doi:10.1093/brain/awm038
Brain (2007), 130, 1089^1104
Meningeal B-cell follicles in secondary progressive
multiple sclerosis associate with early onset of disease
and severe cortical pathology
Roberta Magliozzi,1,2 Owain Howell,2 Abhilash Vora,2 Barbara Serafini,1 Richard Nicholas,2 Maria Puopolo,1
Richard Reynolds2, and Francesca Aloisi1,
1
Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita', Rome, Italy and 2Department of Cellular and
Molecular Neuroscience, Imperial College Faculty of Medicine, London, UK
Correspondence to: Dr Francesca Aloisi, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita', Viale
Regina Elena 299, 00161 Rome, Italy and Prof Richard Reynolds, Department of Cellular & Molecular Neuroscience, Division of
Neuroscience, Imperial College London, Charing Cross Hospital Campus, Fulham Palace Road, London W6 8RF, UK
E-mail: [email protected]; E-mail: [email protected]
These authors share equal credit for senior authorship.
Intrathecal antibody production is a hallmark of multiple sclerosis and humoral immunity is thought to play
an important role in the inflammatory response and development of demyelinated lesions. The presence of
lymphoid follicle-like structures in the cerebral meninges of some multiple sclerosis patients indicates
that B-cell maturation can be sustained locally within the CNS and contribute to the establishment of a
compartmentalized humoral immune response. In this study we examined the distribution of ectopic B-cell
follicles in multiple sclerosis cases with primary and secondary progressive clinical courses to determine their
association with clinical and neuropathological features. A detailed immunohistochemical and morphometric
analysis was performed on post-mortem brain tissue samples from 29 secondary progressive (SP) and 7 primary
progressive (PP) multiple sclerosis cases. B-cell follicles were detected in the meninges entering the cerebral
sulci of 41.4% of the SPMS cases, but not in PPMS cases. The SPMS cases with follicles significantly differed
from those without with respect to a younger age at multiple sclerosis onset, irreversible disability and death
and more pronounced demyelination, microglia activation and loss of neurites in the cerebral cortex. Cortical
demyelination in these SPMS cases was also more severe than in PPMS cases. Notably, all meningeal B-cell
follicles were found adjacent to large subpial cortical lesions, suggesting that soluble factors diffusing from
these structures have a pathogenic role. These data support an immunopathogenetic mechanism whereby
B-cell follicles developing in the multiple sclerosis meninges exacerbate the detrimental effects of humoral
immunity with a subsequent major impact on the integrity of the cortical structures.
Keywords: multiple sclerosis; B cells; ectopic follicles; demyelination; neurodegeneration
Abbreviations: FDC ¼ follicular dendritic cell; GML ¼ grey matter lesion; Ig ¼ immunoglobulins; LFB ¼ Luxol Fast Blue;
MHC ¼ major histocompatibility complex; MBP ¼ myelin basic protein; MOG ¼ myelin oligodendrocyte glycoprotein;
NAGM ¼ normal appearing grey matter; PPMS ¼ primary progressive multiple sclerosis; SPMS ¼ secondary progressive
multiple sclerosis; WML ¼ white matter lesion.
Received December 4, 2006. Revised January 23, 2007. Accepted February 12, 2007
Introduction
Multiple sclerosis is a CNS-specific, putatively autoimmune
disease that causes inflammation in the brain and spinal cord
and results in demyelination, axonal damage and neuronal
loss. As in other chronic inflammatory diseases, the CNS of
patients with multiple sclerosis shows infiltration of activated
T cells and macrophages, dendritic cells, B cells and plasma
cells. This implies potential roles for both cellular and
humoral immune responses and the engagement of different
immunopathological effector mechanisms in CNS tissue
destruction. While the association of multiple sclerosis
with certain MHC class II genes and an extensive literature
in experimental models of multiple sclerosis favour a
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]
1090
Brain (2007), 130, 1089^1104
R. Magliozzi et al.
T-cell-mediated pathogenesis, an important role for humoral
immunity in the development of multiple sclerosis lesions is
also recognized (Sospedra and Martin, 2005; Owens et al.,
2006; Frohman et al., 2006). Elevated CNS levels of
immunoglobulins (Ig) and the presence of oligoclonal IgG
in the CSF are the most consistent immunological abnormalities in multiple sclerosis (Tourtellotte et al., 1984; Archelos
et al., 2000). Such intrathecal immunoglobulin synthesis is
thought to be sustained by long-lived plasma cells recruited
to or differentiating within the CNS (Prineas and Wright,
1978). Neuropathological studies indicate that antibodymediated demyelination is one of the predominant
pathogenetic mechanisms involved in white matter lesion
formation in a substantial proportion of multiple sclerosis
patients (Lucchinetti et al., 2000). Capping of surface IgG on
microglia/macrophages engaged in myelin breakdown and
co-deposition of IgG and activated complement fragments or
complexes at the borders of active multiple sclerosis lesions
strongly implicate antibodies as effectors of demyelination
(Prineas and Graham, 1981; Storch et al., 1988). Despite
detection of antibodies recognizing myelin and neuronal
antigens in serum, CSF and demyelinating lesions of multiple
sclerosis patients, it is still unclear whether such autoantibodies have a pathogenic role (Sospedra and Martin, 2005;
Owens et al., 2006). The recent finding that CSF oligoclonal
IgG bind Epstein–Barr virus proteins indicates that the
compartmentalized B-cell response in multiple sclerosis
could be sustained by a viral infection (Rand et al., 2000;
Cepok et al., 2005).
Recently, molecular analyses of the Ig variable gene region
of B cells and plasma cells isolated from the demyelinated
lesions or CSF of multiple sclerosis patients have provided
evidence that the intrathecal humoral immune response in
multiple sclerosis is antigen-driven and that dominant B-cell
clonotypes persist over time in the CNS compartment (Qin
et al., 1998; Baranzini et al., 1999; Colombo et al., 2000;
Owens et al., 2003; Qin et al., 2003; Colombo et al., 2003).
Remarkably, it was found that a complete recapitulation of
B-cell differentiation resembling a germinal centre reaction
occurs in the CSF of multiple sclerosis patients (Corcione
et al., 2004). By studying the cellular composition and
organization of the inflammatory infiltrates in post-mortem
brain tissue from a limited number of multiple sclerosis
cases, we have shown that in secondary progressive multiple
sclerosis (SPMS) the inflamed cerebral meninges contain
structures that are strikingly similar to secondary B-cell
follicles containing germinal centres (Serafini et al., 2004).
The ectopic follicles found in SPMS comprised proliferating
B cells, plasma cells, T cells and a network of follicular
dendritic cells (FDC), which are essential for B-cell maturation due to their ability to retain antigens on their membrane and to stimulate B-cell proliferation and survival
(Park and Choi, 2005). These findings indicate that the
inflamed CNS is able to sustain B-cell responses through an
inherent predisposition of the meningeal compartment to
favour the organization of ectopic lymphoid tissue. In other
organ-specific autoimmune diseases, such as Hashimoto’s
thyroiditis, myasthenia gravis and Sjogren’s syndrome,
ectopic germinal centres are a source of disease-relevant
antibodies, which suggests that such abnormal formations
may contribute to the pathogenic process through local
amplification of the autoimmune response (Aloisi and
Pujol-Borell, 2006).
