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Migration of Multiple Sclerosis Lymphocytes Through Brain Endothelium
Alexandre Prat, MD, PhD;
Katarzyna Biernacki, MSc;
Jean-Francois Lavoie, BSc;
Josee Poirier, B Inf;
Pierre Duquette, MD;
Jack P. Antel, MD
Arch Neurol. 2002;59:391-397.
ABSTRACT
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Context T-lymphocyte migration through the blood-brain barrier is a central
event in the process of lesion formation in multiple sclerosis (MS).
Objectives To assess the ability of lymphocytes derived from the peripheral blood
of patients with clinically active and inactive MS to migrate across an artificial
model of the blood-brain barrier and to elucidate the molecular mechanisms
involved in such a process.
Design We developed an in vitro model of lymphocyte migration using a Boyden
chamber coated with a monolayer of human brain microvascular endothelial cells.
Results The rates of migration of lymphocytes obtained from patients with acutely
relapsing and active secondary progressive MS was significantly increased
compared with those obtained from healthy controls and patients with inactive
secondary progressive disease. Ribonuclease protection assays and enzyme-linked
immunosorbent assays indicated that monocyte chemoattractant protein 1 and
interleukin 8 were the major chemokines produced by brain endothelial cells
grown under the culture conditions used for the migration assays. The rate
of migration of the MS lymphocytes could be inhibited by 60% with an antimonocyte
chemoattractant protein 1 monoclonal antibody, indicating a functional role
for this chemokine in the migration process. In agreement with previous reports,
we found that the tissue inhibitor of metalloproteinase 1, a matrix metalloproteinase
inhibitor, also reduced migration of MS lymphocytes by 50%.
Conclusions The results demonstrate an increased migration rate of MS T lymphocytes
across the brain endothelium barrier and that such migration is dependent
on chemokine monocyte chemoattractant protein 1 and on matrix metalloproteinases.
INTRODUCTION
MULTIPLE SCLEROSIS (MS) is a disease of the central nervous system characterized
by multifocal infiltration of autoreactive T lymphocytes from the systemic
immune system across the blood-brain barrier. Brain endothelial cells (ECs)
are the first cells of the blood-brain barrier encountered by such leukocytes
and are thought to restrict immune cell access to the central nervous system.
The sequence of cellular events underlying migration include the processes
of lymphocyte-EC adhesion, chemoattraction, and proteolysis of the compact
basal membrane surrounding the blood-brain barrier by matrix metalloproteinases
(MMP).
In previous studies, we used a Boyden chamber coated with fibronectin
as an in vitro model of leukocyte migration.1-4
This assay primarily models the interaction of lymphocytes with the extracellular
matrix proteins. We found that lymphocytes derived from patients with relapsing-remitting
(RR) disease who were in remission or in acute relapse (AR), as well as patients
with secondary progressive (SP) disease, migrated faster than did cells from
healthy donors, and that treatment with interferon (IFN) ß or glatiramer
acetate inhibited migration.1, 3
The tissue inhibitor of metalloproteinase (TIMP)-1, an MMP-9 inhibitor, was
a partial but significant antagonist of lymphocyte migration in this assay,3 suggesting that this enhanced migration of MS T lymphocytes
across fibronectin was dependent on the up-regulation of MMP-9 in MS. Several
investigators have used human umbilical vein ECs or peripheral organ ECs to
study lymphocyte interaction and migration across an EC barrier.5-9
Techniques have recently been developed to isolate and use human brain–derived
microvascular ECs (HBECs), providing a model that more closely reflects lymphocyte-EC
interactions and lymphocyte migration across the human blood-brain barrier.10-19
We have used HBECs grown on fibronectin-coated membranes of Boyden chambers
for our migration studies.
