Aubé et al., J Immunol, 2014

Transcription

Neutrophils Mediate Blood−Spinal Cord
Barrier Disruption in Demyelinating
Neuroinflammatory Diseases
Benoit Aubé, Sébastien A. Lévesque, Alexandre Paré,
Émilie Chamma, Hania Kébir, Roser Gorina, Marc-André
Lécuyer, Jorge I. Alvarez, Yves De Koninck, Britta
Engelhardt, Alexandre Prat, Daniel Côté and Steve Lacroix
J Immunol 2014; 193:2438-2454; Prepublished online 21
July 2014;
doi: 10.4049/jimmunol.1400401
http://www.jimmunol.org/content/193/5/2438
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The Journal of Immunology
Neutrophils Mediate Blood–Spinal Cord Barrier Disruption
in Demyelinating Neuroinflammatory Diseases
Benoit Aubé,*,†,‡,x Sébastien A. Lévesque,*,† Alexandre Paré,*,† Émilie Chamma,‡,x
Hania Kébir,{ Roser Gorina,‖ Marc-André Lécuyer,{ Jorge I. Alvarez,{ Yves De Koninck,‡
Britta Engelhardt,‖ Alexandre Prat,{ Daniel Côté,‡,x and Steve Lacroix*,†
L
eukocyte entry into the healthy mammalian CNS is strictly
controlled by the blood–brain and the blood–spinal cord
barriers (BBB and BSCB, respectively), complex vascular gatekeepers that maintain CNS homeostasis by regulating the
passage of soluble compounds and leukocytes from the periphery
into the parenchyma (1, 2). Dysfunction of the BBB and BSCB is
*Centre de Recherche du Centre Hospitalier Universitaire de Québec–Centre Hospitalier de l’Université Laval, Quebec, Quebec G1V 4G2, Canada; †Département de
Médecine Moléculaire, Faculté de Médecine, Université Laval, Quebec, Quebec G1V
0A6, Canada; ‡Centre de Recherche de l’Institut Universitaire en Santé Mentale de
Québec, Université Laval, Quebec, Quebec G1J 2G3, Canada; xCentre d’Optique,
Photonique et Laser, Université Laval, Quebec, Quebec G1V 0A6, Canada; {Unité de
Neuroimmunologie, Centre d’Excellence en Neuromique, Centre de Recherche du
Centre Hospitalier de l’Université de Montréal, Faculté de Médecine, Université de
Montréal, Montréal, Quebec H3C 3J7, Canada; and ‖Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland
ORCID: 0000-0003-0781-6371 (S.L.).
Received for publication February 11, 2014. Accepted for publication June 27, 2014.
This work was supported by an Emerging Team grant from the Canadian Institutes of
Health Research (to Y.D.K., A. Prat, D.C., and S.L.), an operating grant from the
Multiple Sclerosis Society of Canada (to S.L.), and Swiss National Science Foundation Grant 133092 (to B.E.). Salary support was provided by the Multiple Sclerosis
Society of Canada (to S.A.L., A. Paré, and M.A.L.), the Canada Research Chairs
program (to D.C.), and the Fonds de Recherche du Québec en Santé (to A. Prat and S.L.).
Address correspondence and reprint requests to Dr. Steve Lacroix, Centre de Recherche du Centre Hospitalier Universitaire de Québec—Centre Hospitalier Université Laval, 2705 Boulevard Laurier, Quebec, QC G1V 4G2, Canada. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: Alexa-594, Alexa Fluor 594 hydrazide sodium salt;
BBB, blood–brain barrier; BSCB, blood–spinal cord barrier; d.p.i., day(s) postimmunization; EAE, experimental autoimmune encephalomyelitis; EC, endothelial cell;
ko, knockout; NaFl, fluorescein sodium salt; PMN, polymorphonuclear neutrophil;
MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NMO, neuromyelitis optica; PTX, pertussis toxin.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400401
a common feature of several neurologic conditions, including
traumatic brain and spinal cord injury, stroke, neuromyelitis optica
(NMO), and multiple sclerosis (MS) (3–5). In the animal model of
MS, experimental autoimmune encephalomyelitis (EAE), the
BSCB is compromised, thereby exposing the fragile CNS environment to the immune system and its cellular arsenal. This leads
to immune cell invasion, formation of demyelinated lesions, and
axonal damage (4, 6–8). Early breakdown of the BSCB in EAE
has been widely documented, but the precise timing and triggers
of this disruption are still a matter of debate (9–12). This has
prompted us to look further into the kinetics of BSCB disruption
and the putative cellular candidates involved in this process.
