Cytotoxic effect of neuromyelitis optica antibody (NMO-IgG) to astrocytes: An in vitro study

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Abstract

NMO-IgG is a disease-specific autoantibody for neuromyelitis optica (NMO) that binds selectively to aquaporin-4 (AQ4), an astrocytic water channel. The normal distribution of AQP4 coincides with the sites of immunoglobulin and complement deposits in lesions found in autopsy studies. The underlying mechanisms of cytotoxicity by NMO-IgG on astrocytes are not well known. In this study we show that serum samples from seropositive NMO patients (21) induce a higher rate of cell death compared with sera from seronegative NMO (16), relapsing–remitting MS (20) patients, and healthy controls (24) on primary cultures of astrocytes. Similar results were obtained by two different techniques: lactate dehydrogenase release and tetrazolium-based viability assay. Cell death was only observed in the presence of active complement. The complement-dependent cytotoxicity was not accompanied by caspase-3/7 activation or increase in the percentage of apoptotic cells. Our data show that NMO-IgG induces a complement-dependent cytotoxicity of astrocytes in vitro, and suggest that a mechanism of cellular death by necrosis might be implicated in the pathophysiology of NMO.

Introduction

Neuromyelitis optica (NMO) is a severe inflammatory demyelinating disease that predominantly affects the optic nerve and spinal cord. The finding of a highly specific serum antibody marker (NMO-IgG) (Lennon et al., 2004) has expanded the clinical spectrum of NMO (Lennon et al., 2004, Weinshenker et al., 2006, Matiello et al., 2008), defined a new set of diagnostic criteria (Wingerchuk et al., 2006, Saiz et al., 2007), and helped to distinguish NMO from relapsing–remitting multiple sclerosis (RRMS) (Lennon et al., 2004, Wingerchuk et al., 2006, Saiz et al., 2007, Cabrera-Gómez et al., 2008, Misu et al., 2007, Roemer et al., 2007). NMO-IgG binds selectively to aquaporin-4 (AQP4), the main water channel in the CNS (Lennon et al., 2005). AQP4 is mainly expressed in the plasma membrane of astocytic endfeet associated with microvessels, pia, subpia and Virchow–Robin spaces, the sites of immunoglobulin and complement deposits and loss of AQP4 immunoreactivity in the autopsy studies (Lucchinetti et al., 2002, Lennon et al., 2005, Misu et al., 2007, Roemer et al., 2007).

The pathogenic role of NMO-IgG is supported by in vitro studies using AQP4-transfected cells lines that showed the binding of the antibody to the extracellular domain of AQP4 and the compromise of the membrane integrity in the presence of active complement (Hinson et al., 2007). However, this complement-mediated cytotoxicity by NMO-IgG in the absence of cellular effectors has only been confirmed in one of the two recent studies that analyzed the effect of NMO-IgG on cultures of primary astrocytes, the in vivo target cell (Hinson et al., 2008, Vincent et al., 2008).

In the current study we demonstrate on primary culture of astrocytes that serum samples from seropositive NMO patients induce a significant increase of lactate dehydrogenase release, indicator of cell death associated with loss of plasma membrane integrity, as compared with samples from seronegative NMO and RRMS patients. This cell death depends on the presence of active complement and is not associated with apoptosis.

Section snippets

Patients

The study included serum of 21 patients with NMO-IgG detected by immunohistochemistry as previously described (Saiz et al., 2007) (17 NMO, 3 recurrent longitudinally extensive transverse myelitis [RLETM] and 1 recurrent optic neuritis). The mean age was 34.5 years (range 14–58 years), 91% were female, and the disease duration was 8.3 years (range, months–33 years). Sixteen were seronegative NMO patients (Saiz et al., 2007) (13 NMO and 3 RLETM). The mean age was 36 years (range 17–51 years), 73% were

Immunofluorescence staining on primary astrocytes

Serum from NMO-IgG positive patients, but not from seronegative patients and HC, bound to astrocytes in a pattern that colocalized with that of rabbit IgG anti-AQP4 when the cells were permeabilized (Fig. 1A). In contrast, in non-permeabilized live astrocytes only seropositive NMO-IgG samples produced immunolabeling of the cell membrane in a punctate pattern whereas rabbit IgG anti-AQP4 did not bind to the cell surface because it is specific for AQP4 cytoplasmic epitopes (Fig. 1B). These

Discussion

There is increasing clinical, immunological and pathological evidence supporting a pathogenic role of NMO-IgG in neuromyelitis optica and limited forms of the disease (Lennon et al., 2004, Jarius et al., 2008, Waters et al., 2008, Lennon et al., 2005, Hinson et al., 2007, Hinson et al., 2008, Vincent et al., 2008, Misu et al., 2007, Roemer et al., 2007). Our findings of cytotoxicity dependent on the presence of both the in vivo target cell and active complement reinforce the idea that NMO-IgG

Acknowledgements

This work was supported in part by grant PI060070, Fondo de Investigaciones Sanitarias, Madrid, Spain (AS) and the Instituto de Salud Carlos III (RETICS: Red Española de Esclerosis Múltiple RD07/0060/0012; FG).

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