doi:10.1093/brain/awm027
Brain (2007), 130, 1206 ^1223
Heterogeneity of aquaporin-4 autoimmunity and
spinal cord lesions in multiple sclerosis in Japanese
Takeshi Matsuoka,1 Takuya Matsushita,1 Yuji Kawano,1 Manabu Osoegawa,1 Hirofumi Ochi,1 Takaaki Ishizu,1
Motozumi Minohara,1 Hitoshi Kikuchi,1 Futoshi Mihara,2 Yasumasa Ohyagi1 and Jun-ichi Kira1
1
Department of Neurology, Neurological Institute and 2Division of Neuroradiology, Department of Radiology, Graduate
School of Medical Sciences, Kyushu University, Fukuoka, Japan
Correspondence to: Professor J. Kira, Department of Neurology, Neurological Institute, Graduate School of Medical Sciences,
Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812- 8582, Japan
E-mail: [email protected]
Opticospinal multiple sclerosis (OSMS) in Asians has similar features to the relapsing^remitting form of
neuromyelitis optica (NMO) seen in Westerners. OSMS is suggested to be NMO based on the frequent
detection of specific IgG targeting aquaporin- 4 (AQP4), designated NMO-IgG.The present study sought to clarify the significance of anti-AQP4 autoimmunity in the whole spectrum of MS. Sera from 113 consecutive
Japanese patients with clinically definite MS, based on the Poser criteria, were assayed for anti-AQP4 antibodies
by immunofluorescence using GFP-AQP4 fusion protein-transfected HEK-293T cells. Sensitivity and specificity
of the anti-AQP4 antibody assay, 83.3 and 100%, respectively, were calculated using serum samples with
NMO-IgG status predetermined at the Mayo Clinic. The anti-AQP4 antibody positivity rate was significantly
higher in OSMS patients (13/48, 27.1%) than those with CMS (3/54, 5.6%), other neurological diseases (0/52) or
healthy controls (0/35). None of the 11 patients tested with a brainstem^spinal form of MS were positive. Among
OSMS patients, the antibody positivity rate was highest in OSMS patients with longitudinally extensive spinal
cord lesions (LESCLs) extending over three vertebral segments and brain lesions that fulfilled the Barkhof criteria (5/9, 55.6%). Multiple logistic analyses revealed that emergence of the anti-AQP4 antibody was positively
associated only with a higher relapse rate, but not with optic^spinal presentation or LESCLs. Compared with
anti-AQP4 antibody-negative CMS patients, anti-AQP4 antibody-positive MS patients showed significantly
higher frequencies of severe optic neuritis, acute transverse myelitis and LESCLs while most conditions were
also common to anti-AQP4 antibody-negative OSMS patients. The LESCLs in anti-AQP4 antibody-positive
patients were located at the upper-to-middle thoracic cord, while those in anti-AQP4 antibody-negative
OSMS patients appeared throughout the cervical-to-thoracic cord. On axial planes, the former most frequently
showed central grey matter involvement, while holocord involvement was predominant in the latter. In contrast,
LESCLs in anti-AQP4 antibody-negative CMS patients preferentially involved the mid-cervical cord presenting
a peripheral white matter-predominant pattern, as seen in the short lesions. Anti-AQP4 antibody-positive
MS patients fulfilling definite NMO criteria showed female preponderance, higher relapse rate, greater frequency of brain lesions and less frequent responses to interferon beta-1b than anti-AQP4 antibody-negative
OSMS patients with LESCLs. These findings suggested that LESCLs are distinct in anti-AQP4 antibody positivity and clinical phenotypes. There were cases of anti-AQP4 antibody-positive MS/NMO distinct from CMS, and
anti-AQP4 antibody-negative OSMS with LESCLs in Japanese. This indicated that the mechanisms producing
LESCLs are also heterogeneous in cases with optic^spinal presentation, namely AQP4 autoimmunity-related
and -unrelated.
Keywords: opticospinal multiple sclerosis; neuromyelitis optica; aquaporin- 4; NMO-IgG; Japanese
Abbreviations: ANOVA ¼ analysis of variance; AQP4 ¼ aquaporin-4; ATM ¼ acute transverse myelitis; BSMS ¼ brainstem ^
spinal form of multiple sclerosis; CMS ¼ conventional form of multiple sclerosis; CNS ¼ central nervous system;
CSF ¼ cerebrospinal fluid; EDSS ¼ Expanded Disability Status Scale of Kurtzke; FS ¼ Visual Functional Scale of Kurtzke;
GFP ¼ green fluorescent protein; IRTM ¼ idiopathic recurrent transverse myelitis; LESCL ¼ longitudinally extensive spinal
cord lesion; MRI ¼ magnetic resonance imaging; MS ¼ multiple sclerosis; NMO ¼ neuromyelitis optica; OB ¼ oligoclonal band;
ß The Author (2007). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Aquaporin- 4 autoimmunity in MS
Brain (2007), 130, 1206 ^1223
1207
OND ¼ other neurological disease; OSMS ¼ opticospinal form of multiple sclerosis; PBS ¼ phosphate-buffered saline
Received November 5, 2006. Revised January 10, 2007. Accepted January 31, 2007. Advance Access publication April 17, 2007
Introduction
Multiple sclerosis (MS) is a chronic inflammatory
demyelinating disease of the central nervous system
(CNS). It has been hypothesized to be caused by an
autoimmune mechanism targeting CNS myelin. MS is rare
in Asians. However, when it does appear, the selective
and severe involvement of the optic nerves and spinal
cord is characteristic (Kira, 2003). This form, termed
opticospinal MS (OSMS), has similar features to the
relapsing–remitting form of Devic’s neuromyelitis optica
(NMO) in Western populations (Wingerchuk et al., 1999;
Cree et al., 2002; Lucchinetti et al., 2002). However,
it shows features that are distinct from another form
of MS seen in Asians, the conventional form of multiple
sclerosis (CMS) that shows disseminated lesions in the
central nervous system (CNS) including the cerebrum,
cerebellum and brainstem (Kira et al., 1996). This is similar
to classical MS in Westerners (Kira et al., 1996; Yamasaki
et al., 1999; Kira, 2003; Ishizu et al., 2005). Early studies
on NMO and MS in Japanese demonstrated that from
both clinical and pathological standpoints there were
many intermediate cases between NMO and MS (Okinaka
et al., 1958; Shibasaki and Kuroiwa, 1973). These observations have led most Japanese researchers to consider
that classical MS and NMO represent the opposite
ends of a continuum rather than being two distinct
diseases, with OSMS in between the two (Shibasaki
et al., 1974).
Recently, a specific IgG against NMO, designated NMOIgG, was described (Lennon et al., 2004), and its relevant
antigen was found to be aquaporin-4 (AQP4) (Lennon
et al., 2005). Based on the high specificity of NMO-IgG,
NMO is now claimed to be a distinct disease entity
with a fundamentally different aetiology from MS
(Wingerchuk et al., 2006). Additionally, Pittock and
colleagues (Pittock et al., 2006a) reported that asymptomatic brain lesions are common in NMO and that even
the presence of symptomatic brain lesions does not exclude
a diagnosis of NMO based on the presence of NMO-IgG,
although NMO-IgG was originally discovered in NMO
patients without brain lesions on magnetic resonance
imaging (MRI).
Since Nakashima and colleagues (Nakashima et al., 2006)
reported an NMO-IgG positivity rate of 60% in a selected
series of Japanese patients with OSMS, which is similar
to that for NMO in Western patients (Lennon et al., 2004),
OSMS has been suggested to be NMO (Weinshenker
et al., 2006a). As well, there was a significantly higher
frequency of longitudinally extensive spinal cord lesions
(LESCLs) in NMO-IgG-positive OSMS patients than in
NMO-IgG-negative patients, suggesting that NMO-IgG
may play an important role in the development of extensive
spinal cord lesions in OSMS.
However, although LESCLs are more frequently reported
in OSMS than in CMS, one-fourth of Asian CMS patients
also have LESCLs (Chong et al., 2004; Su et al., 2006;
Minohara et al., 2006), reflecting the severe spinal cord
damage commonly seen in Asians. Thus, the roles of NMOIgG or anti-AQP4 antibodies in the formation of LESCLs
in each MS subtype in Asians is currently uncertain and the
relationship between NMO-IgG-negative OSMS and NMO
remains to be elucidated.
Therefore, we investigated the presence of NMO-IgG and
anti-AQP4 antibodies in a group of Japanese MS patients
in the present study. We covered the whole spectrum of
OSMS and CMS disorders and studied the correlations
of the antibodies with the clinical and laboratory findings
in these patients. Our primary aim was to clarify the
significance of these autoantibodies in the development
of human demyelinating diseases of the CNS.
Material and methods
Patients
At the MS clinic in the Department of Neurology, Kyushu
University Hospital during 1987–2006, 142 consecutive patients,
36 males and 106 females, were diagnosed with clinically definite
MS according to the criteria of Poser and colleagues (Poser et al.,
1983) and subjected to brain and whole spinal cord MRI.
Of these, stock sera from 113 patients, 27 males and 86 females,
were available for antibody assays and used for the present study
while the remaining patients were lost to follow-up or were
deceased. All patients underwent a thorough neurological
examination and routine laboratory tests. All were followed up
and clinically evaluated at regular intervals in the MS clinic. Their
medical records and MRI films were analysed retrospectively for
the present study. All of the patients were residents of Kyushu
Island, the southernmost part of mainland Japan. None of the
patients was seropositive for human T-cell leukaemia virus type I
(during the study period two MS patients were excluded). All
patients had a relapsing–remitting or relapsing–progressive course,
and patients with primary progressive MS were not included
in the present study (during the study period 24 patients were
excluded). Patients with monophasic NMO without subsequent
relapse were also excluded to avoid including patients with acute
disseminated encephalomyelitis (during the study period one
patient was excluded).
The MS patients were clinically classified into the two subtypes
of OSMS and CMS as described previously (Kira et al., 1996).
Briefly, patients who had a relapsing–remitting course and both
optic nerve and spinal cord involvement without any clinical
evidence of disease in either the cerebrum or the cerebellum were
considered to have OSMS. Patients with minor brainstem signs,
such as transient double vision and nystagmus, in addition
to opticospinal involvement were included in this subtype.