Based on the assumption that a better knowledge of the
events associated with ectopic follicle formation in multiple
sclerosis may shed light on the still elusive immunemediated mechanisms underlying CNS tissue destruction,
we have begun to ascertain the pathological relevance of
such abnormal structures by analysing autopsy brain tissue
from a larger sample of multiple sclerosis cases with
progressive disease courses, and to search for any relationship between presence of follicles, clinical course and
neuropathological features.
Material and methods
Demographic and clinical data
This study was performed on autopsy brain tissue from 29 cases
with SPMS and 7 with primary progressive (PP) multiple sclerosis.
All tissues were obtained from the UK Multiple Sclerosis Tissue
Bank at Imperial College, except for MSG1 that was provided
by the Institute of Pathological Anatomy, U.C.S.C. Policlinico
A. Gemelli, Rome, Italy. The multiple sclerosis cases selected
for this study had a range of ages at death (35–81 years), ages
at multiple sclerosis onset (10–56 years) and disease duration
(6–52 years), reflecting the variability of the multiple sclerosis
population. The demographic and clinical data for each multiple
sclerosis group and the 36 individual cases are shown in Tables 1
and 2, respectively. This study includes three SPMS (MS79,
MS80 and MS85) and two PPMS (MS83 and MSG1) cases
that were already examined in Serafini et al. (2004). The UK
Multiple Sclerosis Tissue Bank also provided control postmortem brain tissues from three patients without evidence of
neurological disease or neuropathological alterations: C4, a male
patient (57-year-old) who died from bronchial cancer; C20, a
Table 1 Demographic and clinical data
MS course
Number of cases
Female:male ratio
Age at deatha (years)
Age at onseta (years)
Disease durationa (years)
SPMS
PPMS
n ¼ 29
n¼7
1.42
2.5
50 (35^ 81)
66 (43^78)
28 (10 ^ 42)
36 (21^56)
25 (6 ^ 48)
25 (10 ^52)
a
Values represent medians; the range for values is given in brackets.
Sex/age
at death
(years)
Type of
MS
Age at
onset
(years)
Disease
duration
(years)
Immunotherapy
Cause of deatha
Post-mortem
delay (h)
Degree of
perivascular
inflammationb/
degree of meningeal
inflammationc
Number of brain
tissue blocks with
ectopic follicles/
examined blocks
(number of follicles)
MS 200
MS 163
F/43
F/45
SP
SP
24
39
19
6
Urinary tract infection
Urinary tract infection
20
28
þ/
þ/
0/8
0/8
MS 100
MS 101
MS 42
MS 127
MS 114
MS 104
MS 3
MS 109
MS 56
MS 74
MS 141
MS 80
MS 81
MS 71
MS 99
MS 154
MS 176
MS 92
MS 136
MS 157
M/46
M/50
M/51
M/51
F/52
M/53
M/55
F/60
M/63
F/64
M/66
F/71
M/72
F/78
F/81
F/35
M/37
F/37
M/40
F/40
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
38
19
29
28
37
42
34
35
24
28
29
36
24
36
40
23
10
20
28
18
8
31
22
23
15
11
21
25
39
36
37
35
48
42
41
12
27
17
12
22
Bronchopneumonia
Bronchopneumonia
Urinary tract infection
Bronchopneumonia
Bronchopneumonia
Urinary tract infection
Urinary tract infection
Myocardial infarct
Bronchopneumonia
Respiratory failure
Rectal carcinoma
Heart failure
Bronchopneumonia
Bronchial carcinoma
MS
Bronchopneumonia
Intestinal obstruction
MS
Respiratory failure
Dehydration
7
24
8
21
12
12
44
22
11
7
20
24
23
5
23
12
12
26
10
12
/
þ/
þ/
þ/
/
þ/
þ/
/
/
þ/
/
þ/
/
/
/
þþ/þ
þþ/þ
þþ/þ
þþ/þ
þþ/þ
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
4/8 (6)
3/8 (5)
7/8 (14)
2/8 (3)
3/8 (5)
MS 46
MS 160
MS 180
MS 121
MS 79
MS 153
MS 85
MSG1
MS 83
MS 182
MS 129
MS 102
MS 70
MS 57
M/40
F/44
F/44
F/49
F/49
F/50
F/59
F/43
M/53
F/56
F/66
M/73
F/77
F/78
SP
SP
SP
SP
SP
SP
SP
PP
PP
PP
PP
PP
PP
PP
17
28
26
35
25
18
24
33
36
23
41
21
56
47
23
16
18
14
24
32
35
10
17
33
25
52
21
31
Age 39: azathioprine for 11 months
Age 41: Avonex/immunoglobulin
for 15 months
None
None
None
Age 47: Betaseron for 12 months
Age 41: ACTH for 12 months
None
None
None
None
None
None
Age 51: ACTH for 11 years
None
None
None
None
None
None
Age 39: Rebif for 12 months
Age 31: azathioprine for
8 months
Age 35: methotrexate for
4 months
None
None
None
None
None
Age 42: ACTH for 30 months
None
None
None
None
None
None
None
None
MS
Bronchopneumonia
MS
Bronchopneumonia
Bronchopneumonia
MS
Cerebrovascular accident
Cerebrovascular disease
Bronchopneumonia
Bronchopneumonia
Respiratory failure
Bronchopneumonia
Cerebrovascular accident
Bronchopneumonia
18
18
9
24
7
12
27
24
13
14
8
20
21
9
þþ/þ
þþ/þ
þþ/þ
þþ/þ
þþ/þ
þþ/þ
þþ/þ
/
/
/
/
þ/
þ/
þ/
3/8 (6)
4/8 (9)
4/8 (8)
5/8 (5)
5/8 (6)
6/8 (8)
2/8 (2)
0/4
0/8
0/8
0/8
0/8
0/8
0/8
Brain (2007), 130, 1089^1104
Case
Ectopic follicles in multiple sclerosis
Table 2 Individual clinical, autopsy and neuropathology details
a
1091
In the clinical records, multiple sclerosis is sometimes stated as a cause of death where death occurred as a direct result of multiple sclerosis or from a directly related condition.
Perivascular inflammation was scored as described in section Material and methods: (530 infiltrating cells) ¼ negligible; þ (31^ 60 infiltrating cells) ¼ sparse/moderate; þþ (460
infiltrating cells) ¼ abundant. cMeningeal inflammation was scored as: ¼ absent; ¼ moderate; þ ¼ abundant.
b
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Brain (2007), 130, 1089^1104
R. Magliozzi et al.
female patient (82-year-old) who died from cardiac failure and
C25, a male patient (35-year-old) who died with tongue
carcinoma.
All post-mortem tissues were obtained via a UK prospective
donor scheme with full ethical approval (MREC/02/2/39) except for
case MSG1 where separate ethical approval was obtained from the
Ethics Committee of the Istituto Superiore di Sanità, Rome.
Confirmation of the diagnosis of multiple sclerosis for each case was
provided by Dr F. Roncaroli (Consultant Neuropathologist,
Department of Neuropathology, Imperial College London). A
summary of the clinical history for each case was prepared by a
clinical neurologist with an interest in multiple sclerosis (R.N.).