The abundance of polymorphonuclear neutrophils (PMNs) and
their capacity to be rapidly deployed to sites of inflammation make
these effector cells particularly well suited to participate in inflammatory cascades that may result in early disruption of the
BSCB, in the context of neuroinflammation. Indeed, the prompt
activation of meningeal mast cells in EAE mice was shown to elicit
sustained neutrophil recruitment, alter BSCB integrity, and promote subsequent leukocyte infiltration into the CNS (13). This is
in agreement with data showing that mast cells control early
neutrophil influx via the secretion of chemokines CXCL1 and
CXCL2 (14). Interestingly, Kroenke et al. (15) showed that these
same chemokines were upregulated in the brain and spinal cord
of EAE mice when the disease was induced by adoptive transfer
of IL-23–modulated T cells. This correlated with extensive inflammatory infiltrates predominantly composed of neutrophils.
Segal and colleagues (16) also showed that CXCL1 and CXCL2
were abundantly transcribed in the spinal cord of naive mice
injected with Th17 cells, shedding light on the involvement of
these cells with the ELR+ CXC chemokine pathway in EAE.
Analyses of mRNA levels in the spinal cord of EAE mice suggest
Disruption of the blood–brain and blood–spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early
pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE),
and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study,
we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability
simultaneously with the kinetics of GFP+ myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease
onset, which paralleled the infiltration of GFP+ cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant
proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed
the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the
blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture
model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils
to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill
multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with
regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events
that take place during EAE and that they are intimately linked with the status of the BBB/BSCB. The Journal of Immunology,
2014, 193: 2438–2454.
Materials and Methods
Animals
All animal procedures were approved by the Animal Welfare Committees
of Laval University, the Centre Hospitalier de l’Université de Montréal, and
the Veterinary Office of the Kanton Bern (Bern, Switzerland), in strict
accordance with guidelines of the Canadian and Swiss Council on
Animal Care. Female C57BL/6 mice were purchased from Charles River
Laboratories (St. Constant, QC, Canada) at 8–10 wk of age. Breeders of
the lys-eGFP-ki strain were obtained from Dr. Gregory Dekaban (Robarts
Institute, London, ON, Canada), with authorization from Dr. Thomas Graf
(Barcelona, Spain). Both heterozygous male and female mice of this strain
were used for EAE induction and imaging experiments when they were .8 wk
of age. No differences were observed in terms of induction rate and disease
outcome between either gender or strain used. IL-1R1–knockout (ko) mice
on a C57BL/6 background and their wild-type littermates were purchased
from The Jackson Laboratory (Bar Harbor, ME). All mice had ad libidum
access to food and water.
EAE induction and clinical evaluation
EAE was induced by s.c. injections of 100 mg myelin oligodendrocyte
glycoprotein (MOG)35–55 (MEVGWYRSPFSRVVHLYRNGK; AnaSpec,
Fremont, CA) in CFA (IFA containing 4 mg/ml heat-inactivated Mycobacterium tuberculosis H37Ra; BD Biosciences, Mississauga, ON, Canada). An i.v. injection of 200 ng pertussis toxin (PTX; List Biological
Laboratories, Campbell, CA) was also administered on days 0 and 2 of the
immunization. The severity of EAE was scored daily using a grading scale
of 0–5, following recommendations of Stromnes and Goverman (36): 0,
unaffected; 0.5, partially limp tail; 1, paralyzed tail; 2, hindlimb paresis
and loss in coordinated movement; 2.5, one hindlimb paralyzed; 3, both
hindlimbs paralyzed; 3.5, hindlimbs paralyzed and weakness in forelimbs;
4, forelimbs paralyzed; and 5, moribund/death. Mice displaying a score of
$2 received daily manual bladder evacuation, and those with a score .3
received daily s.c. injections of sterile saline.