The process of chemoattraction to the human central nervous system is
dependent on interactions between chemokines produced by brain ECs or by central
nervous system parenchymal cells and chemokine receptors on the surface of
lymphocytes. Monocyte chemoattractant protein (MCP) 1 is a C-C chemokine that
has preferential affinity for the chemokine receptors CCR2 and CCR4.20-21
Monocyte chemoattractant protein 1 has been shown to increase transendothelial
migration of  /ß (CD4+and CD8+) and  /
lymphocytes in chemotaxis assays using human umbilical vein ECs.5, 9, 22-23
Immunohistochemical examination of central nervous system tissue derived from
patients with MS and from animals with experimental allergic encephalomyelitis
(EAE) has demonstrated increased expression of MCP-1 as well as macrophage
inflammatory protein (MIP) 1, interferon inducible protein
10 (IP-10), and RANTES in astrocytes and perivascular cells located in and
around inflammatory plaques.24-28
Karpus and Kennedy29 showed that blocking MCP-1
could reduce the severity of relapsing EAE. Recently, a sequence polymorphism
identified in the gene encoding MCP-1 was proposed as a candidate for the
locus EAE7, a locus known to control susceptibility
to monophasic EAE in the mouse,30 suggesting
a causative role for MCP-1 in EAE.
We report that migration rates of lymphocytes derived from patients
with RR and SP MS with active disease through HBEC monolayers are increased
compared with patients with inactive disease and controls that include healthy
donors, patients with epilepsy, and individuals with acute systemic viral
infections. This increased migration could be inhibited, at least partially,
by a blocking antibody directed at MCP-1, one of the major chemokines expressed
by HBECs. Migration was also partially inhibited by the MMP inhibitor TIMP-1.
PATIENTS AND METHODS
PATIENTS
The ex vivo lymphocyte studies were conducted on groups of patients
with clinically confirmed MS31 presented in Table 1. Patients were selected from the
databases of our MS clinics (Montréal Neurological Hospital, Montréal,
Québec, and Nôtre-Dame Hospital, Montréal) based on their
clinical profiles and having been classified as having RR or SP disease with
or without intermixed relapses. Patients with RR MS were selected on the basis
of disease activity: patients with new onset of neurological symptoms (consistent
with a new relapse) and an increase in the Expanded Disability Status Scale
score of at least 1.0 were classified having AR MS (n = 5). Patients with
RR MS with no new neurological symptoms for at least 6 months were classified
as having remitting MS (n = 7). Patients with SP MS were further selected
on the basis of disease activity as determined by changes in disability scores
(>0.5) or the occurrence of relapses in the previous 18 months (Table 1). Patients who showed either no signs of progression or
no relapse during this period were considered to have clinically inactive
disease. The active SP group was younger than the inactive SP group (51 ±
8 and 56 ± 3 years, respectively) and had a shorter disease duration
(10.5 ± 2.5 and 17 ± 3.8 years, respectively). The RR MS group
was younger (31 ± 3 years) and had a shorter disease duration (5.6
± 1.2 years) than both SP MS groups. The control group included healthy
donors, patients with epilepsy, and individuals affected with the influenza
virus; the ages of these groups were 29 ± 4, 33 ± 5, and 32
± 4 years, respectively. None of the patients used in this study were
receiving immunosuppressive/modulatory therapy. For patients with AR and patients
affected with the influenza virus, blood was drawn at the time of symptoms.
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Characteristics and Demographics of Patients Enrolled in the Study*
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ISOLATION AND CHARACTERIZATION OF LYMPHOCYTES
Mononuclear cells were isolated from peripheral blood samples from patients
or controls using a Ficoll density gradient (Amersham-Pharmacia-Biotech, Baie
D'Urfee, Québec). Mononuclear cells were depleted of monocytes by a
1-hour culture at 37°C in an Rosewell Park Memorial Institute medium plus
10% fetal bovine serum in a 75-cm2 plastic dish as previously described.32 Anti-CD3, -CD4, -CD8, -CD14, -CD56, -CD45RO, -CD45RA
antibody staining and fluorescent-activated cell sorter analysis was done
to evaluate the lymphocyte populations used in the migration assay.