1208
Brain (2007), 130, 1206 ^1223
Patients with multiple involvement of the CNS, including the
cerebrum and cerebellum, were considered to have CMS. Patients
with only brainstem and spinal cord symptomatology were
difficult to classify into either subtype, but were temporarily
grouped together under the term ‘brainstem–spinal form of MS’
(BSMS). The diagnosis of the different forms of MS was made
before anti-AQP4 antibody and NMO-IgG assays; thereafter,
the diagnosis remained unchanged throughout the study. The
disability status of the patients was scored by one of the authors
(J.K.), according to the Expanded Disability Status Scale (EDSS)
of Kurtzke (Kurtzke, 1983). Severe optic neuritis was defined as
grade 5 or more than 5 on Kurtzke’s Visual Functional Scale (FS)
(Kurtzke, 1983). Acute transverse myelitis (ATM) was defined
according to Fukazawa and colleagues (1990). The response to
interferon beta-1b was evaluated by the changes in the annual
relapse rates in the preceding 2 years and during the therapy. All
sera taken from the patients were stored at 80 C prior to the
present analyses.
Magnetic resonance imaging
All MRI studies were performed using 1.5 T units, Magnetom
Vision and Symphony (Siemens Medical Systems, Erlangen,
Germany) as described previously (Su et al., 2006). The typical
imaging parameters for the brain were as follows: axial
T2-weighted turbo spin-echo imaging using TR/TE ¼ 2800/90 ms,
flip angle ¼ 180 ; axial turbo-fluid-attenuated inversion recovery
(FLAIR) imaging using TI/TR/TE ¼ 2200/9000/110 ms, flip
angle ¼ 180 and sagittal and axial precontrast and axial and
coronal postcontrast T1-weighted spin-echo imaging using TR/TE
range ¼ 400–460/12–17 ms, flip angle range ¼ 80–90 . One excitation, with a matrix of 256 256, slice thickness of 5 mm and slice
gap of 2.5 mm, was used for all brain studies. Gadopentetate
dimeglumine at 0.1 mmol/kg body weight was administered
intravenously for contrast-enhanced studies. The typical
imaging parameters for the spinal cord were as follows: sagittal
T2-weighted turbo spin-echo imaging using TR/TE range ¼ 2500–
2800/90–116 ms, flip angle ¼ 180 , number of excitations ¼ 3 or 4;
sagittal T1-weighted spin-echo imaging using TR/TE range ¼
400–440/11–12 ms, flip angle range ¼ 90–170 , number of
excitations ¼ 2 or 3; axial T2-weighted turbo spin-echo imaging
using TR/TE range ¼ 3200–5360/99–116 ms, flip angle ¼ 180 ,
number of excitations ¼ 3 or 4; axial T1-weighted spin-echo
imaging using TR/TE range ¼ 400–440/12 ms, flip angle
range ¼ 90–170, number of excitations ¼ 2. For sagittal imaging,
a matrix of 256 256 or 512 512, a slice thickness of 4 mm and
a slice gap of 0.4 mm were used; for axial imaging, a matrix
of 256 256 or 512 512, a slice thickness of 5 mm and a slice
gap range of 1.5–5 mm were used.
MRI scans were taken at the time of clinical relapse (within 30
days of the onset of acute exacerbation) or in the remission phase.
Spinal cord MRI was undertaken in 42 patients (26 OSMS and
16 CMS) at relapse presenting with exacerbation of spinal cord
symptomatology and in 97 patients in remission (39 OSMS,
49 CMS and 9 BSMS). In four OSMS patients, four CMS patients
and two BSMS patients, only MRI scans at relapse showing
symptoms other than the spinal cord were available and these
were used for evaluations of the spinal cord lesions for the entire
clinical course. Brain MRI scans were examined in 69 patients
(30 OSMS, 35 CMS and 4 BSMS) at relapse and in 104 patients
(42 OSMS, 52 CMS and 10 BSMS) in remission. Spinal cord MRIs
T. Matsuoka et al.
at relapse and in remission were analysed separately. For statistical
analyses of brain MRIs, relapse MRI findings were used first, but
when only remission MRIs were available, remission MRI findings
were used. Brain and spinal cord MRIs were independently
evaluated by two of the authors (T.M. and F.M.) who were naive
to the diagnoses.
The length of the spinal cord lesions was expressed in terms
of the number of vertebral segments; lesions extending for three or
more than three vertebral segments in length were considered
longitudinally extensive, while lesions of less than three vertebral
segments were regarded as short lesions (Wingerchuk et al., 2006).
Patients who showed LESCLs at either relapse or remission were
classified as LESCL-positive, while those who did not have
LESCLs at relapse or remission as LESCL-negative. Thus, the
number of LESCL-positive patients was potentially underestimated
in the present study, since those who had no LESCL at remission
MRI might have had a LESCL at relapse. Cross-sectional
evaluation was also done for all MRI scans of the spinal cord;
lesions were classified as having a holocord pattern with or
without peripheral sparing, a central grey matter-predominant
pattern, or a peripheral white matter-predominant pattern,
according to Tartaglino and colleagues (1996). Gadoliniumenhancement was evaluated in all available MRI scans taken at
any spinal cord relapses, while fine analysis of the lesion
distribution was assessed for MRI scans taken at the latest relapse
or remission. Brain MRI lesions were evaluated according to either
the Barkhof criteria (Barkhof et al., 1997) or the Paty criteria
(Paty et al., 1988) for MS.
At the time of spinal cord MRI, immunological treatment was
being received by 22 patients [14 on interferon beta-1b and 14 on
high-dose (more than 40 mg/day) corticosteroids] in the 69 scans
of OSMS patients, 34 patients (25 on interferon beta-1b and 13 on
high-dose corticosteroids) in the 69 scans of CMS patients and
one patient (on interferon beta-1b) of the 11 scans of BSMS
patients. At the time of brain MRI, treatment was being received
by 25 of the patients (16 on interferon beta-1b and 12 on highdose corticosteroids) in the 72 scans of OSMS patients, 44 patients
(34 on interferon beta-1b and 14 on high-dose corticosteroids)
in the 87 scans of CMS patients and one patient (on interferon
beta-1b) in the 14 scans of BSMS patients.
NMO-IgG
NMO-IgG was measured at the Mayo Clinic by V. A. Lennon,
as previously described (Lennon et al., 2004). Serum samples from
46 OSMS patients, 51 CMS, 8 with parasitic myelitis (six with
visceral larva migrans of Toxocara canis and two with visceral larva
migrans of Ascaris suum; of whom four had LESCLs and two had
short spinal cord lesions on MRI) and 14 with myelitis and atopic
diathesis (atopic myelitis) (Kira et al., 1998), all of whom had
short spinal cord lesions on MRI, were measured with the
examiners blinded to the origin of the specimens. Parasitic
myelitis and atopic myelitis were chosen as the disease controls
because both usually show eosinophilic myelitis on pathological
examination (Osoegawa et al., 2003), as observed in some cases
of NMO (Lucchinetti et al., 2002).
Anti-AQP4 antibody assay
For the anti-AQP4 antibody assays, serum samples from 113 MS,
4 idiopathic recurrent transverse myelitis (IRTM) and 52 other
neurological disease (OND) patients (11 parasitic myelitis,
Aquaporin- 4 autoimmunity in MS
14 atopic myelitis, 1 HTLV-1-associated myelopathy, 2 viral
encephalitis, 2 neurosarcoidosis, 1 meningitis, 1 neuro-Behcet
disease, 17 spinocerebellar degeneration, 2 Parkinson disease and
1 normal pressure hydrocephalus), and 35 healthy controls were
evaluated.
A full-length cDNA encoding human AQP4 (AQP4 transcript
variant a; GenBank accession number NM_001650) was amplified
from a cDNA library generated from commercially obtained
human spinal cord mRNAs (Clontech, Mountain View, CA, USA).
The PCR product was cloned into the pDONR221 vector
(Invitrogen, Carlsbad, CA, USA) and its sequence confirmed.
After sequencing, the AQP4 cDNA was transferred to the pcDNADEST53 expression vector (Invitrogen). Human embryonic kidney
HEK-293T cells maintained in Dulbecco’s modified Eagle’s
medium containing 10% foetal calf serum were seeded at 5000
cells/well onto 8-well chamber slides (Becton Dickinson, Franklin
Lakes, NJ, USA) at 24 h before transfection. The cells were
transfected with 100 ng/well of the green fluorescent protein
(GFP)-AQP4 fusion protein expression vector using FuGENE6
Transfection Reagent (Roche, Basel, Switzerland) according to the
manufacturer’s instructions. At 48 h after transfection, the cells
were fixed in 10% paraformaldehyde in phosphate-buffered saline
(PBS) for 4 min, washed in PBS and blocked by incubation in
PBS containing 10% goat serum at room temperature for 1 h.
Next, the cells were incubated with human serum samples
(diluted 1 : 400 in accordance with the results of preliminary
experiments) at room temperature overnight, washed and
visualized with an Alexa 594-conjugated goat anti-human IgG
antibody (Invitrogen). Fluorescence was observed with a confocal
laser-scanning microscope (Fluoview FV300; Olympus Optical
Co., Tokyo, Japan). With the scientists blinded to either the
NMO-IgG status or the origin of the specimens, the anti-AQP4
antibody assay was carried out at least twice for each sample and
those that gave a positive result twice were deemed to be positive.
Statistical analyses
Statistical analyses of ages at onset and at examination were done
by an analysis of variance (ANOVA). Relapse rate was analysed
by the Poisson regression analysis. EDSS score and disease
duration were initially performed using the Kruskal–Wallis H
test. When statistical significance was found, the Mann–Whitney
U test was used to determine the statistical differences between
each subgroup. Differences between two subgroups were tested for
significance using Fisher’s exact probability test. For multiple
comparisons, uncorrelated P values (Puncorr) were corrected by
multiplying them by the number of comparisons (Bonferroni–
Dunn’s correction) to calculate corrected P values (Pcorr).