Surgical procedure
Optical access to the spinal cord was achieved by means of a single
laminectomy at the vertebral L1 level, except for the experiment in which
we studied in real time the adhesion of systemically infused neutrophils to
the cervical (C5 level) spinal cord microvasculature (see below). Isoflurane
was used to induce anesthesia (4% v/v) and subsequently throughout the
surgery and imaging session (2% v/v in oxygen). Briefly, a midline incision
∼1 cm in length was performed on the shaved back of mice. The skin was
then retracted to expose the muscles and tissues covering the vertebra of
interest, after which the former were carefully separated from the vertebral
column. Upon removal of the dorsal aspect of the vertebra, the dura mater
was carefully removed. Pilot experiments revealed this step was necessary
in animals displaying heavy clinical burden, because many GFP+ cells
accumulated in the meninges and prevented acceptable image clarity under
the microscope. We confirmed that dura removal did not affect either
BSCB leakage or cellular infiltration during a 90-min observation period
(data no shown). Animals were administered 1% vascular tracer via the tail
vein to visualize the status of the BSCB. C57BL/6 mice were injected with
376 Da fluorescein sodium salt (NaFl; Sigma-Aldrich Canada, Oakville,
ON, Canada, catalog No. F6377), and lys-eGFP-ki mice were injected with
760 Da Alexa Fluor 594 hydrazide, sodium salt (Alexa-594; Life Technologies, Carlsbad, CA, catalog No. A-10438), and both tracers were diluted in sterile saline. Alexa-594 was used because of its size comparable
to NaFl and its emission spectrum not overlapping with GFP as opposed
to the former. Following surgery, mice were placed on a custom-made stabilization device on a heating pad maintained at 37˚C. Gel-Seal (GE
Healthcare, Baie d’Urfé, QC, Canada) was applied on the tissue surrounding
the spinal cord to create a watertight cavity filled with sterile HBSS. Once
the imaging session was completed (∼50 min), the surgical area was carefully cleaned of Gel-Seal. The muscular layers were then sutured and the
cutaneous layers stapled as before (37), after which mice received 150 ml
sterile saline s.c. and were placed in their cage for recovery.
Two-photon intravital microscopy and data acquisition
We used an Olympus FV1000 MPE microscope equipped with a Mai Tai
DeepSee pulsed laser (Spectra-Physics; Newport, Santa Clara, CA) for all intravital imaging experiments. Two-photon excitation was generated at 930 nm
for NaFl in C57BL/6 mice and 840 nm for Alexa-594 in lys-eGFP-ki mice.
Previous independent experiments confirmed that 840 nm was optimally
providing simultaneous excitation of both Alexa-594 and GFP without
spectral overlap. Of note, laser power was kept to a minimum to avoid
photodamage, that is, ∼5–10 mW at the sample. Imaging was performed with
an Olympus Ultra 325 MPE water immersion objective (1.05 numerical
aperture, working distance of 2 mm) at a 512 3 512 resolution, using a pixel
dwell time of 2 ms. The frame rate thus corresponds to ∼2 frames/s. For every
animal, all the visible blood vessels were imaged during the imaging session.
In general, we observed that leakage occurred between 10 and 30 min following tracer administration. Therefore, we made sure to sample all vessels
within this time frame. Interestingly, no noticeable changes in BSCB integrity
were apparent after 30 min. Because animals were not sacrificed following
imaging sessions, the latter did not last .60 min. For time restriction considerations, the dorsal vein was thus partially imaged; its sole purpose was to
provide spatial context for subsequent reconstruction of the vascular arborization. Images were acquired as z-stacks with 2- to 4-mm increments, after
which they were exported in Tiff format for analysis. Time-lapse movies were
processed with the Intravital imaging toolbox using ImageJ (37).
Intravital images analysis
Analyses were carried out on maximal intensity projections using ImageJ
1.46n (Wayne Rasband, National Institutes of Health, Bethesda, MD). For
that neutrophils are among the first inflammatory cells recruited
into the CNS, which is corroborated by studies showing the presence of PMNs in the meninges before the onset of clinical symptoms (13, 17, 18). PMNs are also increasingly recognized as having
pivotal functions in driving inflammatory processes within the
target organ, extending their role beyond that of bystander cells of
the adaptive immune response (19–21). Recent findings present
neutrophils as potent modulators of dendritic cell recruitment and
function, which themselves are responsible for Ag presentation to
encephalitogenic Th1 and Th17 cells following the latter’s transmigration from the perivascular space into the parenchyma (22–
27). The activity of IFN-g–producing Th1 lymphocytes is suppressed by neutrophils in the CNS of EAE mice, and neutrophilderived myeloperoxidase was shown to inhibit dendritic cell activation, therefore dampening T cell–driven inflammation (28, 29).