ADHESION MOLECULES
Lymphocytes were also assessed for expression of adhesion molecules
leukocyte function antigen 1 (LFA-1) and very late antigen 4 (VLA-4). Anti–LFA-1
and anti–VLA-4 monoclonal antibodies conjugated with fluorescein isothiocyanate
or immunoglobulin (Ig) G1isotype were added to 1 x 105 lymphocytes/50 µL for 30 minutes at 4°C. Cells were washed
twice in phosphate-buffered isotonic sodium chloride solution/FCS 2% and fluorescence
intensity was acquired on a fluorescent-activated cell sorter machine (Becton
Dickinson, Mississauga, Ontario) and analyzed by a WinMDI program (Scripps,
La Jolla, Calif).
EC ISOLATION AND CULTURE
Human brain–derived microvascular ECs were isolated and cultured
as previously described11, 15, 18
from temporal lobe specimens resected from young adults undergoing epilepsy
surgery. Tissue was minced and homogenized in phosphate-buffered isotonic
sodium chloride solution, filtered through a 350-µm Nitex mesh, and
then twice through a 112-µm mesh. Filtrate was collected, spun down,
and digested for 10 minutes with collagenase type IV at 37°C. Cells were
collected by centrifugation and seeded on a 0.5% gelatin-coated flask, in
M199 (GIBCO-BRL, Burlington, Ontario) supplemented with 10% FCS, 20% human
normal serum, insulin-transferrin-selenium, and EC growth supplement (all
from Sigma-Aldrich, Oakville, Ontario). On day 10, visible colonies were collected
and expanded in fresh media. As previously demonstrated, these cells express
factor VIII–related antigen, Ulex Agglutinsis Europaeus 1 binding sites,
and HT-7 antigen.15, 18 Immunoreactivity
for -myosin and glial fibrillary acidic protein could not be detected
on the HBECs, suggesting the absence of contaminating astrocytes and smooth
muscle cells. We estimated the percentage of contaminating cells in our cultures
to be less than 1% after 3 passages.
TRANSENDOTHELIAL MIGRATION
All migration assays were conducted in Boyden chambers (3-µm pore-size
membranes) precoated with fibronectin (Collaborative Biomedical Products,
Bedford, Mass). Human brain–derived microvascular ECs (20 000 cells)
were seeded on the membrane 3 days prior to the migration experiment in EC
culture media. On the day of the experiment, permeability of the monolayer
was confirmed by adding fluorothioisocyanate-labelled albumin to the upper
chamber and monitoring levels of fluorescence in the lower chamber. Chambers
that allowed more than 5% of albumine diffusion after 6 hours were discarded.
In uncoated chambers, levels of fluorescence equilibrated between compartments
within 30 minutes. For migration, 106 lymphocytes suspended in
1 mL of RPMI plus 2.5% FBS were added to the top chamber. After 18 hours at
37°C, the contents of the bottom chamber were collected and the number
of cells present were determined by counting aliquots under the microscope.
In some migration experiments, anti–MCP-1 neutralizing monoclonal antibody
(5 µg/mL), anti–IL-8 neutralizing antibody (5 µg/mL), and
IgG1isotype control were added to the upper chamber 10 minutes
prior to the T cells. A similar procedure was used with TIMP-1 (15 ng/mL).
RIBONUCLEASE PROTECTION ASSAY AND ENZYME-LINKED IMMUNOSORBENT ASSAY
FOR CHEMOKINE AND CHEMOKINE RECEPTORS
To detect chemokines produced by HBECs, ribonuclease (RNASE) protection
assays and enzyme-linked immunosorbent assays (ELISAs) were performed using
confluent monolayers of cells grown in 75-cm2flasks. Cells were
either treated or not treated with IFN- (100 U/mL) and tumor necrosis
factor (TNF) (100 U/mL) for 24 hours. RNA from HBEC monolayers was
collected in Trizol (GIBCO-BRL; Invitrogen Life Technologies, Carlsbad, Calif)
and 8 µg of total RNA was used for the chemokine-ribonuclease protection
assay (kit hCK-5; Pharmingen, Mississauga, Ontario) as described in the manufacturer's
instructions. Culture supernatants were collected and assayed for the presence
of IL-8, RANTES, IP-10, and MCP-1 by ELISA as described in the manufacturer's
instructions.