Correlation of NMO-IgG titres with various clinical parameters
was analysed by Spearman’s rank correlation test. Changes in the
relapse rates before and after interferon beta-1b administration
were analysed using the Wilcoxon signed-ranks test. Multiple
logistic analyses were performed to assess possible factors
contributing to the development of anti-AQP4 antibody; such as
gender, age at onset, disease duration, relapse rate, OSMS, EDSS
score, LESCLs and marked CSF pleocytosis (50/ml). In all assays,
statistical significance was set at P50.05. The neurological (J.K.),
neuroimaging (T.M. and F.M.), immunological (T.M. and Y.K.)
and statistical analyses (M.O. and others) were done
independently.
Brain (2007), 130, 1206 ^1223
1209
Results
Demographic features of total MS patients
and each MS subtype
Age at onset and disease duration of all MS patients were
31.9 13.2 years (mean SD) and 12.4 9.9, respectively.
Their average EDSS score was 3.8 3.0. Of the 113 patients,
48 were classified as OSMS, 54 as CMS and 11 as BSMS.
When clinical features were compared between OSMS and
CMS, OSMS showed a significantly higher frequency
of relapse rate than CMS (P50.0001) (Table 1). The
frequencies of severe optic neuritis and ATM were also
significantly higher in OSMS than in CMS (P50.0001).
In CSF, marked pleocytosis (50 cells/ml) was more
common in OSMS than in CMS, while IgG oligoclonal
bands (OB) were more frequently present in CMS than
in OSMS (P ¼ 0.0118). Brain lesions fulfilling either
Barkhof or Paty criteria were significantly more common
in CMS than OSMS (P50.0001 and P ¼ 0.0001, respectively), while LESCLs were more frequently observed in
OSMS than CMS during the entire course (P ¼ 0.0014) or
at the time of relapse. BSMS patients showed the shortest
disease duration, and mildest disease.
Validation of the anti-AQP4 antibody assay
The expressed GFP-AQP4 fusion protein in the transfected
cells was mainly localized on the cell membrane, with an
additional granular expression pattern in the cytoplasm
(Fig. 1A–F). Positive sera stained the cell membrane of the
GFP–AQP4 fusion protein-transfected cells, but not that
of untransfected cells. The positive staining was colocalized
with GFP on the cell membrane but not in the cytoplasm.
Specificity and sensitivity of the anti-AQP4 antibody assay
were calculated with the same serum samples used for the
NMO-IgG assay. In total, 15 of 18 NMO-IgG positive
samples were also positive for the anti-AQP4 antibody,
compared to 0 of 101 NMO-IgG negative samples,
indicating that the sensitivity of the anti-AQP4 antibody
assay was 83.3%, specificity 100%, false-negative rate 16.7%
and false-positive rate 0% (Fig. 1G). The relationships
between the NMO-IgG titres and anti-AQP4 antibody
positivity rates were: anti-AQP4 antibody was positive
in two of two sera with an NMO-IgG titre of 1 : 61 440; two
of two with a titre of 1 : 30 720; three of three with a titre
of 1 : 15 360; three of three with a titre of 1 : 7 680; one of
two with a titre of 1 : 3840; four of four with a titre of
1 : 1920; zero of one with a titre of 1 : 960 and zero of one
with a titre of 1 : 480.
Anti-AQP4 antibody positivity rate
Among the three patients whose sera were NMO-IgGpositive but anti-AQP4 antibody-negative, one gave a
positive result for the anti-AQP4 antibody with serum
taken on other occasion. The remaining two NMO-IgGpositive OSMS patients with LESCLs and without Barkhof
1210
Brain (2007), 130, 1206 ^1223
T. Matsuoka et al.
Table 1 Demographic features of multiple sclerosis subgroups
No. of males/females
Age at onset (years)
Disease duration (years)
Relapse rate
EDSS score
Frequency of symptoms
Optic neuritis
Bilateral optic neuritis
Severe optic neuritis (FS 5)
Myelitis
Acute transverse myelitis
Secondary progression
CSF
Marked pleocytosis (50/ml)
Neutrophilia (5/ml)
OB
IgG index (0.658)a
Barkhof brain lesions
Paty brain lesions
LESCLs during entire course
LESCLs at the relapse
Opticospinal form
of multiple sclerosis
(n ¼ 48)
Conventional form
of multiple sclerosis
(n ¼ 54)
Brainstem ^ spinal form
of multiple sclerosis
(n ¼11)
8/40 (1 : 5.0)
33.6 13.8
13.8 9.5
0.9 0.6*
4.6 3.2
17/37 (1 : 2.2)
29.0 11.4
12.1 10.3
0.7 0.5*
3.5 2.8
2/9 (1 : 4.5)
38.8 15.8
7.5 8.8
0.9 0.7
2.3 1.9
48/48 (100.0%)*
7/48 (14.6%)
35/48 (72.9%)*
48/48 (100.0%)*
28/48 (58.3%)*
3/48 (6.3%)
30/54 (55.6%)*
4/54 (7.4%)
16/54 (29.6%)*
43/54 (79.6%)*
8/54 (14.8%)*
5/54 (9.3%)
0/11 (0.0%)
0/11 (0.0%)
0/11 (0.0%)
11/11 (100.0%)
2/11 (18.2%)
1/11 (9.1%)
7/43 (16.3%)
6/36 (16.7%)
7/38 (18.4%)*
14/33 (42.4%)
13/48 (27.1%)*
29/48 (60.4%)*
31/48 (64.6%)*
15/26 (57.7%)
2/51 (3.9%)
1/32 (3.1%)
21/47 (44.7%)*
27/45 (60.0%)
41/54 (75.9%)*
50/54 (92.6%)*
17/54 (31.5%)*
5/16 (31.3%)
0/10 (0.0%)
0/8 (0.0%)
3/9 (33.3%)
2/9 (22.2%)
6/11 (54.5%)
9/11 (81.8%)
5/11 (45.5%)
0/0
*Statistically significant in comparison between OSMS and CMS (P50.05). Barkhof brain lesions ¼ brain lesions fulfilling the Barkhof criteria
(Barkhof et al., 1997); CSF ¼ cerebrospinal fluid; EDSS ¼ Expanded Disability Status Scale of Kurtzke; FS ¼ Kurtzke’s Visual Functional Scale;
LESCLs ¼ longitudinally extensive spinal cord lesions; OB ¼ IgG oligoclonal bands; Paty brain lesions ¼ brain lesions fulfilling the Paty criteria (Paty et al., 1988).aUpper normal rage of IgG index was derived from our previous study (Kira et al., 1996).
brain lesions constantly showed negative anti-AQP4 antibody assays. Therefore, anti-AQP4 antibody was positive
in 13 of 48 (27.1%) OSMS, 3 of 54 (5.6%) CMS, 0 of
11 BSMS, 1 of 4 (25.0%) IRTM and 0 of 52 OND patients,
including 25 with eosinophilic myelitis and 0 of 35 healthy
controls (Fig. 2). The positivity rate was significantly
higher in OSMS than in CMS (Pcorr ¼ 0.0201), OND
(Pcorr ¼ 0.0069) and healthy controls (Pcorr ¼ 0.0006).
Among OSMS patients without Barkhof brain lesions, the
antibody positivity rates did not differ significantly,
irrespective of the presence or absence of LESCLs either
during the entire clinical course (27.3 versus 15.4%) or at
relapse (42.9 versus 33.3%). The anti-AQP4 antibody
positivity rate was highest in patients with LESCLs and
Barkhof brain lesions (55.6%). In CMS patients with
LESCLs, 2 of 17 (11.8%) were anti-AQP4 antibody-positive
and the antibody was found only in those with extremely
long spinal cord lesions extending over 10 vertebral
segments in length (2/6, 33.3%). It was never found
among the remaining patients with LESCLs of 3–10
vertebral segments in length (0/11, 0%). Three LESCLnegative MS patients with anti-AQP4 antibody were all
examined for spinal cord MRI at relapse. NMO-IgG
gave essentially the same results as anti-AQP4 antibody
(data not shown).
Changes in anti-AQP4 antibody positivity
during the clinical course
Anti-AQP4 antibody positivity was observed in around
20–30% of OSMS patients and 15–20% of the total
MS patients over a wide range of disease durations (from
55 to 15 years) (Supplementary Fig. 1A). Among 13 MS
patients with NMO-IgG whose sera were repeatedly
sampled and examined for anti-AQP4 antibody positivity,
10 were consistently positive (ranging from 0.9 to
5.1 years), 1 was consistently negative for over 5.8 years
and 2 changed from initial negativity to later positivity over
5.0 and 5.9 years, respectively, although their NMO-IgG
titres were relatively low (1 : 480 and 1 : 1920, respectively).
NMO-IgG positivity showed a similar trend over a wide
range of disease duration (data not shown). The NMO-IgG
titre had no correlations with the disease duration, number
of exacerbations or relapse rate, but had an inverse
correlation with both EDSS score and progression index
at the time of blood sampling and at the final follow-up in
NMO-IgG-positive MS patients (P ¼ 0.0076 and P ¼ 0.0517,
respectively, for EDSS score and P ¼ 0.0185 and P ¼ 0.0556,
respectively, for progression index, Spearman’s rank
correlation test) (Supplementary Fig. 1B). We further
examined the effects of immunotherapies on antibody
positivity and found that neither anti-AQP4 antibody nor
Aquaporin- 4 autoimmunity in MS
Brain (2007), 130, 1206 ^1223
GFP-AQP4
Patient sera
Merge
A
B
C
D
E
F
G
1211
NMO-IgG
(+)
(− )
Anti-AQP4
(+)
15
0
antibody
(− )
3
101
Sensitivity
Specificity
83.3%
100.0%
Fig. 1 Immunostaining of HEK-293T cells transfected with a GFP-AQP4 fusion protein expression vector. The expressed GFP-fused AQP4
is mainly localized to the cell membrane (green) but is also observed in the cytoplasm (A). IgG in serum from an NMO-IgG-positive OSMS
patient, which combines with the cell membrane of GFP-AQP4 -transfected cells (B), is detected with an Alexa594-conjugated anti-human
IgG antibody (red). Merged images of the transfected cells (C) show colocalization of the serum IgG from the OSMS patient and the
GFP-AQP4 protein on the cell membrane (yellow). In contrast, serum from an NMO-IgG-negative CMS patient does not stain the
GFP-AQP4 -transfected cells (D ^F). (G) Sensitivity and specificity of the anti-AQP4 antibody assay is determined using sera with NMO-IgG
status predetermined by the Mayo Clinic (V. A. Lennon). AQP4 ¼ aquaporin-4; CMS ¼ conventional form of multiple sclerosis; GFP ¼ green
fluorescent protein; NMO ¼ neuromyelitis optica; OSMS ¼ opticospinal form of multiple sclerosis.