Details of the history included date of symptom onset, date of
diagnosis, number, date and character of relapses, date of onset of
progression, date of wheelchair dependence (EDSS 7 equivalent)
and date of death (EDSS 10 equivalent). Details of any
co-morbidities were noted as well as treatments both for multiple
sclerosis and other co-morbidities (Table 2).
Tissue and lesion classification
For each multiple sclerosis case, eight cerebral tissue blocks
(4 cm3), including cortical and periventricular areas with white
matter (WM) demyelination and an intact meningeal compartment were examined. In order to sample as widely as possible
from the cerebrum and to avoid sample bias, tissue blocks from
frontal, temporal, parietal, occipital and central (insula) lobes were
selected from the digital images of coronal brain slices acquired at
the time of dissection at the UK Multiple Sclerosis Tissue Bank.
Other CNS regions (mamillary bodies, basal ganglia, pons,
medulla oblongata, cerebellum and spinal cord) were also analysed
in 19 SPMS and 5 PPMS cases. The post-mortem delay ranged
between 5 and 44 h (median time, 16 h). Four tissue blocks from
MSG1 were formalin fixed and paraffin embedded. Tissue blocks
from the remaining cases (eight blocks per case) were snap frozen
in isopentane on dry ice (four blocks) or fixed in 4%
paraformaldehyde (mean fixation time ¼ 17 6 days), cryoprotected in a 30% sucrose, frozen in cooled isopentane and stored
at 75 C (four blocks).
Inflammatory cell infiltrates were studied using haematoxylin–
eosin staining. The degree of WM inflammation was evaluated
manually by counting the number of DAPI-positive nuclei in four
randomly selected perivascular infiltrates for each multiple
sclerosis case (Table 2). The extent of demyelination and degree
of lesion activity were evaluated by combining Luxol Fast Blue
(LFB) histochemical staining with major histocompatibility
complex (MHC) class II molecule immunostaining (see later).
Because grey matter lesions (GML) are difficult to identify in
sections stained with LFB, we also used myelin basic protein
(MBP) and myelin oligodendrocyte glycoprotein (MOG) immunohistochemistry (Table 3). Since the extent of GM demyelination
appeared almost identical when probed with anti-MBP and
anti-MOG antibodies (data not shown), MOG immunostaining
was used to quantify the areas of both cortical and WM
demyelination. The inflammatory activity of GML and WML
was defined by the pattern of MHC class II staining: active lesions
contained numerous MHC class IIþ cells in the lesion core and at
the lesion border; chronic active lesions had a border of MHC
class IIþ cells and a lower number in the core; chronic inactive
lesions had a very low MHC class IIþ cell density throughout
the lesion.
Immunohistochemistry
Air dried, acetone fixed (þ4 C), 10 mm-thick cryosections, cut
from the 4% paraformaldehyde fixed and snap frozen tissue
blocks, were rehydrated with PBS and immunostained with the
monoclonal or polyclonal antibodies listed in Table 3. Post-fixed
sections were subjected to an antigen retrieval procedure, either
microwave treatment in citrate buffer 10 mM (pH 6.0) or
incubation in target retrieval solution (Dako; 90 C) or permeabilization with cold methanol (10 min at –20 C). Sections were
Table 3 Primary antibodies used for immunohistochemistry
Antigen
Cell specificity
Clone
Dilution
Source
CD3
CD20
CD138 (Syndecan-1)
CD35(C3b/C4b receptor, CR1)
CD68
CXCL13
Fluorescein-conjugated
Ig-A, -G, -M
Ki67
MHC class II
MOG
MBP
T lymphocytes
B lymphocytes
Plasma cells
Stromal cells/FDC
Macrophages
Stromal cells/FDC
Plasmablasts and plasma cells
PS1
L26
B-B4
Ber/MAC/DRC
KP1
Goat polyclonal
Rabbit polyclonal
Pre-diluted
Pre-diluted
1:100
1:25
1:50
1:20
1:300
Immunotech, Marseille, France
Immunotech
Serotec, Oxford, UK
Dako, Carpinteria, CA
Dako
R&D System, Minneapolis, Minn
Dako
Proliferating cells
Antigen presenting cells
Myelin and oligodendrocytes
Myelin and oligodendrocytes
Rabbit polyclonal
CR3/43
Z12
Rabbit polyclonal
1:400
1:50
1:50
1:500
SMI32
Dephosphorylated neurofilaments Smi32
1:500
Neurofilament 200 kDa
Neurofilaments
1:500
Novocastra Laboratories, Newcastle, UK
Dako
Kind gift of S. Piddlesden, Cardiff, UK
R. Reynolds, Imperial College
London, UK
Sternberger Monoclonals Inc.,
Berkeley, CA
Chemicon International, Temecula, CA
RT97
Antigen retrieval procedures for CD3, CD20, CD68, CXCL13 and Ki67 immunostainings utilized microwave of sections in citrate buffer; for
CD35 immunostaining, heat treatment with Dako target retrieval solution was performed. For MBP and MOG immunostaining, treatment
with cold methanol was performed.
Ectopic follicles in multiple sclerosis
then incubated for 30 min with 0.1% H2O2 in PBS to eliminate
endogenous peroxidase activity, for 1 h with 10% of normal sera,
and overnight at 4 C with the primary antibodies diluted in PBS
containing 0.2% Triton 100-X and 1% BSA or 1% normal
sera. Binding of biotinylated secondary antibodies (Jackson
Immunoresearch Laboratories) was visualized with the avidin–
biotin horseradish peroxidase complex (ABC Vectastain Elite kit;
Vector Laboratories, Burlingame, CA) followed by 3,30 -diaminobenzidine (DAB) (Sigma Chemical Co., St Louis, MO) as
substrate. All sections were counterstained with haematoxylin,
sealed with Depex Polystyrene (DPX) and viewed with a Nikon
E1000M microscope. Images were captured with a QICAM digital
camera (QImaging Inc.), and analysed using Image Pro Plus
software (Media Cybernetics Inc.). Negative controls included IgG
isotype controls or preimmune serum, or omission of the primary
antibody.
Immunofluorescence
Cryosections of multiple sclerosis and control brain tissues,
prepared as earlier, were also stained using a double immunofluorescence technique employing anti-MBP polyclonal antibody
in combination with anti-MHC class II, SMI-32 or RT97
monoclonal antibodies, and anti-CD20 monoclonal antibody in
combination with anti-CXCL13 or Ki67 polyclonal antibodies
(Table 3). After an initial post-fixation in cold acetone and
blockade with 5–10% normal sera in PBS, sections were incubated
overnight at 4 C with the primary antibodies (listed in Table 3),
diluted in PBS containing 0.2% Triton X-100 and 1% normal sera.
Sections were then washed, treated with Cy3-, rhodamineor fluorescein-conjugated anti-rabbit or anti-goat IgG and
fluorescein- or rhodamine-conjugated anti-mouse IgG in PBS
containing 1% normal sera for 1 h, washed again in PBS and then
in distilled water. Some sections were counterstained with 40 , 6diamidino-2-phenylindole (DAPI, Sigma) for the localization of
cell nuclei. Finally, sections were coverslipped with aqueous
mounting medium Vectashield (Vector Laboratories). For
RT97 immunostaining, a streptavidin-conjugated Alexafluor-546
amplification (Invitrogen) was performed after incubation
with the biotinylated anti-mouse Ig secondary antibody. Single
immunostaining with a fluorescein-labelled anti-Ig-A, -G, -M
polyclonal antibody was also performed. For negative controls,
the primary antibodies were replaced with preimmune serum or
IgG isotype controls. Slides were viewed under epi-fluorescence
with the Nikon E1000M microscope and images captured as
described.