Aside from their capacity to secrete a vast array of proinflammatory
mediators, neutrophils are now recognized for their ability to
prime Ag-specific Th1 and Th17 responses (30, 31). It was also
reported that the depletion of circulating granulocytes led to
a marked reduction in the number of relapses, attenuated disease
severity, and abrogated the increase in BBB permeability typically
observed during EAE in SJL mice (16, 32). Collectively, these
data suggest an important role for neutrophils in the events that
occur early in the course of EAE, among which BSCB breakdown
is a crucial component warranting closer examination.
Simultaneous analyses of the time course of immune cell recruitment into the CNS and BSCB disruption are hampered in EAE
by the paucity of tools available for tracking cellular populations
with minimal disturbance of their physiological environment. Intravital optical imaging enables sampling/monitoring of a substantial
volume of tissue within the anatomical and functional cell microenvironment (33–35). In this study, we performed intravital imaging
in lys-eGFP-ki mice to investigate the kinetics of BSCB disruption
and CNS myeloid cell infiltration during active EAE. Our data
show that leakage of small fluorescent tracers (,800 Da) across the
BSCB precedes the onset of neurologic deficits and is transient.
Interestingly, the temporal pattern of infiltration of LysM+ cells
coincides with an increase in BSCB permeability. In the present
study, we demonstrate that these infiltrated GFP+ cells are majorly
composed of neutrophils and that their depletion leads to a marked
decrease of vascular leakage in EAE mice as compared with control
animals. Moreover, and in accordance with previous reports, we
found the severity of the disease to be reduced in neutrophildepleted animals (16, 27, 32). Finally, immunohistochemical
analysis of tissue sections from MS brain and NMO spinal cord
reveals that neutrophils are closely apposed to the CNS vasculature, in areas associated with increased BBB/BSCB leakage.
2439
2440
NEUTROPHILS DISRUPT BSCB INTEGRITY IN NEUROINFLAMMATION
Immunohistochemistry on murine EAE spinal cords
Immunohistochemical analyses were performed on lys-eGFP-ki and
C57BL/6 EAE mice that did not undergo surgery or imaging (with the
exception of the experiment in Supplemental Fig. 2C, 2D). In brief, mice
were overdosed with ketamine/xylazine and transcardially perfused with
4% paraformaldehyde (pH 7.4) in PBS. Spinal cords were dissected out,
postfixed overnight at 4˚C, and then transferred for 1 d into PBS containing
20% sucrose. Cervical, thoracic, and lumbar spinal segments, corresponding respectively to spinal levels C4–6, T5–12, and L1–4, were isolated and cut in seven to nine series of 35-mm-thick coronal sections using
a cryostat or microtome, as described before (38).
Immunoperoxidase labeling of Ly6G and CD3 was used to visualize
neutrophils and lymphocytes, respectively, in spinal cord tissue sections
from EAE mice. Because none of the tested Abs against F4/80, Iba1, CD68,
and Galectin-3 could successfully distinguish macrophages from activated
microglia in immunohistochemistry, the infiltration of monocyte-derived
macrophages was assessed using flow cytometry, taking advantage of the
fact that the latter cells express higher levels of CD45 than do their
microglial counterparts (39, 40). Primary Abs used in this study are from
the following sources: rat anti-mouse Ly6G (1:2500, BD Biosciences), rat
anti-mouse CD3 (1:500, BD Biosciences), rat anti-mouse F4/80 (1:200,
AbD Serotec, Raleigh, NC), rabbit anti-mouse Iba1 (1:750, Wako Chemicals, Richmond, VA), rat anti-mouse CD68 (1:2500, AbD Serotec), and rat
anti-mouse Galectin-3 (1:500, American Type Culture Collection, Manassas,
VA). Primary Abs were detected using a biotinylated anti-rat or anti-rabbit
secondary Ab (Vector Laboratories, Burlington, ON, Canada) in conjunction
with an avidin-biotin-peroxidase amplification system (VectaStain ABC Kit,
Vector Laboratories) and 3,3-diaminobenzidine.
