Introduction
Most neuromyelitis optica (NMO) patients have IgG against aquaporin-4 (AQP4), here termed AQP4-IgG[
1,
2]. AQP4 is a water channel protein found in astrocytes throughout the central nervous system (CNS), especially in perivascular astrocyte foot processes[
3]. In cultured cells, AQP4-IgG binds extracellular conformational domains of AQP4 and activates complement, causing cell lysis[
4]. In mice, intracerebral injection of AQP4-IgG activates co-injected human complement (C
hu) and damages the astrocytes[
5,
6]. Inflammatory cells then enter the lesion causing further tissue injury including demyelination and axonal damage. In AQP4-IgG NMO patients, recovery after an attack is usually limited[
7‐
9].
A few NMO patients without AQP4-IgG have IgG against myelin oligodendrocyte glycoprotein (MOG-IgG), which recognize extracellular conformational domains of MOG[
10‐
13]. MOG is expressed on the outer surface of CNS myelin sheaths and comprises about 0.05% of total myelin protein[
14]. There is mounting evidence that MOG-IgG NMO has more favorable clinical outcome than AQP4-IgG NMO, with resolution of imaging abnormalities[
10,
11,
15,
16]. It is currently unclear whether MOG-IgG plays any role in NMO by causing lesions in the CNS
in vivo. Here we compared the effects of MOG-IgG with those of AQP4-IgG in the intracerebral injection mouse model. We used total IgG from a normal subject (IgG
CON) and from NMO patients with AQP4-IgG (IgG
AQP4) or MOG-IgG (IgG
MOG).
Materials and methods
IgG and complement
NMO patients with MOG-IgG or AQP4-IgG were identified using live cell-based assays. Briefly, AQP4-IgG and MOG-IgG positivity was determined by visualization of binding to human embryonic kidney cells, transfected with the extracellular and trans-membrane domains of MOG or with full-length M23-AQP4. Details of the assays are given elsewhere[
5,
10,
11,
15]. IgG was purified using Protein G from sera or plasmas of five patients with AQP4-IgG NMO, five MOG-IgG NMO or one healthy volunteer. The effect of injecting IgG and C
hu from healthy volunteers into mouse brain was extensively investigated in our earlier studies[
5,
17,
18]. The purified, dialysed and pooled total IgG preparations (6 – 38 mg/ml) are termed IgG
AQP4, IgG
MOG and IgG
CON. Clinical details of the 5 AQP4-IgG + [
5] and 5 MOG-IgG + [
11,
15] patients are given elsewhere. To deplete MOG-IgG, the IgG
MOG was adsorbed by incubation with MOG-HEK cells until MOG-IgG became undetectable (IgG
MOG(AdsMOG-HEK)). IgG
MOG adsorbed against untransfected HEK cells (IgG
MOG(AdsHEK)) was used as control. A chimeric mouse-human recombinant monoclonal anti-mouse MOG antibody, MOG-IgG
2B7, was produced as described[
19]. Human recombinant monoclonal anti-AQP4 IgG
1, termed AQP4-IgG
53, was also generated[
20]. A measles virus-specific antibody termed CON-IgG
2B4 was used as isotype control[
20]. The source of C
hu was fresh serum from healthy volunteers[
5].
Mice
Experiments were performed at St. George’s, University of London using CD1 mice 8 – 12 30 – 35 g, 8 – 12w old. Protocols were approved by the British Home Office (Project Licence, PPL 70/7081). After administering 2,2,2-tribromoethanol i.p., mice were mounted onto a stereotactic frame (Benchmark, Neurolab, St Louis, MO, USA). Four burrholes were made on the right side using a high speed drill (0.7 mm burr, Foredom, Bethel, CT, USA) at the following coordinates in millimetres from the bregma (lateral, anterior): (1, 0), (1, −1), (1, −2), (2, −1). Mice were allocated to the different experimental groups by a person unaware of the aim of the study. A 30 g needle attached to 50 ml gas-tight glass syringe (Hamilton, Reno, NV, USA) was inserted 3 mm deep to micro-infuse (1 μL/min) into the right hemisphere 16.8 μL IgG
MOG, IgG
AQP4 or IgG
CON or 16.8 μL (20 μg) MOG-IgG
2B7 or AQP4-IgG
53 + 11.2 μL C
hu (or normal saline) as described[
5]. Rectal temperature was kept 37 – 38°C with a heating lamp. After regaining the righting reflex, mice were returned to their cages, kept in 12 hour light/dark cycle and given water and normal chow
ad libitum. Mice (5 per group) were killed at 24 hours, seven days or two weeks. Investigators were unaware of which antibody was injected.
Mouse brain histology and immunohistology
Mice were anaesthetized and perfused-fixed by injecting 4% formaldehyde through the left cardiac ventricle. Brains were removed, post-fixed in 4% formaldehyde overnight and processed into paraffin. Coronal tissue sections (7 μm thick) through the injection tract were stained with H + E, Luxol Fast Blue (LFB)[
5] or immunostained.