Expression of chemokine receptors by T lymphocytes derived from patients
with MS or control donors was also assessed by ribonuclease protection assay.
RNA from T lymphocytes was collected in Trizol (GIBCO-BRL) and 10 µg
of total RNA was used for the chemokine-ribonuclease protection assay (kit
hCR-5, 6 and 8; Pharmingen) as described in the manufacturer's instructions.
STATISTICS
Data are given as mean ± SEM unless otherwise indicated. For
each donor, lymphocyte migration was performed in duplicate. Results are presented
as column scatter plots, with the mean for each subgroup. Chemokine determination
by ELISA was done in duplicate in 3 individual experiments using 3 different
HBEC primary cell strains. Statistical comparison between the groups was performed
using an analysis of variance test and a Bonferroni posttest.
RESULTS
MS-DERIVED T-LYMPHOCYTE MIGRATION ACROSS THE HBEC MONOLAYER
Migratory behavior of T lymphocytes obtained from patients with MS and
control donors was assessed in a Boyden chamber coated with HBECs grown under
basal culture conditions. As shown in Figure
1, T cells obtained from AR, RR, and active SP MS groups had significantly
higher migration rates compared with cells from healthy controls or patients
with epilepsy (P<.05, P<.01,
respectively). The migration rates of the active SP MS group were also significantly
increased compared with the inactive SP MS group (P<.05).
Donors affected with the influenza virus did not show an increased migration
rate compared with healthy donors and patients with epilepsy.
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Figure 1. Lymphocyte migration across human
brain endothelial cells. Ex vivo lymphocytes derived from the peripheral blood
of healthy controls (n = 12), patients with epilepsy (n = 7), and controls
affected by influenza virus (influenza virus, n = 4) or relapsing-remitting
(RR) (n = 7), acutely relapsing (AR) (n = 5), active secondary progressive
(SP) (n = 8), and inactive SP (n = 6) patients with multiple sclerosis were
subjected to migration for 18 hours in a Boyden chamber coated with a human
brain endothelial cell monolayer. Asterisk indicates AR and RR compared with
controls, P<.05; dagger, active SP compared with
controls, P<.01; and double dagger, inactive SP
compared with active SP, P<.05.
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Anti-CD3, -CD4, -CD8, -CD14, -CD56, -CD45RO, and -CD45RA antibody staining
and fluorescent-activated cell sorter analysis revealed that 95% of the leukocytes
used in the migration assays were CD3+, 61% were CD4+,
29% were CD8+, less than 3% were CD14+, and less than
3% were CD56+. We could not observe a difference in CD45RA/R0 staining
between the control and each MS group (controls: CD45RA+ 49% ±
6%, CD45RO+ 42% ± 8%; overall MS groups: CD45RA+
56% ± 8%, CD45RO+ 42% ± 4%).
ADHESION MOLECULE EXPRESSION ON LYMPHOCYTES
We analyzed the expression of VLA-4 and LFA-1 on MS and control T cells
before migration, using immunostaining and flow cytometry. We found no difference
in the proportion of CD3+ cells expressing these antigens when
cells obtained from patients with AR, RR, and SP MS were compared with control
T cells (LFA-1: 81% ± 2%, 84% ± 2%, 83% ± 3%, and 82%
± 2%, respectively; VLA-4: 77% ± 7%, 79% ± 6%, 77% ±
4%, and 79% ± 8%, respectively).