Quantitative analysis of demyelinated lesion size
and neurite loss
To determine the number and size of WML and GML, one section
from each paraformaldehyde fixed tissue block (four blocks per
case) was immunostained for MOG. Low magnification images
(four frames per section) were acquired at 0.5 magnification.
The areas of demyelination and the total area of WM and GM
were manually outlined for each section and then automatically
analysed using the Image Pro Plus software. GML were
characterized into type I (grey matter/white matter border), type
II (intracortical) or type III (subpial) according to the criteria of
Peterson et al. (2001).
Neurite density was analysed in chronic active type III GML
and equivalent areas of subpial normal appearing grey matter
Brain (2007), 130, 1089^1104
1093
(NAGM). Two serial sections for each tissue block from 12 B-cell
follicle-positive SPMS, 12 B-cell follicle-negative SPMS (both
groups containing chronic active GML and NAGM) and 3 control
cases were immunostained with anti-MBP polyclonal antibody
and anti-MHC class II monoclonal antibody (for classification of
the lesion activity) and with anti-MBP polyclonal antibody and
RT97 monoclonal antibody recognizing the phosphorylated
200 kDa neurofilament protein. For each multiple sclerosis case,
we analysed one subpial (type III) chronic active cortical lesion
and the NAGM distant (51 cm) from the lesion. Our analysis was
limited to layers II and III of the cerebral cortex as these were
always fully demyelinated and contained activated microglia in the
chronic active GML group. For each lesion and NAGM area, four
images were acquired at 400 magnification and the number of
RT97þ neurites was manually counted by two investigators
blinded to the case number (R.M. and O.H., inter-individual
variability 52%). Total neurite density (per 0.1 mm2) was
calculated from four counting squares (0.0036 mm2) located at
the corners of each image to ensure that no RT97þ structures
were counted twice.
Statistical analysis
Data are expressed as median and half the interquartile range
(IQR/2), as a measure of variability, or as mean standard error
of the mean (SEM). Comparisons between groups of multiple
sclerosis patients were carried out by the Mann–Whitney U-test
for continuous variables and by the chi-square test or Fisher’s
exact probability test for categorical variables. Comparisons within
each multiple sclerosis subgroup for continuous variables were
carried out by the Wilcoxon test for repeated measures.
Spearman’s rank correlation coefficients were calculated to
investigate the linear relationship between pairs of variables.
Survival curves for age at which the patient became a wheelchair
user were established by the Kaplan–Meier method and comparisons between SPMS subgroups were assessed by the generalized
Wilcoxon test. Bonferroni’s correction has been adopted to
control for multiple comparisons.
Results
Ectopic B-cell follicles are detected in SPMS,
but not in PPMS
In agreement with previous findings (Serafini et al., 2004),
prominent inflammatory cell infiltrates comprising CD3þ
T cells, CD20þ B cells, CD138þ or Igþ plasmablasts/
plasma cells, CD68þ macrophages and large perivascular
B-cell aggregates were detected in the cerebral leptomeninges of 12 out of 29 cases with SPMS, hereafter denoted
Fþ SPMS (Table 2). In all Fþ SPMS cases, the meningeal
B-cell aggregates were characterized as ectopic follicles with
germinal centres based on the presence of a reticulum of
CD35þ and CXCL13þ cells (bona fide stromal cells and
FDC), proliferating Ki67þ B cells and Igþ plasmablasts/
plasma cells (Fig. 1A–D). Only in one case (MS85), did we
observe one out of two meningeal follicles that lacked
CD138þ plasma cells and a well-developed FDC network,
indicating incomplete germinal centre formation.
Meningeal follicles were present in 48 out of 96 cerebral
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Brain (2007), 130, 1089^1104
tissue blocks examined, with a mean of 6.4 3.1 follicles
(range 2–14) found in sections from the eight blocks
studied for each Fþ SPMS case (Table 2). In most
Fþ SPMS cases, numerous B cells and plasma cells were
also observed in the perivascular cuffs of WML (Fig. 1E).
The ectopic follicles were mainly found in the frontal,
temporal and parietal lobes and in the cingulate gyrus.
We found only one tissue block containing a meningeal
B-cell follicle in the occipital lobe and one in the insula.
Although most of the selected tissue blocks (71%)
contained the external surface of the brain, the follicles
were always found along, in the depth and, more rarely, at
the entrance of the cerebral sulci, in particular in the central
and lateral sulci (Fig. 1H–J). Only sparse inflammatory
R. Magliozzi et al.
infiltrates were detected in the meninges covering the
external surface of the brain.
In the remaining 17 SPMS cases that lacked ectopic
follicles (denominated F SPMS), meningeal immune cell
infiltration was absent (6 cases) or much less prominent
(11 cases) than in the Fþ SPMS group (Table 2). Only
sparse B cells and plasma cells were detected in the
moderately inflamed meninges of the F SPMS cases,
particularly at the tip of the cerebral sulci (Fig. 1F).
The perivascular cuffs of WML were also less prominent in
the F SPMS group than in the Fþ SPMS group and
contained only occasional B cells and plasma cells (data not
shown). Counts of DAPI-stained nuclei in four randomly
selected WM perivascular cuffs for each case revealed
Fig. 1 Characterization of ectopic B-cell follicles and inflammatory cell infiltrates in post-mortem brain tissue from cases with SPMS and
PPMS. Immunostainings of serial brain sections from a Fþ SPMS case (A^D) shows an intrameningeal ectopic B-cell follicle in a cerebral
sulcus containing CD20þ B cells (A), ramified stromal cells/FDC expressing CD35 (inset in A) and CXCL13 [B, double immunofluorescence
staining with monoclonal anti-CD20 (red) and polyclonal anti-CXCL13 (green) antibodies], proliferating B cells [C, double immunofluorescence staining with monoclonal anti-CD20 (green) and polyclonal anti-Ki67 (red) antibodies] and plasmablasts/plasma cells stained with an
anti-Ig-G, -A, -M polyclonal antibody (D; the inset shows two intrafollicular plasma cells at high-power magnification). Panel E shows prominent perivascular accumulation of CD20þ B cells in a periventricular WML from a Fþ SPMS case. Several scattered CD20þ B cells are
present in the scarcely inflamed meninges entering a cerebral sulcus in a F SPMS case (F) and in a PPMS case (G). The lower, composite
panel illustrates the localization of ectopic B-cell follicles in the multiple sclerosis brain. The schematic drawing shows that ectopic B-cell
follicles develop along (H) and in the depth (I) of the cerebral sulci, whereas scattered B lymphocytes (J) are detected in the meninges
covering the external brain surface. The micrographs in panels H^J show representative fields from a Fþ SPMS case out of the 12
examined. Original magnifications: E^G ¼100 ; A, D, H^J ¼ 200, B, C and insets in A and D ¼ 400.
Ectopic follicles in multiple sclerosis
that the number of perivascular cells in the F SPMS group
was significantly lower than in the Fþ SPMS group
[median values (range), 34 (29–50) versus 94 (65–103),
respectively; P50.0001].