For diaminobenzidine immunostaining, the sections were unmasked in citrate, incubated with primary antibody (one hour, 25°C), biotinylated secondary antibody (1:500, one hour, 25°C) and visualized using the Vectastain HRP kit (Vector Labs, Peterborough, UK). We counterstained nuclei with haematoxylin. Primary antibodies were rabbit anti-AQP4 (1:100), rabbit anti-glial fibrillary acidic protein (GFAP, 1:200), mouse anti-NeuN (1:200), (Millipore, Livingstone, UK), mouse anti-myelin basic protein (MBP, 1:400, Leica, Newcastle, UK), mouse anti-neurofilament-70 (1:600, DAKO, Ely, UK), rabbit anti-C5b-9 (1:100, Abcam, Cambridge, UK) and rat anti-CD45 (1:200, BD Bioscience, Oxford, UK). Samples were then incubated with the appropriate species biotinylated secondary antibody (1:500, Vector Laboratories). Immunostaining was visualized brown using the Vectastain horseradish peroxidase kit (Vector Laboratories) followed by diaminobenzidine/H2O2. Nuclei were counterstained blue with haematoxylin.
For immunofluorescence staining, we used rabbit anti-Ankyrin G (AnkG, InsightBio, Wembley, UK) or rabbit anti-Contactin associated protein (Caspr) from Abcam (1:200, 12 hours, 25°C) followed by Alexafluor-linked anti-rabbit antibody (1:200, one hour, 25°C, Invitrogen, Paisley, UK).
To determine MOG-IgG binding to mouse brain sections, brains were removed, immersed in 30% sucrose overnight, embedded in OCT and cut into 7 μm sections. These were fixed in acetone and exposed to IgGMOG(AdsHEK), IgGMOG(AdsMOG-HEK), IgGCON (1:100) or MOG-IgG2b7 (1 mg/mL) ± rabbit anti-MOG (1:100, InsightBio) for one hour at 25°C followed by Alexafluor-linked anti-human ± anti-rabbit IgG (1:200, one hour, 25°C, Invitrogen) and DAPI.
Photomicrographs were taken using an Olympus BX-51 microscope.
Data analysis
Coded photomicrographs were analysed with ImageJ (v1.45S, NIH). Neurofilament immunoreactivity in the injected hemisphere was quantified as mean staining intensity minus background. AnkG and Caspr expression was the number of fluorescent spots/mm2 in four photomicrographs, 90 μm × 67 μm, taken from the injected hemisphere 0.5 mm from the needle tract. After subtracting background, formatting images to 8-bit, adjusting threshold, the ‘analyse particles’ function of Image J was used. Spots < 0.01 μm2 were excluded as noise.
Statistics
Data are mean ± standard error. We used Student t-test or ANOVA with Student-Newman-Keuls post-hoc analysis. Significance is P < 0.05*, 0.01**, 0.001***.
Discussion
Although there is growing interest in the potential pathogenicity of MOG antibodies in NMO, the effects of NMO MOG-IgG have not been explored
in vivo. Our results indicate that MOG-IgG directly damages myelin. The detrimental effects of MOG-IgG markedly differ from those of AQP4-IgG and are reversible (see Table
1).
Table 1
Comparison of MOG-IgG with AQP4-IgG lesions in mouse brain
Target cell
| Oligodendrocyte (myelin) | Astrocyte (foot process) |
Lesion onset
| Within hours of exposure to MOG-IgG | Within hours of exposure to AQP4-IgG |
Effect on astrocytes
| No major effect (normal AQP4 and GFAP) | Astrocyte death (loss of AQP4 and GFAP) |
Effect on neurons
| No major effect | Neuronal death (loss of NeuN, FJ-C staining[ 5]) |
Effect on oligodendrocytes
| Change in myelin (loss of LFB) | Loss of myelin (loss of LFB) |
Effect on axons
| Myelin (transient change in MBP) | Permanent loss of myelin |
| Intact axons (normal nfil) | Axonal degeneration (β-APP expression[ 5]) |
| Node of Ranvier (transient change in casp and ankG) | |
Inflammatory cell infiltration
| No | Yes |
Complement activation
| Slight (in white matter tracts) | Marked (perivascular) |
| Not required for lesion to develop | Essential for lesion to develop |
Recovery
| Yes, within 2 weeks | No, pan-necrosis followed by glial scarring |
AQP4-IgG lesions are characterized by astrocyte damage followed by leukocyte infiltration that entirely depend on complement activation[
5]. We showed that recovery of myelin loss in AQP4-IgG lesions is limited, with gliosis and neuronal death. This finding may explain why clinical recovery after AQP4-IgG NMO attacks is often limited[
7‐
9]. By contrast, MOG-IgG, as examined here, damages myelin and axons temporarily, with little complement activation, and no leukocyte infiltration. The myelin and axonal recovery and lack of neuronal death mirror the reported good outcomes of MOG-IgG NMO patients[
10,
11,
15,
16].