The spatial location of GFP+ cells with respect to the BSCB in lyseGFP-ki mice with EAE and lys-eGFP-ki mice treated with PTX was
determined by confocal immunofluorescence labeling of the endothelial
and parenchymal basement membranes using a rabbit anti–pan-laminin
polyclonal Ab (1:500, Dako Canada, Burlington, ON, Canada). The
identity of GFP+ cells as to whether they are neutrophils (or M1 monocytes) or not was verified using the rat anti-mouse 7/4 Ab (1:1000, AbD
Serotec). A goat anti–IL-1R1 polyclonal Ab (1:100, R&D Systems, Minneapolis,
MN) was used to identify cells expressing the IL-1R1 in the normal spinal cord.
Alexa Fluor secondary Ab conjugates (1:200, Life Technologies) were used as
secondary Abs, whereas DAPI (Life Technologies) was used for nuclear counterstaining. Immunofluorescence labeling was performed according to our previously published methods (41). Sections were observed and imaged on a IX81
inverted confocal microscope system equipped with Ar 488, HeNe1 543, and
HeNe2 633 laser lines (Olympus Canada).
Blood sample preparation
Flow cytometric analysis of whole blood was performed in both naive and
immunized (at 8 d postimmunization [d.p.i.]) lys-eGFP-ki mice to identify
leukocyte populations expressing GFP. Additionally, peripheral blood
leukocytes were analyzed by flow cytometry 1 d prior to neutrophil depletion and 1, 2, and 3 d later to confirm the effectiveness and specificity of
the depletion strategy in C57BL/6 mice. Blood was harvested with heparinized syringes via cardiac puncture in mice deeply anesthetized with
a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Blood
samples were immediately placed in EDTA-coated tubes (Sarstedt, Montreal, QC, Canada) in an agitator at room temperature until further processing. Cervical dislocation was performed to ensure euthanasia. Cell
suspensions were washed in PBS/2% FBS, after which they were incubated on ice with mouse Fc block (i.e., purified anti-mouse CD16/CD32;
BD Biosciences) for 15 min to prevent nonspecific binding. They were
then incubated on ice for 30 min with the following fluorochromeconjugated Abs (all from BD Biosciences, except where noted) for multicolor analysis: anti-CD45 PerCP (dilution: 1:100), anti-CD11b Alexa
Fluor 700 (1:100), anti-7/4 PE (1:40, AbD Serotec), anti-Ly6C V450
(1:150), anti-Ly6G PE-Cy7 (1:100), and anti-B220 Alexa Fluor 488
(1:100). After a 5-min wash, erythrocytes were lysed (Beckman Coulter
Canada, Mississauga, ON, Canada, catalog No. 6603152), and the Live/
Dead fixable yellow dead cell stain kit (Life Technologies) was used to
distinguish live from dead cells. Cells were then washed twice in PBS
without serum before data collection.