Ectopic follicles were not detected in the seven cases with
PPMS, of which only three showed moderate meningeal
inflammation with scattered B cells and plasma cells
(Fig. 1G and Table 2). In PPMS cases, the size of the
inflammatory cell infiltrates in WML was significantly lower
than that observed in Fþ SPMS cases [median value
(range) of DAPI-stained nuclei counted in four WM
perivascular cuffs ¼ 29 (19–34); P ¼ 0.0007]. Only a few
B cells and plasma cells were detected in the perivascular
cuffs of WML in the PPMS cases (data not shown).
To investigate whether ectopic follicles could form in
CNS areas away from the cerebral cortex, we also analysed
tissue blocks containing mamillary bodies, dentate nucleus,
pons, medulla oblongata, cerebellum and spinal cord from
9 Fþ SPMS, 10 F SPMS and 5 PPMS cases. Using both
haematoxylin/eosin staining and CD20 immunostaining, no
B-cell follicles were found in any of these areas, with the
exception of one Fþ SPMS case (MS 121) which had
prominent meningeal inflammation and one B-cell follicle
in the brainstem (data not shown).
Earlier age at clinical onset and at death in
the SPMS cases with ectopic B-cell follicles
When comparing the 12 cases in the Fþ SPMS group with
the 17 cases in the F SPMS group, we found that the two
groups differed with regard to the age at clinical onset,
which was significantly earlier for Fþ than F SPMS cases;
[median value ¼ 23.5 (4.7) versus 34 (5.7) years;
P ¼ 0.0019] (Fig. 2A). Interestingly, the proportion of Fþ
SPMS cases progressively decreased from 83% (5/6) to 46%
(6/13) to 10% (1/10) in the SPMS population studied here
developing disease in the second, third and fourth decade
Brain (2007), 130, 1089^1104
1095
of life, respectively (chi-square test for trend, P ¼ 0.0035),
supporting a link between early disease onset and the
formation of ectopic B-cell follicles in the late relapsing or
progressive phase of the disease. In the SPMS patient
population examined here the female : male ratio was 1.42.
However, a greater female preponderance was seen in the
Fþ SPMS group when compared to the F SPMS group
(female : male ratio ¼ 3.0 versus 0.88; Fisher exact
probability test, P ¼ 0.2510). Although this difference did
not reach statistical significance due to the small sample
size, it shows a trend towards a higher probability for
women to develop ectopic B-cell follicles. Age at death was
also significantly lower in the Fþ than in the F SPMS
group [median value ¼ 42 (5.6) versus 55 (9.0) years;
P ¼ 0.0003] (Fig. 2B). The median age at death of males
was 40 (1.5) years in the Fþ SPMS group and 53 (7.0)
years in the F SPMS group and that of females was 44
(5.5) years in the Fþ SPMS group and 62 (14.8) years in
the F SPMS group, indicating that in both genders the Fþ
SPMS cases died earlier than the F SPMS cases. It should
be noted that the main causes of death in both the F
SPMS and Fþ SPMS groups were directly related to MS.
Only 3 out of 12 cases in the Fþ SPMS group and 5 out of
17 cases in the F SPMS group received treatment with
immunosuppressive agents or interferon-b for a short
period, indicating that the therapeutic regimen is very
unlikely to account for the different clinical features in the
F and Fþ SPMS subgroups (Table 2).
Because an assessment of Kurtzke Expanded Disability
Status scale (EDSS) scores was not consistently present in
the clinical notes of the multiple sclerosis cases examined,
we used the age at which the patient became a wheelchair
user (approximately equivalent to EDSS 7) as an indication
of chronic motor dysfunction. We observed that the Fþ
SPMS cases (12 out of 12) became wheelchair bound at
a significantly earlier age than the F SPMS cases (15 out
of 17) [median age ¼ 33 (0.49) versus 47 (8.2) years,
Fig. 2 Clinical characteristics of the Fþ SPMS, F SPMS and PPMS cases. A: Age at onset; B: age at death. Dot points represent
values for each multiple sclerosis case; the bars represent median values for each multiple sclerosis subgroup (n ¼17 for F SPMS, n ¼12
for Fþ SPMS; n ¼ 7 for PPMS); P values are indicated where statistically significant.
1096
Brain (2007), 130, 1089^1104
respectively; P ¼ 0.0011] (Fig. 3A), which is consistent with
the earlier age at disease onset.
Although the F and Fþ SPMS groups did not differ
significantly with respect to number of relapses during the
first 3 years after multiple sclerosis onset (mean SEM,
2.3 0.5 versus 3.1 0.6), a negative correlation between
number of relapses in the first 3 years of the disease and the
age at death was found in the F SPMS group (r ¼ –0.54;
P ¼ 0.0267; not significant after Bonferroni’s correction),
but not in the Fþ SPMS group (r ¼ 0.11; P40.05)
(Fig. 3B). These findings suggest that disease progression in
the latter group might be influenced by local inflammatory
events that are not associated with high relapse frequency in
the early phase of disease, a known predictor of more severe
disease.
Due to the relatively small number of cases examined, no
major conclusions could be made concerning the clinical
features of PPMS cases as compared to those of F and Fþ
SPMS cases. However, it is interesting to note that PPMS
cases were more similar to F SPMS than to Fþ SPMS
cases with respect to age at disease onset and at death
(Fig. 2A and B).
R. Magliozzi et al.
More severe grey matter pathology in the
SPMS cases with ectopic B-cell follicles
We next examined whether the F and Fþ SPMS groups
differed with respect to the neuropathological pattern.
Because of the proximity of meningeal follicles to the
cerebral cortical grey matter, we paid particular attention to
the characterization of GML and NAGM.
GM and WM demyelination
Using MOG immunostaining to evaluate the number and
extent of GML, we found that both the number of GML and
the percentage of the GM that had been demyelinated were
significantly higher in the Fþ SPMS group (3.4- and 5.7-fold,
respectively) than in the F SPMS group (Fig. 4A and B).
When compared to PPMS cases, the Fþ SPMS and F SPMS
cases had a significantly higher and lower cortical involvement, respectively (Fig. 4A and B). Conversely, neither the
number of WML nor the area of demyelinated WM differed
significantly between the F SPMS and Fþ SPMS groups. In
the PPMS group, the area of demyelinated WM was similar
to that in the Fþ and F SPMS groups, but the number of
WML was significantly lower compared to the F SPMS
group (Fig. 4A and B).
Localization of GML
Fig. 3 (A) Kaplan^Meier curve comparing the age at which the
patient became a wheelchair user in the F and Fþ SPMS groups.
Generalized Wilcoxon test, P ¼ 0.0001. (B) Correlation between
relapse frequency during the first 3 years of disease and age at
death in the F and Fþ SPMS groups. The continuous and dashed
lines represent the linear trends in the F and Fþ SPMS groups,
respectively.
Following a descriptive system used in previous studies
(Peterson et al., 2001; Bö et al., 2003) GML were classified
as type I (leucocortical), type II (intracortical) and type III
(subpial) lesions. In the Fþ SPMS group, type-III lesions
predominated (accounting for 70% of total GML), whereas
in the F SPMS group GML had no preferential
distribution (Fig. 4C). The Fþ SPMS cases had 5.3-fold
more type III lesions, but only 2.1- and 1.4-fold more type
I and type II lesions, respectively, than the F SPMS cases.