HBECs PRODUCE IL-8 AND MCP-1
We analyzed chemokine messenger RNA (mRNA)and protein production by
HBECs grown under basal culture conditions using RNASE protection assays and
ELISAs. As described in Figure 2A,
resting HBECs expressed both IL-8 and MCP-1 mRNA transcripts (lane 1). We
could not detect transcripts for RANTES, IP-10, MIP-1, or MIP-1ß
on HBECs under basal conditions. When HBECs were exposed to IFN- and
TNF-, there was an up-regulation of mRNA transcripts for RANTES and
IP-10 (lane 2) but not for MIP-1 or MIP-1ß. Similar data could
be obtained by ELISA (Figure 2B),
namely, that MCP-1 and IL-8 could be found in culture supernatants of resting
HBECs whereas levels of IP-10 and RANTES were below detectability. Monocyte
chemoattractant protein 1, IL-8, and RANTES could be detected in the supernatants
of IFN- and TNF-–treated HBEC cultures.
CHEMOKINE RECEPTOR EXPRESSION BY MS LYMPHOCYTES
To evaluate the expression of specific chemokine receptors for IL-8
and MCP-1 by lymphocytes obtained from patients with MS and healthy donors,
we performed ribonuclease protection assay analysis using RNA samples extracted
from lymphocytes from each of these donor groups. As shown in Figure 3, lymphocytes did not express mRNA for either the IL-8 chemokine
receptors CXCR1 and CXCR2,
or the MCP-1 receptor CCR2A and CCR2B. We could not detect differences in the mRNA expression of the
MCP-1 receptor CCR4 between patients with MS and
controls. While CXCR4 mRNA was equally expressed
in healthy donors and MS-derived lymphocytes, levels of CXCR3 mRNA were higher in MS-derived samples, as previously reported.33 Messenger RNA levels of the orphan chemokine receptor GPR15 were also elevated in MS lymphocytes compared with
controls.
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Figure 3. Chemokine receptor expression
by multiple sclerosis (MS) lymphocytes. Ribonulease protection assay (10 µg
of total RNA per lane) revealed that while CCR4
messenger RNA (mRNA) is expressed at similar levels by MS- and control-derived
lymphocytes, CCR2a+b, CXCR1,
and CXCR2 mRNA are not expressed. Both CXCR3 and GPR15 mRNA were found to be elevated
in acutely relapsing (AR) and secondary progressive (SP) MS-derived lymphocytes
and low in healthy donor lymphocytes. These gels are representative of 3 independent
experiments using lymphocytes derived from 12 patients with MS and 10 healthy
controls. GADPH indicates chloroplast glyceraldehyde-3-phosphate dehydrogenase.
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ANTIMIGRATORY EFFECTS OF ANTI–MCP-1 NEUTRALIZING ANTIBODY AND
TIMP-1
To evaluate the contribution of MCP-1, IL-8, and MMPs in the transendothelial
migration of healthy control- and MS-derived lymphocytes, we added anti–MCP-1
or anti–IL-8 neutralizing antibody (5 µg/mL), or TIMP-1 (15 ng/mL)
to our assay system. As shown in Figure 4A-C, the addition of either anti–MCP-1 neutralizing monoclonal
antibody (5 µg/mL) or TIMP-1 (15 ng/mL) significantly inhibited the
migration of lymphocytes derived from patients with both active and inactive
SP MS as well as those obtained from patients with AR MS (either treatment
compared with untreated cells, P<.05). Anti–MCP-1
antibody and TIMP-1 treatment also reduced healthy control T-cell migration
(Figure 4A and B), although the
data did not reach statistical significance (anti–MCP-1 compared with
untreated cells, P>.30; TIMP-1 compared with untreated
cells, P>.20). Treatment with the appropriate isotype
control (IgG1) or with anti–IL-8 antibody did not affect
healthy control- or MS-derived lymphocyte migration in this assay.