NMO-IgG positivity was significantly affected by treatment.
Specifically, the NMO-IgG positivity rates were: 3/21
(14.3%) in patients undergoing any of the immunotherapies versus 15/76 (19.7%) without; 2/16 (12.5%) with
interferon beta-1b versus 16/81 (19.8%) without; and 1/6
(16.7%) with corticosteroids versus 17/91 (18.7%) without,
while the anti-AQP4 antibody positivity rates were: 2/23
(8.7%) in patients with any of the immunotherapies versus
14/90 (15.6%) without; 1/18 (5.6%) with interferon beta-1b
versus 15/95 (15.8%) without; and 0/5 (0.0%) with
corticosteroids versus 16/108 (14.8%) without (P40.1 in
all cases).
Relationships between anti-AQP4 antibody
positivity and clinical findings
We compared the clinical and laboratory findings among
anti-AQP4 antibody-positive MS, anti-AQP4 antibodynegative OSMS and anti-AQP4 antibody-negative CMS
patients, excluding two OSMS patients who were positive
1212
Brain (2007), 130, 1206 ^1223
T. Matsuoka et al.
(%)
100
90
*
80
*
13/48
(27.1%)
70
60
*
5/9
(55.6%)
50
40
30
1/3
(33.3%)
6/22
(27.3%)
20
3/54
(5.6%)
2/13
(15.4%)
2/15
(13.3%)
10
0
LESCLs
Barkhof
1/26
(3.8%)
NA
NA
+
+
−
−
+
+
−
−
+
+
−
−
+
−
NA
NA
+
−
+
−
+
−
+
−
+
−
+
−
−
−
(n = 9) (n = 22) (n = 4) (n = 13) (n = 15) (n = 2) (n = 26) (n = 11) (n = 2) (n = 3) (n = 4) (n = 2) (n = 3) (n = 1) (n = 52) (n = 35)
OSMS
CMS
BSMS
IRTM
OND
HC
Fig. 2 Anti-AQP4 antibody seropositivity rates, according to the presence or absence of either LESCLs or brain lesions fulfilling the
Barkhof criteria (Barkhof brain lesions) (Barkhof et al., 1997), among patients with OSMS, CMS, BSMS, IRTM and other neurological
diseases including eosinophilic myelitis as well as healthy controls. Uncorrected P values are corrected by multiplying the number
of comparisons by the original P value to calculate the corrected P values. AQP4 ¼ aquaporin-4; BSMS ¼ brainstem ^ spinal form of MS;
CMS ¼ conventional form of multiple sclerosis; EDSS ¼ Expanded Disability Status Scale of Kurtzke; HC ¼ healthy controls;
IRTM ¼ idiopathic recurrent transverse myelitis; LESCLs ¼ longitudinally extensive spinal cord lesions; NA ¼ not applicable; OND ¼ other
neurological diseases; OSMS ¼ opticospinal form of multiple sclerosis. *Significant difference between the linked groups (Pcorr50.05).
for NMO-IgG but negative for anti-AQP4 antibody
(Table 2). The anti-AQP4 antibody-positive MS patients
were all females. Female preponderance was only statistically significant in anti-AQP4 antibody-positive MS
compared with anti-AQP4 antibody-negative CMS patients
(Pcorr ¼ 0.0204). The age at onset was also significantly
higher in anti-AQP4 antibody-positive MS than in antiAQP4 antibody-negative CMS patients (Pcorr ¼ 0.0331).
Although disease duration did not differ significantly
among the three groups, anti-AQP4 antibody-positive MS
patients showed a significantly higher relapse rate than antiAQP4 antibody-negative OSMS and CMS ones
(Pcorr50.0001, both) while among anti-AQP4 antibodynegative MS patients, OSMS had a significantly higher
relapse rate than did CMS ones (Pcorr ¼ 0.0121). Both antiAQP4 antibody-positive MS and the anti-AQP4 antibodynegative OSMS patients showed greater EDSS scores than
anti-AQP4 antibody-negative CMS ones, but the difference
did not reach statistical significance. Compared with
anti-AQP4 antibody-negative CMS patients, severe optic
neuritis and ATM were significantly more frequent
in anti-AQP4 antibody-positive MS (Pcorr ¼ 0.0078 and
Pcorr ¼ 0.0039, respectively) and anti-AQP4 antibodynegative OSMS (Pcorr ¼ 0.0042 and Pcorr ¼ 0.0006, respectively) patients. However, the frequency of bilateral
optic neuritis was more than 2-fold higher in anti-AQP4
antibody-negative OSMS in comparison to anti-AQP4
antibody-positive MS patients. None of the antiAQP4 antibody-positive MS patients showed secondary
progression, compared to around 10% of those in the
anti-AQP4 antibody-negative CMS group.
Marked CSF pleocytosis and CSF neutrophilia were
more common in anti-AQP4 antibody-positive MS and
anti-AQP4 antibody-negative OSMS than in anti-AQP4
antibody-negative CMS patients, while CSF OB were more
frequently observed in anti-AQP4 antibody-negative CMS
than in anti-AQP4 antibody-positive MS and anti-AQP4
antibody-negative OSMS patients, yet the differences were
Aquaporin- 4 autoimmunity in MS
Brain (2007), 130, 1206 ^1223
1213
Table 2 Comparison of the clinical findings among multiple sclerosis subtypes according to the anti-AQP4 antibody status
Clinical subtype: OSMS/CMS
No. of males/females
Age at onset (years)
Disease duration (years)
Relapse rate
EDSS score
Frequency of symptoms
Optic neuritis
Bilateral optic neuritis
Severe optic neuritis (FS 5)
Myelitis
Acute transverse myelitis
Secondary progression
CSF
Marked pleocytosis (50/ml)
Neutrophilia (5/ml)
OB
IgG index (0.658)b
ANA
SSA/SSB
Anti-AQP4 Ab(þ)
multiple sclerosis
(n ¼16)
Anti-AQP4 Ab()
opticospinal form of
multiple sclerosis (n ¼ 33)a
Anti-AQP4 Ab()
conventional form of
multiple sclerosis (n ¼ 51)
13 : 3
0/16*
38.2 13.6*
14.0 8.9
1.1 0.6*,**
5.1 2.9
33 : 0
8/25 (1: 3.1)
32.0 13.4
13.9 9.8
0.8 0.5*,***
4.2 3.2
0 : 51
17/34 (1: 2.0)*
28.6 11.5*
12.1 10.5
0.6 0.5**,***
3.4 2.8
16/16 (100.0%)*
1/16 (6.3%)
12/16 (75.0%)*
16/16 (100.0%)
9/16 (56.3%)*
0/16 (0.0%)
33/33 (100.0%)**
5/33 (15.2%)
22/33 (66.7%)**
33/33 (100.0%)*
18/33 (54.5%)**
2/33 (6.1%)
27/51 (52.9%)*,**
4/51 (7.8%)
15/51 (29.4%)*,**
40/51 (78.4%)*
7/51 (13.7%)*,**
5/51 (9.8%)
2/16 (12.5%)
2/14 (14.3%)
1/11 (9.1%)
4/10 (40.0%)
6/16 (37.5%)
5/16 (31.3%)
5/28 (17.9%)
4/22 (18.2%)
6/28 (21.4%)
10/24 (41.7%)
5/30 (16.7%)
3/24 (12.5%)
2/48 (4.2%)
1/30 (3.3%)
21/44 (47.7%)
26/42 (61.9%)
8/45 (17.8%)
4/38 (10.5%)
*,**,***Significant difference between the linked values (Pcorr50.05). Ab ¼ antibody; AQP4 ¼ aquaporin-4; CMS ¼ conventional form of
multiple sclerosis; CSF ¼ cerebrospinal fluid; EDSS ¼ Expanded Disability Status Scale of Kurtzke; FS ¼ Kurtzke’s Visual Functional Scale;
OB ¼ IgG oligoclonal bands; OSMS ¼ opticospinal form of multiple sclerosis. aTwo OSMS patients who were positive for NMO-IgG but
negative for anti-AQP4 antibody were excluded. bUpper normal rage of IgG index was derived from our previous study (Kira et al., 1996).
not statistically significant. The positivity rates for ANA
and SSA/SSB were 2–3-fold higher in anti-AQP4 antibodypositive MS than in anti-AQP4 antibody-negative OSMS
and CMS patients.
Relationships between anti-AQP4 antibody
positivity and magnetic resonance imaging
findings
Representative MRI findings of anti-AQP4 antibodypositive MS, anti-AQP4 antibody-negative OSMS and
anti-AQP4 antibody-negative CMS patients are shown in
Fig. 3. Brain lesions fulfilling either the Barkhof or Paty
criteria were most common in anti-AQP4 antibodynegative CMS patients and the differences were significant
between anti-AQP4 antibody-negative CMS and OSMS
(Pcorr50.0001 and Pcorr ¼ 0.0003, respectively) patients
(Table 3). For all items of the Barkhof criteria, anti-AQP4
antibody-negative CMS showed significantly higher
frequencies than the antibody-negative OSMS patients
(Pcorr50.05 in all), except for juxtacortical lesions. AntiAQP4 antibody-positive MS patients showed a similar
high frequency of brain lesions fulfilling the Paty criteria
to that of anti-AQP4 antibody-negative CMS ones.
As well, the frequency of Barkhof brain lesions was about
2-fold higher than in anti-AQP4 antibody-negative
OSMS patients. The frequency of ovoid lesions was
significantly higher in anti-AQP4 antibody-negative CMS
than in anti-AQP4 antibody-negative OSMS (Pcorr50.0001)
and anti-AQP4 antibody-positive MS (Pcorr ¼ 0.0399)
patients.
Atypical brain lesions, previously reported in NMO-IgGpositive NMO patients (Pittock et al., 2006a, b), such as
extensive cerebral white matter lesions (3 cm), bilateral
diencephalic (thalamic/hypothalamic) lesions, cavity formation and extension from the cervical cord into brainstem
were not significantly different among the three groups.