Remarkably, all ectopic follicles were found adjacent to
subpial type-III lesions, suggesting a causal relationship
between follicle formation and cortical damage (Fig. 5A
and B). In most of the Fþ SPMS cases, subpial
demyelination extended over large cortical areas (Fig. 5C),
whereas in the F SPMS cases it was more limited and
mainly localized in the depth of the cerebral sulci (Fig. 5D).
In the PPMS group, type I and type III lesions tended to
predominate over type II lesions (Fig. 4C). However, the
number of type III lesions was significantly lower in the
PPMS cases than in the Fþ SPMS cases [median (range),
1.0 (0–3) versus 5.5 (3–12) lesions/case, respectively;
P ¼ 0.0006], indicating that the prominent inflammatory
process localized in the meninges of Fþ SPMS cases has a
key role in the development of subpial demyelination.
Lesion inflammatory activity
In agreement with previous studies (Peterson et al., 2001;
Bö et al., 2003), no perivascular immune infiltrates were
Ectopic follicles in multiple sclerosis
Brain (2007), 130, 1089^1104
1097
Fig. 4 Degree of GM and WM demyelination and localization of GML in Fþ SPMS, F SPMS and PPMS cases. The number of GML and
WML (A), the percentage of demyelinated area in the GM and WM (B) and the percentage of type-I, type-II and type-III lesions in the
cerebral GM (C) were evaluated in four paraformaldehyde fixed brain tissue blocks for each multiple sclerosis case. For each tissue block,
one section was examined, as described in the section Material and methods. Dot points represent values for each multiple sclerosis case;
the bars represent median values for each multiple sclerosis subgroup (n ¼17 for F SPMS, n ¼12 for Fþ SPMS; n ¼ 7 for PPMS); P values
are indicated where statistically significant.
observed in purely cortical GML (type-II and type-III
lesions) of all the multiple sclerosis groups examined, and
MHC class II immunostaining, a measure of lesion activity,
was restricted to activated microglia. Conversely, leucocortical type-I lesions consistently displayed inflammatory
infiltrates, although at lower levels than WML (data not
shown). In the Fþ SPMS group, the number of active,
chronic active and chronic inactive GML was 12.7-, 3.3-
and 3.4-fold higher than in the F SPMS group,
respectively (Fig. 6A). The Fþ SPMS group also had a
significantly higher number of chronic active GML than the
PPMS group, whereas the F SPMS group had less active
GML than the PPMS group (Fig. 6A). The number of
active, chronic active and chronic inactive WML did not
differ significantly among the different multiple sclerosis
groups (Fig. 6B).
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Brain (2007), 130, 1089^1104
R. Magliozzi et al.
Fig. 5 Subpial demyelination in the GM of Fþ and F SPMS cases. Immunostaining with anti-CD20 and anti-MOG antibodies in
serial brain sections of a Fþ SPMS case shows the presence of an intrameningeal B-cell follicle (f) (A) which is adjacent to a large type-III
lesion (B; the arrows point to the lesion border). In panels A and B, layers I to III of the cerebral cortex are indicated. MOG
immunostaining shows an extended type-III lesion along a cerebral sulcus which affects most of the GM in a Fþ SPMS case (C, arrows)
and a more circumscribed subpial demyelinated area in a F SPMS case (D, arrow). In panels C and D, the GM:WM border is indicated
by arrowheads. Original magnifications: A ¼100; B ¼ 40 (composed of four tiled frames); C, D ¼ 5 (C is composed of two tiled
frames).
In active and chronic active GML, MHC class IIþ
microglia displayed for the most part a radial ramified
morphology with thick and shortened processes (Fig. 7).
Consistent with the larger cortical demyelination observed
in Fþ SPMS cases, microglial activation was more
prominent in Fþ SPMS cases (Fig. 7A and C) than in
F SPMS (Fig. 7B and D) and PPMS cases (not shown).
MHC class IIþ amoeboid macrophage-like cells were rarely
seen in active type-III lesions in the Fþ SPMS group and
more frequently in type-I lesions (data not shown).
Remarkably, in the Fþ SPMS group diffuse microglial
activation was also detected in the NAGM, not necessarily
adjacent to the demyelinated areas (Fig. 7E). Conversely,
microglial activation was rarely observed in the NAGM of
F SPMS (Fig. 7F) and PPMS cases (data not shown).
In the WM, microglial activation was lower in PPMS cases
compared to SPMS cases, whereas no differences were
evident between the F and Fþ SPMS groups (data not
shown).
Neuronal damage in type-III lesions and NAGM
of Fþ and F SPMS cases
Because all ectopic follicles in Fþ SPMS cases were found
adjacent to extensive subpial type-III demyelinated lesions,
we investigated whether neuronal damage in type-III lesions
and subpial NAGM differed between the F and Fþ SPMS
cases. Immunostaining for SMI32, a marker of
dephosphorylated neurofilaments, revealed the presence of
diffuse axonal injury in most type-III cortical lesions
examined and in the peri-plaque regions in both the Fþ
and F SPMS groups, also in the early stages of
demyelination (data not shown). To more precisely
quantitate the extent of neuronal loss, double immunofluorescence with anti-MBP polyclonal antibody and RT97
monoclonal antibody, which labels both phosphorylated
and de-phosphorylated neurofilament protein-containing
neurites (predominantly axons), was performed in 12 F
SPMS, 12 Fþ SPMS and 3 control cases (Fig. 8A). We
observed that the density of RT97-positive neurites in
NAGM and chronic active type-III lesions of Fþ and F
SPMS cases was considerably lower than in control GM
(Fig. 8A–D). Within both the F SPMS and Fþ SPMS
groups, the numerical density of RT97þ neurites in type-III
lesions was significantly lower than in the NAGM (Fig. 8A).
Moreover, the reduction in the number of RT97þ neurites
within type-III chronic active lesions was significantly more
pronounced in the Fþ SPMS cases than in the F SPMS
cases (Fig. 8A). A trend towards a greater reduction of
RT97 immunostaining was also observed in the NAGM of
Fþ SPMS cases as compared to F SPMS cases (Fig. 8A).
Although decreased RT97 immunostaining does not
necessarily reflect neuritic loss but may result from
downregulation of neurofilament expression, these findings
indicate that such a reduction was significantly more
prominent in the cerebral cortex of the Fþ SPMS group.
Ectopic follicles in multiple sclerosis
Brain (2007), 130, 1089^1104
1099
Fig. 6 Inflammatory activity in GML and WML of F SPMS, Fþ SPMS and PPMS cases. A, B: The number of active, chronic active and
chronic inactive GML (including all cortical lesion types) and WML was evaluated using MHC class II immunostaining in four brain tissue
blocks for each multiple sclerosis case; for each tissue block, one section was examined, as described in the section Material and methods.
Dot points represent values for each multiple sclerosis case; the bars represent median values for each multiple sclerosis subgroup (n ¼17
for F SPMS, n ¼12 for Fþ SPMS; n ¼ 7 for PPMS). P values are indicated where statistically significant.
Discussion
The presence of ectopic B-cell follicles in the cerebral
meninges of a substantial proportion of the multiple
sclerosis cases analysed in the present study is an important
finding that represents a step forward, both in our
understanding of the underlying pathogenetic mechanisms
and the design of targeted immunomodulatory therapies.