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Figure 4. Antimigratory effects of anti–monocyte
chemoattractant protein 1 (MCP-1) antibodies and tissue inhibitor of metalloproteinase
(TIMP-1). A, Ex vivo peripheral blood lymphocytes from patients with inactive
secondary progressive (SP) multiple sclerosis (MS) (n = 5) as well as from
control donors (n = 4) were subjected to migration for 18 hours in a Boyden
chamber coated with a human brain endothelial cell monolayer in the presence
or absence of antihuman–MCP-1 neutralizing antibody (5 µg/mL)
or TIMP-1 (15 ng/mL). B and C, Identical experiments were conducted using
T lymphocytes obtained from patients with active SP (n = 5) and acutely relapsing
(AR), (n = 4) MS and compared with independent groups of healthy donors. IL
indicates interleukin; asterisk, anti–MCP-1 and TIMP-1 compared with
untreated cells; P<.05. Horizontal bars represent
the mean of number of cells recovered from the lower chamber.
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COMMENT
We have previously studied lymphocyte migration in patients with MS,
using a Boyden chamber in which fibronectin was the only barrier to migrating
cells. We have shown that lymphocytes derived from patients with MS with relapsing
and actively progressive disease migrated faster than healthy control cells.1 This increased migration of MS lymphocytes was found
to be inhibited by the pretreatment of cells with IFN-ß and TIMP-1, an
inhibitor of MMPs.3-4
In the present study, we have used HBECs grown on Boyden chamber membranes
to evaluate transendothelial lymphocyte migration in MS. We used lymphocytes
obtained from patients with active MS, either RR or SP, and from patients
with inactive MS. These groups were defined based on data recorded at our
MS clinics on relapses and disability scores (EDSS) during a 2-year period.
Results presented here show that T cells obtained from patients with RR and
active SP MS migrated faster through HBECs than cells derived from controls
or patients with inactive MS. We have previously shown that patients with
active and inactive SP MS differ in another in vitro immune assay, namely,
functional suppressor activity assay.34 Using
our HBEC-based migration assay, we also demonstrated that the migration rate
of lymphocytes through brain endothelium is not solely dependent on the activation
state of the peripheral immune system since cells obtained from donors affected
with the influenza virus migrated at the same rate as those obtained from
healthy donors. This observation does not, however, allow us to conclude that
our findings are specific for MS since other organ-specific inflammatory diseases
were not included.
The role of adhesion molecules, such as VLA-4 and LFA-1 ligation to
vascular cell adhesion molecule and intercellular adhesion molecule, is known
to be critical in leukocyte adhesion and migration through the endothelium.
We did not detect differences in expression of VLA-4 and LFA-1 on lymphocytes
between lymphocytes of patients with MS and those of control donors, consistent
with previous reports.35-36 We
could also not correlate differences in rates of migration in our assay with
levels of VLA-4 or LFA-1 expressed on lymphocytes.
To evaluate the potential contribution of chemokines produced by HBECs
to the lymphocyte migration process, we determined which of these molecules
were expressed by HBECs under the culture conditions in which we performed
our migration assay. Our findings demonstrate that HBECs grown under these
basal culture conditions produce IL-8 and MCP-1. When exposed to IFN-
and TNF-, HBECs also up-regulate additional chemokines, IP-10 and RANTES.