However, anterior periventricular rim-like lesions lining
along with the third and lateral ventricles, which did not
deeply extend into the white matter and were not enhanced
by contrast media (Fig. 3A-3), were more common in antiAQP4 antibody-positive MS and anti-AQP4 antibodynegative OSMS patients than anti-AQP4 antibody-negative
CMS ones, though the difference did not reach statistical
significance. However, LESCL frequency was higher in
anti-AQP4 antibody-positive MS and anti-AQP4 antibodynegative OSMS patients than in anti-AQP4 antibodynegative CMS ones during the entire course
(Pcorr ¼ 0.0012 and Pcorr ¼ 0.0741, respectively), and the
similar trend was also observed at relapse (P40.1, because
of the small sample sizes).
Relationships between anti-AQP4 antibody
positivity and distribution of spinal cord
lesions on MRI
We compared the distribution and location of each spinal
cord lesion between anti-AQP4 antibody-positive MS,
1214
Brain (2007), 130, 1206 ^1223
R
T. Matsuoka et al.
R
Th4/5
C3
Th4
C3
B-2
A-2
A-1
A-3
R
R
B-3
B-4
B-1
Fig. 3 Representative MRIs of anti-AQP4 antibody-negative (A and B) and -positive (C^E) MS patients with LESCLs. MRI of a 50 -year-old
female patient with OSMS at relapse (A-1^3) shows an LESCL but no Barkhof brain lesions. Disease duration was 0.7 years at the time of
the MRI scan and EDSS was 7.0. The patient had neither NMO-IgG nor the anti-AQP4 antibody. The LESCL shows a holocord pattern at the
thoracic level. Periventricular rim-like lesions (arrow in A-3) are seen around the anterior horns. MRI of a 37-year-old male patient with
CMS at relapse (B-1^4) shows an LESCL at the C3^ C7 level and Barkhof brain lesions. Disease duration was 17.2 years and EDSS was 3.5.
The patient had neither NMO-IgG nor the anti-AQP4 antibody. The LESCL shows a peripheral white matter-predominant pattern
(arrow in B-2). Spinal cord and brain MRI of an OSMS patient (72-year-old female with a disease duration of 16.2 years and an EDSS
score of 7.5 at the time of the MRI scans) with LESCLs presenting the central grey matter-predominant pattern involving the upper-tomiddle thoracic cord and subclinical brain lesions fulfilling the Barkhof criteria at relapse (C-1-5). The patient initially showed short spinal
cord lesions with gadolinium-enhancement when serum was negative for both NMO-IgG and anti-AQP4 antibodies. LESCLs developed
later and serum became positive for NMO-IgG (titre, 1 : 480) and anti-AQP4 antibodies. Spinal cord and brain MRI of a CMS patient
(42-year-old female with a disease duration of 17.1 years and an EDSS score of 7.5 at the time of the MRI scans) with LESCLs presenting the
central grey matter-predominant pattern, marked spinal cord atrophy and brain lesions fulfilling the Barkhof criteria in remission (D-1^5).
The arrow (D- 4) indicates a cavity-like lesion. The patient’s NMO-IgG titre is 1 : 15 360. Spinal cord and brain MRI of a CMS patient
(43-year-old female with a disease duration of 4.0 years and an EDSS score of 7.0 at the time of the MRI scans) with LESCLs (E-1^4).
The patient had multiple gadolinium-enhanced lesions in the cerebrum and cerebellum at the initial attack, all of which have almost
completely disappeared in the follow-up scans. Four years later, the patient developed severe left hemiparesis and cortical sensory
impairment. Methylprednisolone pulse therapy almost completely resolved her symptoms; NMO-IgG titre is 1 : 61440. A huge confluent
brain lesion in the right cerebral hemisphere and an LESCL involving mainly the upper-to-middle thoracic cord with the central grey
matter-predominant pattern were also found. No gadolinium-enhancement is visible in the spinal cord lesion or the huge brain lesion.
The huge brain lesion (E-3) showed increased diffusivity (increased ADC values) on the ADC map of the diffusion-weighted sequence
(Supplementary Fig. 3A and B) and increased choline and decreased N-acetylaspartate on magnetic resonance spectroscopy
(Supplementary Fig. 3C). All these findings are compatible with acute demyelination. The sagittal and axial spinal cord MRI areT2 -weighted
images and the brain MRI are fluid-attenuated inversion recovery images except for (E-4), which is a gadolinium-enhanced T1-weighted
image. AQP4 ¼ aquaporin-4; CMS ¼ conventional form of multiple sclerosis; EDSS ¼ Expanded Disability Status Scale of Kurtzke;
LESCLs ¼ longitudinally extensive spinal cord lesions; MRI ¼ magnetic resonance imaging; NMO ¼ neuromyelitis optica;
OSMS ¼ opticospinal form of multiple sclerosis.
anti-AQP4 antibody-negative OSMS and anti-AQP4 antibody-negative CMS patients in detail on MRI. On sagittal
planes, in anti-AQP4 antibody-positive MS patients, 31.3%
(10/22) and 39.3% (11/28) of the total lesions observed in
acute and remission phases, respectively, were LESCLs,
while others were short spinal cord lesions (Supplementary
Fig. 2). In anti-AQP4 antibody-negative patients,
40.0% (6/15) of the lesions in acute phase and 17.9%
(7/39) in remission phase were LESCLs in OSMS while in
CMS it was 17.6% (6/34) and 13.3% (10/75) in the acute
and remission phases, respectively.
LESCLs in anti-AQP4 antibody-positive MS patients
preferentially involved the upper-to-middle thoracic cord,
while those in anti-AQP4 antibody-negative OSMS patients
were extremely long, extending from the upper cervical
cord through to the mid-thoracic cord even during relapse
and in remission (Fig. 4A and B, upper panels).
Distribution of the lesions significantly differed between
the two (Pcorr ¼ 0.0333 at relapse and Pcorr ¼ 0.0084 in
remission). In anti-AQP4 antibody-negative CMS patients,
both LESCLs and short spinal cord lesions most frequently
affected the cervical cord either at relapse or remission
Aquaporin- 4 autoimmunity in MS
Brain (2007), 130, 1206 ^1223
R
R
Th1
1215
Th3
Th2
Th2
D-3
C-3
R
Th3
C-4
D-4
Th2
Th2
C-1
C-2
C-5
D-1
D -2
D-5
Fig. 3 Continued.
R
Th3
E-2
R
E-3
R
E-4
E-1
Fig. 3 Continued.
Th3
(Fig. 4A and B, lower panels). On the axial plane,
distribution of LESCLs in remission tended to differ
between anti-AQP4 antibody-positive MS and anti-AQP4
antibody-negative
OSMS
patients
(Puncorr ¼ 0.0218,
corr
P 40.1) and between anti-AQP4 antibody-positive MS
and anti-AQP4 antibody-negative OSMS patients
(Puncorr ¼ 0.0274, Pcorr40.1) (Fig. 4C and D). In antiAQP4 antibody-positive MS patients, LESCLs consistently
showed the central grey matter-predominant pattern
through relapse and remission phases; even short lesions
most frequently involved the central grey matter at
relapse (Figs. 3 and 4C and D). However, in anti-AQP4
antibody-negative OSMS patients, LESCLs most frequently
revealed the holocord pattern at either relapse or remission, while most of the short lesions demonstrated
the peripheral white matter-predominant pattern during
relapse and in remission. In anti-AQP4 antibody-negative
OSMS patients, distribution of the lesions significantly
differed between LESCLs and short lesions in remission
(Pcorr50.0001) although the difference was not significant due to the small sample size at relapse
(Puncorr ¼ 0.0117, Pcorr40.1). In anti-AQP4 antibodynegative CMS patients, LESCLs most frequently showed
the central grey matter-predominant pattern at relapse but
the peripheral white matter-predominant pattern in remission, while short lesions constantly had the peripheral white
matter-predominant pattern in relapse and remission
phases. When patients undergoing interferon beta-1b
treatment at the time of the MRI studies were excluded,
essentially the same profiles were seen, though differences
in the relapse phase lost statistical significance due to the
small sample size.
1216
Brain (2007), 130, 1206 ^1223
T. Matsuoka et al.
Table 3 Comparison of the brain and spinal cord magnetic resonance imaging findings among multiple sclerosis subtypes
according to the anti-AQP4 antibody status
Brain MRI findings
Axial images
Fulfillment for Barkhof criteria
1 Gd-enhanced lesions or 9 T2 brain lesions
9 T2 lesions
1 Gd-enhanced lesions
1 juxtacortical lesion
3 periventricular lesions
1 infratentorial lesion
Fulfillment for Paty criteria
Atypical brain lesions
Extensive white matter lesions (43 cm)
Bilateral diencephalic lesions
Cavity formation
Extension from cervical cord into brainstem
Sagittal FLAIR images
Ovoid lesions
Anterior rim-like lesionsb
Spinal cord MRI findings
LESCLs during entire course
LESCLs at the relapse
Anti-AQP4 Ab(þ)
multiple sclerosis
(n ¼16)
Anti-AQP4 Ab()
opticospinal form of
multiple sclerosis (n ¼ 33)a
Anti-AQP4 Ab()
conventional form of
multiple sclerosis (n ¼ 51)
8/16 (50.0%)
8/16 (50.0%)
7/16 (43.8%)
1/16 (6.3%)
10/16 (62.5%)
7/16 (43.8%)
9/16 (56.3%)
13/16 (81.3%)
5/16 (31.3%)
1/16 (6.3%)
0/16 (0.0%)
3/16 (18.8%)
1/16 (6.3%)
7/33 (21.2%)*
9/33 (27.3%)*
9/33 (27.3%)*
2/32 (2.9%)*
13/33 (39.4%)
10/33 (30.3%)*
10/33 (30.3%)*
18/33 (54.5%)*
2/33 (6.1%)
0/33 (0.0%)
0/33 (0.0%)
1/33 (3.0%)
2/33 (6.1%)
37/51 (72.5%)*
42/51 (82.4%)*
36/51 (70.6%)*
17/50 (34.0%)*
33/51 (64.7%)
39/51 (76.5%)*
36/51 (70.6%)*
47/51 (92.2%)*
9/51 (17.6%)
1/51 (2.0%)
1/51 (2.0%)
6/51 (11.8%)
1/51 (2.0%)
10/14 (71.4%)*
10/14 (71.4%)
16/27 (59.3%)**
18/27 (66.7%)
39/40 (97.5%)*,**
19/40 (47.5%)
13/16 (81.3%)*
9/13 (69.2%)
18/33 (54.5%)
6/14 (42.9%)
15/51 (29.4%)*
4/14 (28.6%)
*,**Statistically significant in comparison between each subgroup indicated by Bonferroni ^Dunn’s correction (Pcorr50.05). Ab ¼ antibody;
AQP4 ¼ aquaporin-4; Barkhof brain lesions ¼ brain lesions fulfilling the Barkhof criteria (Barkhof et al., 1997); FLAIR ¼ fluid-attenuated
inversion recovery; Gd ¼ gadolinium; LESCLs ¼ longitudinally extensive spinal cord lesions; Paty brain lesions ¼ brain lesions fulfilling the
Paty criteria (Paty et al., 1988). aTwo OSMS patients who were positive for NMO-IgG but negative for anti-AQP4 antibody were excluded.
b
Anterior rim-like lesions are defined as anterior periventricular lesions lining along with the third and lateral ventricles (Fig. 3A^3).