By analysing post-mortem brain specimens from a larger
sample of multiple sclerosis cases, we have confirmed and
extended our previous finding (Serafini et al., 2004) to
show that formation of ectopic follicles in the cerebral
meninges accompanies disease progression in a proportion
of SPMS cases in which the initial RR phase is followed by
a progressive phase, but not in PPMS cases in which the
progressive phase is present from the onset of the disease.
Most importantly, by grouping the SPMS cases according
to the presence or absence of meningeal follicles we show
that, compared to the F SPMS group, the Fþ SPMS
group is characterized by an earlier age at multiple sclerosis
onset, irreversible disability and death, and by a more
severe cortical pathology. Because the SPMS cases with age
at death 450 years are over-represented in the multiple
sclerosis population examined here (51%) when compared
to the general multiple sclerosis population (estimated to be
13% from the UK Multiple Sclerosis Tissue Bank data
collection), the real frequency of follicles in the multiple
sclerosis population with an initial relapsing–remitting
onset may be lower than that observed in this study
(41%). However, it is clear that only rarely did we observe
the presence of follicles in multiple sclerosis cases with an
age of death over 50 years.
The younger age at onset in the Fþ SPMS group
compared to the F SPMS group, and the finding that the
proportion of Fþ SPMS cases progressively decreases as the
age at clinical onset increases, support the idea that a more
aggressive inflammatory process favours the establishment
of a permissive environment for ectopic follicle formation.
The present data are in line with the results obtained in
other organ-specific autoimmune diseases. In myasthenia
gravis, ectopic thymic follicles form mainly in patients with
early-onset disease (Roxanis et al., 2002), while in
rheumatoid arthritis ectopic follicles are found in the
synovial tissues with the highest degree of inflammation
(Magalhães et al., 2002), indicating that formation of
ectopic lymphoid tissue requires strong immune activation.
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Brain (2007), 130, 1089^1104
R. Magliozzi et al.
Fig. 7 Microglia activation in the GM of Fþ and F SPMS cases. Combination of LFB staining and MHC class II immunostaining in the GM
of Fþ and F SPMS cases (A and B, respectively). Activated MHC class IIþ microglia in a subpial, type-III lesion of a Fþ SPMS case
adjacent to an intrameningeal follicle (f) is shown in A; serial brain sections stained for CD20 and MOG are shown in Fig. 5A and B,
respectively. The inset in A shows a high-power magnification of a cluster of activated microglia in the same lesion (arrow). Activated MHC
class IIþ microglia in a type-III lesion at the tip of a cerebral sulcus in a F SPMS case (B). Double immunostaining for MHC class II (red)
and MBP (green) shows enhanced microglia reactivity in type-III lesions (C) and NAGM (E) of a Fþ SPMS case compared to type-III GML
(D) and NAGM (F) of a F SPMS case. m ¼ meninges. Original magnifications: A ¼100; B (two tiled frames) ¼ 40; C^F ¼ 200.
Because relapsing–remitting multiple sclerosis cases were
not included in our analysis and only one relapsing–
remitting multiple sclerosis case lacking follicles was
examined in a previous study (Serafini et al., 2004), it is
not known whether ectopic follicles form before or after the
transition to the SPMS phase. In a chronic relapsing model
of murine EAE, meningeal follicles were detected during the
relapsing phase and increased in number and size during
disease progression (Magliozzi et al., 2004; ColumbaCabezas et al., 2006), indicating that repeated CNS
inflammatory events are required to induce formation of
ectopic follicles.
Remarkably, the large difference in the age at which the
patients became wheelchair dependent and in the age at
death between the Fþ and F SPMS groups indicates that
formation of ectopic follicles is associated with a more
severe disease course. Most importantly, females in the
Fþ SPMS group died nearly 20 years earlier than those in
the F SPMS group. Due to their potential relevance for
multiple sclerosis prognosis and therapy, these findings
need to be confirmed in a larger data set. Another finding
that indirectly supports an association between ectopic
follicle formation and rapid disease progression is the
negative correlation between number of relapses during the
first 3 years of disease and age at death in the F SPMS
group, but not in the Fþ SPMS group. We hypothesize that
while a high frequency of intermittent acute relapses
contributes to the accumulation of disability in the F
SPMS group, the same progressive phase could be reached
in the Fþ SPMS group as a consequence of the persistent
inflammatory milieu associated with meningeal follicles.
Failure to detect ectopic follicles in the cerebral meninges
of a subgroup of SPMS cases and of PPMS cases could have
several explanations. The first and more obvious one is that
ectopic follicles have been missed due to the small number
of brain tissue blocks analysed. However, we think that this
is unlikely because ectopic follicle formation was generally
accompanied by prominent meningeal inflammation and
B-cell/plasma-cell infiltration in the WM, two features that
were not observed in the F SPMS and PPMS cases
analysed in this study. A second explanation is that ectopic
follicles might have developed transiently during a more
Ectopic follicles in multiple sclerosis
Brain (2007), 130, 1089^1104
1101
Fig. 8 Lower neurite density in type-III chronic active lesions of Fþ SPMS cases as compared to F SPMS cases. A: The density of
neurofilament proteinþ neurites was evaluated using the RT97 monoclonal antibody, in combination with anti-MBP polyclonal antibody to
distinguish lesioned GM from NAGM. The density of RT97þ neurites in both the NAGM and type-III lesions of F and Fþ SPMS cases was
lower than in control GM. In this case, statistical analysis was not performed due to the low number of control cases analysed (n ¼ 3).
The values represent means SEM of RT97þ neurites per 0.1mm2 for 3 control, 12 F SPMS and 12 Fþ SPMS cases. P values are indicated
where statistically significant. B^D: Visualization of the density of RT97þ structures in the cerebral cortex of a control brain (B) and of its
reduction in the NAGM (C) and in a type-III lesion (D) of a representative Fþ SPMS case. The inset in B shows a high-power magnification
of a myelinated axon in the control case, double immunostained for MBP (green) and RT97 (red). In panels B ^D, the cell nuclei are counterstained with DAPI (blue). Original magnifications: 400 (B ^D), 600 (inset in B).
active phase of disease and had disappeared by the time of
death. This scenario would be consistent with the
disappearance of the antigenic stimulus triggering the
formation of meningeal follicles, possibly an infectious
agent, whose persistence would instead be essential for
the maintenance of the germinal centre reaction in the
Fþ SPMS cases. A third explanation is that in some cases of
multiple sclerosis ectopic follicles might develop at sites
different from the cerebral meninges. To date, we have
failed to detect ectopic follicles at the inferior surface of the
brain, around the lateral ventricles (caudate nucleus and
subependymal WM of the cerebral hemispheres) and in the
cerebellum, brainstem and spinal cord. However, we do not
rule out that ectopic follicles in multiple sclerosis might
localize in still unidentified brain regions close to the third
and fourth ventricles, as shown in the EAE-affected CNS
(Magliozzi et al., 2004; Columba-Cabezas et al., 2006). If we
postulate that ectopic follicles are where the intrathecal
humoral immune response, which is a hallmark of multiple
sclerosis, is sustained, their formation in the cerebral
meninges could be viewed as the extreme manifestation of
a common pathological mechanism that spreads to the
cortical surface only in a subset of patients in which a more
severe inflammatory process has been induced.