While IL-8 is known for its ability to attract primarily neutrophils, MCP-1
is a monocyte-lymphocyte chemoattractant molecule.6, 9, 22-23,37
Monocyte chemoattractant protein 1 immunoreactivity has been demonstrated
in reactive astrocytes and macrophages in and around inflammatory plaques
in MS and EAE.24-26,38
Monocyte chemoattractant protein 1 immunoreactivity can be detected in the
brain27 and specifically in brain ECs28 in EAE at the onset of inflammation and prior to
clinical expression of the disease. Anti–MCP-1 antibodies can reduce
the severity of relapsing EAE.29 Furthermore, CCR2 (MCP-1 receptor) knock-out mice did not develop clinical
or pathologic features of EAE.39 In our migration
assay, the addition of anti–MCP-1 neutralizing antibody but not anti–IL-8,
significantly reduced MS lymphocyte migration. This observation suggests a
functional role for MCP-1 in the recruitment of MS lymphocytes across the
blood-brain barrier. Anti–MCP-1 antibodies have also been shown to inhibit
monocyte migration across human umbilical vein ECs.6
Our finding that TIMP-1 inhibits MS lymphocyte migration supports previous
reports showing that MMP inhibitors can reduce lesion formation and clinical
scores in EAE.40
To evaluate the chemokine receptor by which MCP-1 could modulate lymphocyte
migration across HBECs, we performed ribonuclease protection analysis of chemokine
receptor mRNA expression by lymphocytes obtained from controls and patients
with MS. CCR2 mRNA and CCR4
mRNA are the chemokine receptors that are established to bind to MCP-1. CCR2 mRNA was not detected in MS and control donor lymphocytes. CCR4 mRNA was detected at comparable levels. We showed
an up-regulation of the recently identified GPR15
receptor in MS-derived lymphocytes. Since the chemotactic ligands for GPR
molecules (orphan receptors) have not yet been identified, it is possible
that MCP-1 exerts its chemotactic activity through such a receptor. This hypothesis
remains to be tested. We also confirmed a previous report showing increased
expression of the IP-10 receptor CXCR3 on T lymphocytes
in MS.33 In our hands, lymphocytes derived
either from patients with MS or healthy controls did not express mRNA for CXCR1 and CXCR2, the known receptors
for IL-8.
In this study, we present functional data that show increased migration
of T lymphocytes derived from patients with active MS across a human brain-endothelium
barrier. The migration of MS-derived T cells could not be attributed to a
differential expression of VLA-4 and LFA-1 on the surface of MS T lymphocytes.
We showed that brain ECs grown under basal culture conditions used to assess
migration produce the chemokine MCP-1 and that MCP-1 chemoattraction contributed
to the migratory process. Additional experiments will be needed to define
the migratory behavior of MS T cells across activated HBECs and the role of
IP-10 and RANTES in such a process.
AUTHOR INFORMATION
Accepted for publication October 12, 2001.
Author contributions: Study
concept and design (Drs Prat, Duquette, and Antel, and Ms Biernacki); acquisition of data (Drs Prat and Duquette, and Mss Biernacki
and Poirier); analysis and interpretation of data
(Drs Prat and Antel, and Ms Biernacki); drafting of the
manuscript (Dr Prat and Ms Biernacki); critical revision
of the manuscript for important intellectual content (Drs Prat, Duquette,
and Antel, and Mss Biernacki and Poirier); statistical expertise (Drs Prat and Antel, and Ms Biernacki); obtained
funding (Dr Antel); administrative, technical, and
material support (Dr Prat, and Mss Biernacki and Poirier); study supervision (Dr Prat and Ms Biernacki).
This study was supported by the Multiple Sclerosis Society of Canada,
Toronto, Ontario, the Medical Research Council of Canada, Ottawa, Ontario,
a fellowship from the Medical Research Council of Canada (Dr Prat), and a
studentship from the Fonds pour la Formation de Chercheurs et l'Aide a la
Recherche, Québec City, Québec (Ms Biernacki).
Corresponding author and reprints: Alexandre Prat, MD, PhD, Neuroimmunology
Unit, Montréal Neurological Institute, 3801 University St, Montréal,
Québec, Canada H3A 2B4 (e-mail: aprat{at}po-box.mcgill.ca).
From the Neuroimmunology Unit, Montréal Neurological Institute,
McGill University, Montréal, Québec (Drs Prat and Antel, Mss
Biernacki and Poirier, and Mr Lavoie); and the Department of Neurology, Hopital
Nôtre-Dame, Université de Montréal, Montréal (Drs
Prat and Duquette).
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