Multiple logistic analyses for possible factors
contributing to anti-AQP4 antibody
production
To further identify possible factors contributing to the
production of anti-AQP4 antibodies, we divided MS
patients into anti-AQP4 antibody-positive and -negative
groups and performed multiple logistic analyses. Among
the clinical and laboratory parameters examined, only the
relapse rate was significantly related to the occurrence of
anti-AQP4 antibodies (OR ¼ 6.612, P ¼ 0.0229) (Table 4).
Comparison of clinical and laboratory
features between anti-AQP4 antibodypositive MS/NMO patients and anti-AQP4
antibody-negative OSMS patients with
LESCLs
Among 16 MS patients who were seropositive for both NMOIgG and anti-AQP4 antibodies, 14 (87.5%) fulfilled the new
NMO criteria (Wingerchuk et al., 2006) and could be
regarded as definite NMO (MS/NMO). Therefore, we finally
compared the clinical and laboratory findings between these
14 NMO-IgG- and anti-AQP4 antibody-positive MS/NMO
patients and 18 anti-AQP4 antibody-negative OSMS
patients with LESCLs from whom NMO-IgG-positive but
anti-AQP4 antibody-negative patients were excluded.
Both groups demonstrated similar clinical features, such
as higher EDSS scores, higher frequencies of severe optic
neuritis and ATM and lower frequencies of CSF OB,
compared with the antibody-negative CMS patients
(Tables 2 and 5). However, female preponderance was
more marked in anti-AQP4 antibody-positive MS/NMO
patients, although the difference between the two groups
did not reach statistical significance. Relapse rate was
significantly higher in anti-AQP4 antibody-positive
MS/NMO patients than in anti-AQP4 antibody-negative
OSMS ones with LESCLs (P ¼ 0.0017). The mean age at
onset was 4 years older in anti-AQP4 antibody-positive
MS/NMO than in anti-AQP4 antibody-negative OSMS
patients with LESCLs.
Concerning laboratory findings, ANA and SSA/SSB were
more frequent in anti-AQP4 antibody-positive MS/NMO
than in anti-AQP4 antibody-negative OSMS patients with
LESCLs, while marked CSF pleocytosis and CSF neutrophilia were more common in the latter than in the former,
although the difference was not significant. Brain lesions
fulfilling either Barkhof or Paty criteria were 2-fold more
commonly seen in anti-AQP4 antibody-positive MS/NMO
than in anti-AQP4 antibody-negative OSMS patients
Aquaporin- 4 autoimmunity in MS
A
Brain (2007), 130, 1206 ^1223
B
At relapse
16
(%)
P corr
= 0.0333
LESCLs
Anti-AQP4 Ab(+) MS (n=10)
Anti-AQP4 Ab(−) OSMS (n=6)
In remission
16
(%)
P corr
12
12
8
8
4
4
0
C4
Th1
Th5
Th9
L1
0
0
0
4
4
8
8
12
Anti-AQP4 Ab(−) CMS
LESCLs (n=6)
Short lesions (n=28)
16
1217
C4
12
= 0.0084
Th1
LESCLs
Anti-AQP4 Ab(+) MS (n=11)
Anti-AQP4 Ab(−) OSMS (n=7)
Th5
Th9
L1
Anti-AQP4 Ab(−) CMS
LESCLs (n=10)
Short lesions (n=65)
16
Fig. 4 A comparison of the distributions of LESCLs between anti-AQP4 antibody-positive MS and anti-AQP4 antibody-negative OSMS
patients at relapse (A, upper panel) and in remission (B, upper panel), and between LESCLs and short lesions in anti-AQP4 antibodynegative CMS at relapse (A, lower panel) and in remission (B, lower panel). Comparisons of the frequencies of the patterns in the lesions on
axial planes are also shown according to the presence of LESCLs and short spinal cord lesions in each group at relapse (C) and in remission
(D). The distributions of the lesions are classified as central grey matter-predominant, peripheral white matter-predominant and holocord
patterns, according to Tartaglino and colleagues (Tartaglino et al., 1996). Uncorrected P values are corrected by multiplying the number
of comparisons with the original P value to calculate corrected P values (Mann ^Whitney U test with Bonferroni ^Dunn’s correction).
Ab ¼ antibody; AQP4 ¼ aquaporin-4; CMS ¼ conventional form of multiple sclerosis; LESCLs ¼ longitudinally extensive spinal cord
lesions; Ls ¼ lesions; MS ¼ multiple sclerosis; n ¼ numbers of lesions; OSMS ¼ opticospinal form of multiple sclerosis. *Significant
difference between the linked groups (Pcorr50.05).
(P ¼ 0.0751 for the frequency of Paty brain lesions). Spinal
cord lesions were even longer in anti-AQP4 antibodynegative OSMS with LESCLs than in anti-AQP4 antibodypositive MS/NMO patients. When the frequency of
gadolinium-enhancement of the spinal cord lesions
was evaluated at spinal cord relapses during the entire
clinical course, it was significantly lower for 4 relapses
than for 3 relapses in anti-AQP4 antibody-negative OSMS
patients with LESCLs (P ¼ 0.0172). No such trend was
found in anti-AQP4 antibody-positive MS/NMO patients.
The frequency of spinal cord swelling at relapse and spinal
cord atrophy during remission was similar in both groups.
Although interferon beta-1b was administered for some
periods during the clinical course in about half of the
patients with anti-AQP4 antibody-positive MS/NMO or
anti-AQP4 antibody-negative OSMS with LESCLs, the
drug was frequently discontinued in all groups. The
reduction in the calculated annual relapse rates was larger
in anti-AQP4 antibody-negative OSMS with LESCLs than
in anti-AQP4 antibody-positive MS/NMO patients, while
the on-treatment relapse rate was significantly lower than
the pre-treatment relapse rate only in anti-AQP4 antibodynegative OSMS patients with LESCLs (P ¼ 0.0103)
(Table 6). Moreover, the proportion of patients showing
more than a 50% reduction in the relapse rate on treatment
was also higher in anti-AQP4 antibody-negative OSMS with
LESCLs than in anti-AQP4 antibody-positive MS/NMO
patients (72.7 versus 14.3%, P ¼ 0.0498), yet the frequency
of other immunotherapies did not differ significantly
between the two groups before or during interferon beta1b therapy.
Discussion
The present study, by assaying anti-AQP4 and NMO-IgG
antibodies in an unbiased series of Japanese MS patients,
revealed: (i) anti-AQP4 antibody and NMO-IgG are
positive in about 15% of total MS patients and in 30%
1218
C
(%)
100
Brain (2007), 130, 1206 ^1223
T. Matsuoka et al.
D
At relapse
In remission
Central lesions
Peripheral lesions
Holocord lesions
90
Central lesions
Peripheral lesions
Holocord lesions
*
*
80
70
60
50
40
30
20
10
0
LESCLs Short Ls
(n=10)
(n=22)
Anti-AQP4 Ab (+)
MS
LESCLs Short Ls
(n=6)
(n=9)
Anti-AQP4 Ab (−)
OSMS
LESCLs Short Ls
(n=6)
(n=28)
Anti-AQP4 Ab (−)
CMS
LESCLs Short Ls
(n=11)
(n=17)
Anti-AQP4 Ab (+)
MS
LESCLs Short Ls
(n=7)
(n=32)
Anti-AQP4 Ab (−)
OSMS
LESCLs Short Ls
(n=10)
(n=65)
Anti-AQP4 Ab (−)
CMS
Fig. 4 Continued.
Table 4 Multiple logistic analyses for possible factors contributing to anti-AQP4 antibody production
Possible factors
Anti-AQP4 Ab(þ)
multiple sclerosis
(n ¼16)
Anti-AQP4 Ab()
multiple sclerosis
(n ¼ 97)
Odds ratio
95% CI
P-value
Sex (male/female)
Age at onset (years)
Disease duration (years)
Relapse rate
OSMS
EDSS score
LESCLs
Marked CSF pleocytosis ( 50/ml)
0/16
38.2 13.6
14.0 8.9
1.1 0.6
13/16 (81.3%)
5.1 2.9
13/16 (81.3%)
2/16 (12.5%)
27/70 (1 : 2.6)
30.9 12.9
12.1 10.1
0.7 0.6
35/97 (36.1%)
3.6 2.9
40/97 (41.2%)
7/88 (8.0%)
1
1.060
1.106
6.612
3.505
0.946
2.666
0.897
(0.000 ^)
(0.996 ^1.127)
(0.999^1.225)
(1.299^33.664)
(0.754 ^16.296)
(0.702^1.274)
(0.408 ^17.406)
(0.112^7.153)
0.9943
0.0656
0.0528
0.0229
0.1096
0.7129
0.3057
0.9182
Ab ¼ antibody; AQP4 ¼ aquaporin-4; CI ¼ confidence interval; CSF ¼ cerebrospinal fluid; EDSS ¼ Expanded Disability Status Scale of
Kurtzke; LESCLs ¼ longitudinally extensive spinal cord lesions; OSMS ¼ opticospinal form of multiple sclerosis.
of OSMS patients over a wide range of disease durations.