Our findings do not allow us to determine whether
follicle formation is causative or is the consequence of a
more severe disease process. However, the observation that,
when compared to the F SPMS cases, the Fþ SPMS cases
showed a more extensive subpial demyelination, an
increased number of active cortical lesions, and a more
pronounced microglia activation and neurite loss within the
areas of subpial demyelination and in the surrounding
NAGM, indicates that ectopic follicles, or the inflammatory
milieu favouring their formation, are involved in the
exacerbation of cortical damage. In contrast to WML and
leucocortical type-I lesions, purely cortical lesions (type-II
and type-III) are devoid of inflammatory cell infiltrates and
foamy macrophages, and show sparse deposition of Ig and
complement activation products (Bö et al., 2003; Brink
et al., 2005), indicating that the mechanisms mediating
WM and GM damage might differ quantitatively and/or
qualitatively. The proximity of ectopic follicles to large
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Brain (2007), 130, 1089^1104
subpial demyelinated lesions, a consistent finding in all the
Fþ SPMS cases examined in this study, strongly supports
the idea that these abnormal structures have a direct role in
cortical injury, most likely by releasing soluble factors that
diffuse into the subarachnoid space and through the pial
membrane (Rennels et al., 1985). These may include
pathogenic antibodies, pro-inflammatory cytokines and/or
proteolytic enzymes that directly cause, or amplify, tissue
damage. In contrast, no major differences in the number,
extension and activity of WML were detected between
F and Fþ SPMS cases, which indicates that the
inflammatory process localized in the cerebral meninges
mainly affects the adjacent cortical GM. These findings
are in agreement with recent studies showing that there
is no correlation between the extent of GM demyelination
and focal or diffuse WM demyelination in multiple
sclerosis (Kutzelnigg et al., 2005; Bö et al., 2007). Our
observation that ectopic B-cell follicles localize in the
cerebral sulci and do not develop at the external brain
surfaces suggests that these anatomical sites represent ‘hot
spots’ for accumulation of, or accessibility to, the putative
triggering antigens and/or for B-cell migration and
activation.
Previous neuropathological studies have shown the
presence of demyelination, axonal damage and neuronal
loss, in the cortical and deep GM of multiple sclerosis
patients (Brownell and Hughes, 1962; Lumdsen et al., 1970;
Kidd et al., 1999; Peterson et al., 2001; Bö et al, 2003).
GML may represent a substantial proportion of the total
lesions in the multiple sclerosis brain and are more
prominent in SPMS and PPMS than in relapsing–remitting
multiple sclerosis (Kutzelnigg et al., 2005). Our finding that
the numerical density of neurofilament proteinþ structures
in subpial cortical lesions was significantly lower in Fþ than
in F SPMS cases, despite an earlier age at death, indicates
that the presence of ectopic B-cell follicles accelerates
neuronal degeneration and/or dysfunction. Axon loss in
WML has been linked with inflammatory episodes occurring during relapses, in addition to a longer term slower
loss that is thought to predominate during the progressive
phase (Trapp et al., 1998; Kornek et al., 2000). Because
neuronal loss and brain atrophy are the most significant
magnetic resonance imaging variables in determining the
final disability in multiple sclerosis patients (Filippi and
Rocca, 2005; Tedeschi et al., 2005), the present findings
suggest that the extensive neurite loss associated with
meningeal inflammation and ectopic follicles could be a
major factor determining the earlier progression to
irreversible disability and the more limited survival of Fþ
SPMS cases as compared to F SPMS cases.
The finding that the Fþ SPMS cases also showed a higher
number of cortical demyelinated lesions with a predominance of subpial lesions and more chronic active GML
compared to the PPMS cases examined in this study further
supports an important role for meningeal inflammation
accompanied by formation of ectopic follicles in
R. Magliozzi et al.
exacerbating cortical damage. Although extensive subpial
cortical demyelination was previously described in a limited
number of PPMS and SPMS cases (Bö et al., 2003), to date
no studies have directly compared the distribution of
cortical lesions in different multiple sclerosis groups.
Kutzelnigg et al. (2005) reported similar cortical demyelination in the forebrain of PPMS and SPMS cases, but did not
determine whether the extent of subpial demyelination was
also comparable. Although these authors showed that
median values for lesioned cortical area were similar in
the two groups, the highest percentage of demyelinated
cortical area was found in SPMS (68.63 versus 38.68% in
PPMS) (Table 1 in Kutzelnigg et al., 2005). This indicates
that at least a subset of SPMS cases develops more extensive
cortical damage than PPMS cases, which is in keeping with
our observations in the Fþ SPMS subgroup.
Formation of ectopic lymphoid tissue has been observed
in several chronic inflammatory diseases with an autoimmune or infectious aetiology and is thought to be
induced by the persistent inflammatory milieu of the target
tissue (Aloisi and Pujol-Borrell, 2006). In multiple sclerosis,
an immune response directed against CNS antigens
(Owens et al., 2006) or infectious agents (Gilden, 2005) is
thought to arise in peripheral lymphoid organs leading to
the intracerebral migration of memory and effector
lymphocytes. It can be envisaged that if antigen-specific,
CNS-infiltrating B cells find a favourable environment in
the inflamed meninges that allows them to initiate a
germinal centre reaction and to undergo proliferation,
somatic hypermutation, selection of high-affinity clones and
differentiation into antibody-producing plasma cells, all
downstream events mediated by the humoral immune
response would be amplified locally, resulting in exacerbation of the destructive inflammatory process (Uccelli et al.,
2005). As discussed earlier, several issues remain to be
solved, in particular whether ectopic follicles develop in the
multiple sclerosis brain already during early disease stages
and localize at sites other than the cerebral meninges, and
whether they are stable or transient structures whose
formation is linked to environmental triggers.
In conclusion, this study is the first one to show an
association between ectopic lymphoid tissue formation,
clinical course and extent of tissue destruction in the target
organ during a chronic inflammatory CNS disease.
The identification of the antigenic stimuli driving ectopic
follicle formation in multiple sclerosis and of the factors
mediating the extensive cortical damage associated with
meningeal follicles must be considered as a primary goal of
future multiple sclerosis research and could shed light into
the still elusive immune mechanisms that mediate CNS
tissue injury. The present findings also suggest that
prevention or eradication of lymphoid microenvironments
nested within the CNS should be identified as an important
goal for therapeutic intervention in multiple sclerosis
patients.
Ectopic follicles in multiple sclerosis
Acknowledgements
All tissue samples were supplied by the UK Multiple
Sclerosis Tissue Bank (www.ukmstissuebank.imperial.ac.uk),
funded by the Multiple Sclerosis Society of Great Britain
and Northern Ireland (registered charity 207495). The
authors would like to thank members of the UK multiple
sclerosis Tissue Bank Team (S. Gentleman, M. Graeber, F.
Roncaroli, S. Fordham, I. Ghebrenegus and N. Patel) for
assistance in the collection and characterization of the
material used in this study and Ms Estella Sansonetti for
graphical work. This work was supported by the Multiple
Sclerosis Society of Great Britain and Northern Ireland
(grant No 747/02), a fellowship from the Italian Multiple
Sclerosis Foundation to R.M. Programme of Collaboration
between Istituto Superiore di Sanità and National Institutes
of Health, and 6th Framework Program of the European
Union NeuroproMiSe LSHM-CT-2005-01863 to F.A.
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