One study suggested that IgG
MOG obtained from children with demyelination does not bind mouse MOG[
23], but another study showed that human MOG-IgG binds mouse MOG[
24]. Our IgG
MOG samples obtained from adult NMO patients, and the anti-mouse MOG-specific monoclonal antibody, both recognized mouse MOG in frozen brain sections, and produced comparable LFB loss without inflammation. This discrepancy may be due to differences in MOG-IgG levels and specificity or differences in MOG glycosylation state, which plays a key role in MOG-IgG binding[
24], between children and adults.
The effects of MOG-IgG on cultured oligodendrocytes have already been studied. MOG-IgG binds extracellular epitopes on MOG[
23] and can cause crosslinking[
25] and internalization[
12] of MOG molecules and reversible retraction of oligodendrocyte processes[
25]. At high concentration, MOG-IgG causes complement-mediated lysis of MOG-expressing cells[
12]. Passive transfer of MOG-IgG antibodies exacerbates CNS damage in experimental autoimmune encephalomyelitis rodent models in which cellular immunity is the predominant pathogenic mechanism[
26,
27]. Using the intracerebral injection mouse model, we have shown unequivocally that NMO MOG-IgG directly damages myelin
in vivo independent of pre-existing cellular immunity and complement.
MOG-IgG changed MBP architecture and reduced expression of axonal proteins. Caspr and AnkG are required for the integrity of the nodes of Ranvier and normal action potential firing[
21,
22]. Mice that lack MBP have a characteristic motor dysfunction including tremor and seizures[
28], mice that lack Caspr have severe motor paresis[
22] whereas mice lacking cerebellar ankG develop progressive ataxia[
21]. Therefore, the altered MBP expression and reduced Caspr and AnkG expression produced by MOG-IgG are predicted to produce a neurological deficit if the NMO lesion is in an eloquent region of the CNS. Unlike AQP4-IgG, MOG-IgG did not produce axonal disintegration or neuronal death. Given the 96% homology between mouse and human MOG[
14], our findings raise the possibility that MOG-IgG may also cause similar reversible lesions in the human CNS.
MOG-IgG has been reported in other non-NMO diseases including multiple sclerosis, acute disseminated encephalomyelitis and even some normal subjects[
29]. Does MOG-IgG from these non-NMO subjects also cause the same reversible CNS changes, as described here for NMO MOG-IgG? This question is difficult to answer at present because of the variety of assays used to detect MOG-IgG. For example, the assay used here, which employs C-terminal truncated rather than full-length MOG, did not detect MOG-IgG in adult multiple sclerosis patients and normal individuals[
11], which suggests that different assays detect different subpopulations of MOG-IgG. It is important to first standardize the assays before determining which subpopulations of MOG-IgG can cause CNS damage and in which diseases.
The mechanism of MOG-IgG-induced myelin damage in vivo is unknown. Our data show that MOG-IgG – mediated myelin damage is a direct effect of MOG-IgG and that complement activation is not necessary. MOG-IgG binding may cause MOG conformational changes or internalization that disrupts the myelin structure and secondarily alters axonal protein expression. To explain the lack of complement involvement, we hypothesize that, after MOG-IgG binding, MOG might not aggregate (because of its low abundance) or MOG might become internalized (thus prohibiting C1q activation). The full recovery within two weeks of the MOG-IgG-induced LFB, MBP, ankG and Caspr changes suggests that MOG-IgG does not kill the oligodendrocytes, but causes a reversible damage.
Our findings raise the possibility that MOG-IgG contributes to pathology in some NMO patients. If MOG-IgG is pathogenic, antibody depletion (plasmapheresis) or suppression with steroids should be effective, as indeed appears to be the case[
10,
11,
15,
16]. Conversely, some of the newly proposed therapies for AQP4-IgG NMO, such as sivelestat for inhibiting neutrophils[
17], or eculizumab for inhibiting complement[
30], are less likely to be needed in MOG-IgG NMO. Examining lesions from MOG-IgG NMO patients may help elucidate the pathogenicity of MOG-IgG in the human CNS.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SS designed and carried out the animal experiments, analyzed the data, produced the figures, contributed key ideas and helped draft the manuscript. PW prepared the IgG patient samples and contributed key ideas. GPO produced the recombinant MOG-IgG antibody. JLB produced the recombinant AQP4-IgG antibody and contributed key ideas. AV contributed key ideas and helped to draft the manuscript. MCP participated in the design and coordination of the study and wrote manuscript. All authors read and approved the final manuscript.