Anti-AQP4 antibody emergence had a significant positive
association with a higher relapse rate but with neither
LESCLs nor OSMS presentation by multiple logistic
analyses; (ii) although both anti-AQP4 antibody-positive
MS and antibody-negative OSMS patients with LESCLs had
many common clinical features, LESCLs in anti-AQP4
antibody-positive MS preferentially involved the upperto-middle thoracic cord with the central grey matterpredominant pattern through relapse and remission phases.
Whereas, anti-AQP4 antibody-negative OSMS with LESCLs
had extremely long spinal cord lesions from the upper
cervical to mid-thoracic cord with the holocord pattern;
(iii) in anti-AQP4 antibody-negative CMS patients, LESCLs
still occurred in about 30% and showed a predilection to the
cervical cord with the peripheral white matter-predominant
pattern, which was more evident in remission; (iv) antiAQP4 antibody-positive MS patients who fulfilled the new
NMO criteria had higher ages at onset, female preponderance, higher relapse rate, higher frequencies of severe optic
neuritis and ATM and higher EDSS scores compared with
antibody-negative CMS patients. While most were also
common to antibody-negative OSMS patients with LESCLs,
anti-AQP4 antibody-positive MS/NMO patients more commonly had brain lesions fulfilling either the Barkhof or Paty
criteria and less frequent responses to interferon beta-1b.
As compared with anti-AQP4 antibody-negative CMS,
LESCLs were quite distinct for not only frequency, but also
distribution in anti-AQP4 antibody-positive MS and antiAQP4 antibody-negative OSMS. Importantly, although
Aquaporin- 4 autoimmunity in MS
Brain (2007), 130, 1206 ^1223
1219
Table 5 Comparison of clinical findings between anti-AQP4 antibody-positive multiple sclerosis patients fulfilling the new
criteria for definite neuromyelitis optica and anti-AQP4 antibody-negative opticospinal form of multiple sclerosis patients
with longitudinally extensive spinal cord lesions
Anti-AQP4 Ab(þ) MS/NMO (n ¼14)
Clinical subtype: OSMS/CMS
12 : 2
No. of males/females
0/14
Age at onset (years)
38.0 14.6
Disease duration (years)
14.8 9.2
Relapse rate
1.2 0.6*
EDSS score
5.6 2.7
Frequency of symptoms
Optic neuritis
14/14 (100.0%)
Bilateral optic neuritis
1/14 (7.1%)
Severe optic neuritis (FS 5)
11/14 (78.6%)
Myelitis
14/14 (100.0%)
Acute transverse myelitis
9/14 (64.3%)
Secondary progression
0/14 (0.0%)
CSF
Marked pleocytosis (50/ml)
2/14 (14.3%)
Neutrophilia (5/ml)
2/12 (16.7%)
OB
1/9 (11.1%)
IgG index (0.658)b
4/8 (50.0%)
ANA
6/14 (42.9%)
SSA/SSB
4/14 (28.6%)
Barkhof brain lesions
7/14 (50.0%)
Paty brain lesions
11/14 (78.6%)
Ovoid lesions
7/14 (50.0%)
LESCLs
13/14 (92.9%)
Length of LESCLs
8.4 3.9
Gadolinium-enhancement at spinal cord relapsec
Total
15/54 (27.8%)
3 relapses
5/16 (31.3%)
4 relapses
10/38 (26.3%)
Spinal cord swelling at relapse
1/11 (9.1%)
Spinal cord atrophy at remission
5/13 (38.5%)
Anti-AQP4 Ab() OSMS with LESCLs (n ¼18)a
18 : 0
4/14 (1 : 3.5)
33.9 15.4
14.0 9.4
0.8 0.5*
6.4 2.3
18/18 (100.0%)
3/18 (16.7%)
15/18 (83.3%)
18/18 (100.0%)
15/18 (83.3%)
1/18 (5.6%)
5/15 (33.3%)
4/11 (36.4%)
2/16 (12.5%)
5/13 (38.5%)
3/16 (18.8%)
2/12 (16.7%)
4/18 (22.2%)
8/18 (44.4%)
7/18 (38.9%)
18/18 (100.0%)
11.0 6.5
14/49 (28.6%)
i
9/19 (47.4%)
5/30 (16.7%)
2/9 (22.2%)
6/13 (46.2%)
*Significant difference between the linked values (P50.05). Ab ¼ antibody; AQP4 ¼ aquaporin-4; CMS ¼ conventional form of multiple
sclerosis; CSF ¼ cerebrospinal fluid; EDSS ¼ Expanded Disability Status Scale of Kurtzke; FS ¼ Kurtzke’s Visual Functional Scale;
LESCLs ¼ longitudinally extensive spinal cord lesions; NMO ¼ neuromyelitis optica; OB ¼ IgG oligoclonal bands; OSMS ¼ opticospinal form
of multiple sclerosis. aTwo OSMS patients who were positive for NMO-IgG but negative for anti-AQP4 antibody were excluded. bUpper
normal rage of IgG index was derived from our previous study (Kira et al., 1996). cNumber of gadolinium-enhanced lesions/number of total
T2 lesions.
fundamental clinical features, such as severe optic neuritis
and ATM, were seen at similar frequencies in anti-AQP4
antibody-positive MS and -negative OSMS, LESCLs were
significantly different between the two. There was upper to
middle thoracic cord involvement in the former versus the
cervical to thoracic cord involvement in the latter in a
sagittal plane, while in the axial plane there was central grey
matter involvement in the former versus holocord involvement in the latter. As well, there were several distinctive
features of anti-AQP4 antibody-positive MS/NMO relative
to anti-AQP4 antibody-negative OSMS with LESCLs; such
as exclusive occurrence in females, higher age at onset,
higher relapse rate and an absence of secondary progression
in the former. These patients also showed poor responses to
interferon beta-1b, although this was not decisive due to
the limitations of the small size of the retrospective study.
However, CSF inflammatory cell response was more
marked and brain lesions were the least in the latter,
suggesting that the latter has more restricted involvement of
the CNS accompanied with graver inflammatory cell
responses. These observations, together with the constant
anti-AQP4 antibody negativity over the long clinical course,
may indicate that a mechanism apart from anti-AQP4
antibody production is operative in anti-AQP4 antibodynegative OSMS patients with LESCLs. Various unusual
features of MS seen in anti-AQP4 antibody-positive MS/
NMO may indicate that the disease is fundamentally
distinct from MS. However, since anti-AQP4 antibodypositive MS/NMO frequently shows brain lesions, while
antibody-negative OSMS with LESCLs is compatible with
NMO in Western populations, patients carrying anti-AQP4
antibody do not appear to perfectly overlap with OSMS or
NMO in Asians, and so may be separately classified as an
autoimmune aquaporinopathy of the CNS. Further studies
1220
Brain (2007), 130, 1206 ^1223
T. Matsuoka et al.
Table 6 Comparison of responses to interferon beta-1b between anti-AQP4 antibody-positive multiple sclerosis patients
fulfilling the new criteria for definite neuromyelitis optica and anti-AQP4 antibody-negative opticospinal form of multiple
sclerosis patients with longitudinally extensive spinal cord lesions
Administration of IFN beta-1b
Cessation of IFN beta-1bb
Periods of IFN beta-1b administration (mean SD, years)
Annual relapse rate (mean SD, years)
2 years prior to IFN beta-1b
2 years on IFN beta-1bc
% changes
More than 50% reduction in relapse rated
Immunotherapies in 2 years prior to IFN beta-1b
Methylprednisolone pulse therapy
Corticosteroids
Azathioprine
Immunotherapies in 2 years on IFN beta-1b
Methylprednisolone pulse therapy
Corticosteroids
Azathioprine
Anti-AQP4 Ab(þ)
MS/NMO (n ¼14)
Anti-AQP4 Ab()
OSMS with LESCLs (n ¼18)a
7/14 (50.0%)
5/7 (71.4%)
2.2 1.8
11/18 (61.1%)
7/11 (63.6%)
2.4 3.0
1.8 0.6
2.3 1.9
þ39.3 122.0
1/7*
i
1.4 0.83 0.6 0.7
58.2 49.8
8/11*
6/7 (85.7%)
3/7 (42.9%)
0/7 (0.0%)
10/11 (90.9%)
6/11 (54.5%)
0/11 (0.0%)
7/7 (100.0%)
4/7 (57.1%)
1/7 (14.3%)
6/11 (54.5%)
6/11 (54.5%)
0/11 (0.0%)
*Significant difference between the linked values (P50.05). Ab ¼ antibody; AQP4 ¼ aquaporin-4; IFN ¼ interferon; LESCLs ¼ longitudinally
extensive spinal cord lesions; NMO ¼ neuromyelitis optica; OSMS ¼ opticospinal form of multiple sclerosis. aTwo OSMS patients who were
positive for NMO-IgG but negative for anti-AQP4 antibody were excluded. bAmong 11 anti-AQP4 antibody-negative OSMS patients with
LESCLs who received interferon beta-1b, seven discontinued the drug (two due to exaggeration of lower limb spasticity, two because of
drug eruption, one for injection site pain, one for depression and one lost to follow-up). Among seven anti-AQP4 antibody-positive MS/
NMO patients, five discontinued the drug (three due to claims of exacerbation after introduction of the drug, one for injection site pain
and one for the intention of getting pregnant). cAnalyses were done by intention to treat. dThe relapse rates were compared between
pretreatment and on-treatment in each group. Analyses were done by intention to treat.
are required to determine whether or not the two
conditions are indeed the same, irrespective of the presence
or absence of the anti-AQP4 antibody.
Earlier pathological studies (Shiraki et al., 1958; Tabira
and Tateishi, 1982) and ours (Ishizu et al., 2005) disclosed
variability in the degree of inflammatory cell infiltration in
Asian OSMS patients; some showed marked perivascular
inflammatory cell cuffing or heavy infiltration of T cells and
neutrophils in the parenchyma of the spinal cord lesions,
while others showed scant inflammatory cell infiltrates.
In pathological studies of Western NMO cases, eosinophil
infiltration has frequently been observed in lesions
(Lucchinetti et al., 2002), whereas we did not detect any
eosinophils in our previous studies (Ishizu et al., 2005).
