxicity (CDC) [12–17] or antibody-dependent cell-mediated cytotoxicity [18–20], and AQP4 loss without astrocyte lysis might be caused by AQP4-antibody alone (Supplement table 1). For CDC, “homologous restriction” cannot be ignored.
Neuromyelitis 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).
Purified 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.
AQP4-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.
Taken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.

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NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement

Abstract

Introduction

Section snippets

Antibodies and sera

Establishment of hAST-AQP4 cell lines

Discussion

Conflict of interest

Acknowledgments

Lancet Neurol

Lancet

J Biol Chem

J Neuroimmunol

J Clin Neurosci

Prog Neuropsychopharmacol Biol Psychiatry

Int Rev Cell Mol Biol

J Neuroimmunol

J Biol Chem

J Neuroimmunol

Mol Cell Neurosci

IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel

J Exp Med

Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance

Proc Natl Acad Sci U S A

Heterotetrameric composition of aquaporin-4 water channels

Biochemistry

Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms

Proc Natl Acad Sci U S A

Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica

Neurology

Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2

J Exp Med

Oligodendrocytes are damaged by neuromyelitis optica immunoglobulin G via astrocyte injury

Brain

Molecular outcomes of neuromyelitis optica (NMO)-IgG binding to aquaporin-4 in astrocytes

Proc Natl Acad Sci U S A