Such differences in pathology may suggest the heterogeneity
of an effector arm in OSMS and NMO. It is plausible that
anti-AQP4 antibody-negative OSMS patients with LESCLs
may have predominantly T-cell-mediated immune mechanisms, while anti-AQP4 antibody-positive MS/NMO may be
prone to humoral immune mechanisms, as indicated by the
higher frequencies of ANA and SSA/SSB, though these are
not mutually exclusive and may operate in different stages.
AQP4 is located in astrocyte foot processes surrounding
capillaries (Aoki-Yoshino et al., 2005) and its defects
prolong the resolution of vasogenic oedema, as shown
in AQP4-knockout mice (Papadopoulos et al., 2004).
Blood–brain barrier damage strongly induces AQP4 expression in perivascular and parenchymal astrocytes, which in
turn contributes to efficient clearance of vasogenic oedema
(Tomás-Camardiel et al., 2005). Upregulation of AQP4 in
MS lesions has been reported in Japanese MS patients by
some authors (Aoki-Yoshino et al., 2005), while others
(Misu et al., 2006) reported the disappearance of AQP4
around capillaries in one Japanese autopsied case of OSMS.
If the latter is actually a widespread occurrence, anti-AQP4
antibodies may prolong the resolution of tissue oedema
associated with blood–brain barrier destruction through the
perturbation of water channels in anti-AQP4 antibodypositive MS/NMO. Since NMO-IgG immunostaining produces diffuse labelling of CNS tissues (Lennon et al., 2004)
and AQP-4 is present ubiquitously not only in the spinal
cord, but also in the brain (Aoki-Yoshino et al., 2005), the
occurrence of brain lesions in NMO-IgG-positive patients
(Pittock et al., 2006a; Nakashima et al., 2006) may be a
natural outcome, which explains the higher frequency of
brain lesions seen in this condition compared with antiAQP4 antibody-negative OSMS. Moreover, the preferential
involvement of the central grey matter of the spinal
cord, which shows abundant expression of AQP-4
(Misu et al., 2006), may also suggest the involvement
of AQP-4 autoimmunity in lesion development in this
condition.
Aquaporin- 4 autoimmunity in MS
In the present series, anti-AQP4 antibodies and NMOIgG were detected at nearly constant rates over a wide range
of disease durations from the early course to the late stage,
when secondary destruction of optic nerves and spinal cord
occurs repeatedly. These observations are in good accord
with the findings of Weinshenker and colleagues (2006b)
that NMO-IgG appears during the initial attack in onethird of longitudinally extensive transverse myelitis patients.
Therefore, secondary production of the antibody following
destruction of optic nerves and spinal cord tissue may not
occur in the late stage of illness in most cases. However,
this does not necessarily exclude the possibility that the
antibody is produced secondarily as a response to severely
disrupted optic nerves and spinal cord tissue at the very
beginning of the initial insult in susceptible individuals.
Since two patients in our series later developed NMO-IgG
and anti-AQP4 antibody positivity, albeit with relatively
low titres, a longitudinal follow-up study of the antibodies
in high-risk patients would be needed to clarify this point.
If AQP-4 autoimmunity is the sole cause of NMO, it is
hard to fully explain the preferential involvement of the
optic nerves and spinal cord in most NMO patients.
Moreover, from the results of our multiple logistic analyses,
the presence of anti-AQP4 antibodies was not linked to
long spinal cord lesions, disability or even OSMS presentation. Rather, it was most significantly associated with higher
relapse rate. Therefore, it is also possible that the antiAQP4 antibody is a modifying factor contributing to
frequent relapses and prolongation of vasogenic oedema,
but does not have a role in the severity of tissue destruction
or predispose a patient to optic nerve and spinal cord
involvement. Paradoxically, NMO-IgG titres tended to have
an inverse correlation with the progression index. It is
possible that the antibody titres may decrease in chronic
burnt-out cases. However, in AQP4 deficient mice,
cytotoxic oedema following either ischaemic stroke or
water intoxication was decreased and neurological outcomes significantly improved (Manley et al., 2000). Thus,
although anti-AQP4 antibody appears a good marker of
this condition, the antibody may in some cases be
neuroprotective through a reduction in cytotoxic oedemas
by water channel blockade. Further studies on the in vivo
actions of the antibody are necessary to clarify its exact
roles in MS/NMO.
On the other hand, LESCLs in antibody-negative CMS
patients showed the same predilection for peripheral white
matter and the cervical cord as with short lesions. These
characteristics are the same as those reported in Western MS
(Tartaglino et al., 1995). In anti-AQP4 antibody-negative
CMS, when clinical characteristics were compared between
LESCL-positive and -negative patients, LESCL-positive ones
had significantly higher relapse rate, greater EDSS scores, and
higher frequencies of ATM and secondary progression than
LESCL-negative ones; also, Barkhof brain lesions were more
common in LESCL-negative patients (Supplementary
Table 1). Therefore, anti-AQP4 antibody-negative CMS
Brain (2007), 130, 1206 ^1223
1221
patients with LESCLs seem to have classical MS that is
similar to Western MS with high disease activity. This
suggests a possibility that most of the LESCLs in antibodynegative CMS patients are of a similar nature to short
demyelinated lesions and may indeed be conglomerations
of short ones.
In the present study, as classifications of clinical
phenotypes were based on clinical symptomatology,
subclinical or silent brain lesions on MRI could possibly
be found in OSMS patients. However, it has repeatedly
been reported in Asians, even in CMS patients, that long
spinal cord lesions are not infrequent (Chong et al., 2004;
Su et al., 2006; Minohara et al., 2006). This is consistent
with the present result that up to 30% of anti-AQP4
antibody-negative CMS patients demonstrated LESCLs.
Existence of cases intermediate between OSMS and CMS
is in part attributable to the limitation of clinical
classifications; however, such intermediate cases have
frequently been reported in Asians clinically and pathologically (Okinaka et al., 1958; Shibasaki and Kuroiwa, 1973;
Chong et al., 2004; Su et al., 2006). It remains to be
determined whether or not the higher frequency of LESCLs
in anti-AQP4 antibody-negative CMS patients in Japanese
as well as in other Asians (Chong et al., 2004; Su et al.,
2006; Minohara et al., 2006) in comparison to those
in Western MS series (Tartaglino et al., 1995) is caused
by genetic differences.
Finally, to exclude the possibility that anti-AQP4 antibody-positive MS/NMO and anti-AQP4 antibody-negative
OSMS and CMS are a continuum, we considered the
following. First, the anti-AQP4 antibody-positive MS/NMO
and the antibody-negative OSMS patients with LESCLs also
had the periventricular ovoid lesions that are typically seen
in CMS. Also, pathologically sharply demarcated demyelinated lesions in extra-opticospinal regions have repeatedly
been reported in NMO in Western (Cone et al., 1934;
Balser, 1936; Stansbury, 1949) and OSMS in Asian
(Shiraki et al., 1958; Okinaka et al., 1958; Shibasaki
and Kuroiwa, 1973; Shibasaki et al., 1974) populations.
Even a huge atypical brain lesion in one of our anti-AQP4
antibody-positive MS/NMO patients (shown in Fig. 3F-3)
demonstrated increased diffusivity [increased apparent
diffusion coefficient (ADC) values] on the ADC map
of the diffusion-weighted sequence, along with increased
choline and decreased N-acetylaspartate on magnetic
resonance spectroscopy (Supplementary Fig. 3). These
findings are compatible with acute demyelination. Second,
we could not detect any statistically significant difference
in the frequency of atypical brain lesions previously
reported in NMO-IgG-positive NMO patients (Pittock
et al., 2006a, b) among the three above-mentioned
subgroups; yet periventricular rim-like lesions were more
common in OSMS irrespective of presence or absence
of the anti-AQP4 antibody. Third, some antibody-negative
CMS patients with Barkhof brain lesions also had extremely
long spinal cord lesions with central grey matter
1222
Brain (2007), 130, 1206 ^1223
involvement (an intermediate case in Supplementary
Fig. 4). Fourth, recent clinico-epidemiological studies
showed changes in the clinical phenotypes from CMS to
OSMS in Japanese (Kira et al., 1999; Nakashima et al.,
1999) and NMO to classical MS in French West Indies
(Cabre et al., 2005). Finally, interferon beta-1b was shown
to be beneficial for OSMS in Japanese by a double blind
randomized controlled trial (Saida et al., 2005). Unless we
consider that these conditions constitute a spectrum, it is
difficult to explain the frequent occurrence of such
additional demyelinated lesions in the brain of anti-AQP4
antibody-positive MS/NMO patients, the existence of
intermediate cases, and the phenotypic changes of CNS
demyelinating diseases in the same races over time.
However, demyelinated lesions indistinguishable from MS
may tend to develop secondarily under CNS inflammatory
conditions such as found with NMO (autoimmune
aquaporinopathy). The beneficial effects of interferon beta
on OSMS as well as the phenotypic changes from OSMS
to CMS may also be explained by splitting OSMS into antiAQP4 antibody-positive NMO (autoimmune aquaporinopathy) distinct from MS, and anti-AQP4 antibody-negative
OSMS that forms part of MS.
In summary, anti-AQP4 antibodies are present in 30%
of Japanese OSMS patients over a wide range of disease
durations. Patients with these antibodies have various
distinct features. However, there are also cases of antiAQP4 antibody-negative OSMS with extremely long spinal
cord lesions and few brain lesions, indicating that the
mechanisms of LESCL production in OSMS are heterogeneous, i.e. both anti-AQP4 antibody-related and
-unrelated. Further characterization of the precise actions
of anti-AQP4 antibodies in anti-AQP4 antibody-positive
MS/NMO patients, and searches for other autoantigens in
anti-AQP4 antibody-negative OSMS patients may shed light
on the relationships among NMO, OSMS and CMS at the
molecular level.
Acknowledgements
We wish to thank Professors Allan G. Kermode and
William Carroll (Australian Neuromuscular Research
Institute, Sir Charles Gairdner Hospital), and Professor
Brian G. Weinshenker (Department of Neurology,
Mayo Clinic College of Medicine), for valuable comments
on the manuscript. This work was supported in part by
a Neuroimmunological Disease Research Committee
grant from the Ministry of Health, Labour and Welfare,
Japan.
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