Spinal cords preparation
Mice of the lys-eGFP strain were immunized for EAE and their lumbar
spinal cord was harvested shortly before or at disease onset for flow
cytometric analysis of infiltrated leukocytes to confirm the presence of
GFP+ neutrophils, following our previously published method (42). Briefly,
animals were transcardially perfused with cold HBSS to remove immune
cells from the vasculature, their spinal cords were dissected out, and
lumbar spinal cords were homogenized with a Potter–Elvehjem tissue
grinder. The tissue was then digested with an enzymatic mixture (0.25%
[w/v] collagenase type IV (Worthington Biochemical, Lakewood Township, NJ), 1 U/ml elastase (Worthington Biochemical), 0.025 U/ml DNAse
I (Worthington Biochemical), 0.1 mg/ml Na-tosyl-L-lysine chloromethyl
ketone hydrochloride (Sigma-Aldrich Canada), and 20 mM HEPES in
HBSS) at 37˚C for 30 min. After a wash in HBSS, cells were filtered
through a 70-mm nylon mesh cell strainer (BD Biosciences), centrifuged at
300 3 g for 10 min, and washed again with HBSS. Specific removal of
myelin debris was performed by incubating single-cell suspensions with
Myelin Removal Beads II (Miltenyi Biotec, Auburn, CA), according to the
manufacturer’s instructions. For multicolor immunofluorescent labeling,
cells were incubated on ice with mouse Fc block (i.e., purified anti-mouse
CD16/CD32; BD Biosciences) for 15 min to prevent nonspecific binding,
followed by labeling for 30 min on ice with the following fluorescently
conjugated primary Abs (all from BD Biosciences except where noted):
anti-CD45 PerCP (dilution, 1:100), anti-CD11b Alexa Fluor 700 (1:100),
anti-Ly6C BD Horizon v450 (1:167), anti-Ly6G PE-Cy7 (1:100), anti-F4/80
allophycocyanin (1:25), anti-CD3e PE-CF594 (1:100), and anti-7/4 PE (1:40,
AbD Serotec) (for a full description of some of these primary Abs, please
refer to our published work; see Ref. 43). As for the blood samples, the
Live/Dead fixable yellow dead cell stain kit (Life Technologies) was used
to distinguish live from dead cells.
Flow cytometry and gating strategy
Cells were first excluded from debris and erythrocytes according to their
forward and side scatter characteristics, after which doublets and dead cells
were discarded. Myeloid cells were identified as follows: neutrophils
(CD45hi, B2202, CD11b+, 7/4dim, Ly6Cdim, Ly6G+), proinflammatory M1
monocytes (CD45hi, B2202, CD11b+, 7/4hi, Ly6Chi, Ly6G2), and antiinflammatory M2 monocytes (CD45hi, B2202, CD11b+, 7/42, Ly6Clo,
Ly6G2). B cells were unaffected by the depletion of neutrophils (see
Fig. 3B and Ref. 44), and therefore cell counts were normalized relative to
this population, identified as (CD45hi, B220+, CD11b2, 7/42). Of note, the
Alexa Fluor 488–conjugated anti-B220 was only used in mice of the
C57BL/6 strain in neutrophil depletion experiments. Data were acquired
for 1 min with an LSR II special order flow cytometer (BD Biosciences)
and analyzed with FlowJo software (version 9.2; Tree Star, Ashland, OR).
Fluorescence minus one controls were used to establish gating boundaries
for every fluorochrome of the staining mixture (45).
Neutrophil depletion and anakinra treatment
The 1A8 mAb was used to deplete neutrophils in EAE mice, whereas the 2A3
mAb served as the isotype control (both from BioXCell, West Lebanon, NH).
The 1A8 clone was chosen over the anti–Gr-1 mAb (clone RB6-8C5) because in addition to the Ly6G Ag the latter binds Ly6C, expressed on
monocytes, dendritic cells, and lymphocytes as well as on neutrophils (46,
47). In contrast, 1A8 recognizes only Ly6G and as such is specific to neutrophils (48, 49). As described in Results, compounds were administered i.p.
in 100 ml sterile saline because the i.v route resulted in significant mortality
of EAE animals. For flow cytometry experiments an additional control
group was injected with 100 ml saline. The depletion treatment was initiated
either at 5 or 7 d.p.i. with an injection of 100 mg Ab or control IgG in sterile
saline. Afterward mice received 10 mg every other day up to 10 d later.
evaluation of BSCB leakage, every vessel was manually outlined with the
freehand line tracer tool, and leaky vessels were identified based on the
presence of dye outside blood vessels. The extent of permeability was
calculated as the ratio of “leaky vessels length” over the “total vessel
length” for a given animal. We routinely imaged .10,000 mm in total
vessel length. For cellular infiltration studies, the analysis strategy was
based on the parenchymal surface occupied by GFP+ cells. Channels from
RGB images were split, after which the red channel (corresponding to the
vasculature) was thresholded so that blood vessels were assigned a pixel
value of 0 (black) and the parenchyma was assigned a value of 255 (white).
In doing so, a vasculature mask was created and the number of white pixels
was used as the measure of parenchymal surface that could potentially be
occupied by GFP+ cells. Then the thresholded red channel was subtracted
from the green channel of the original RGB image so that the latter
was devoid of blood vessels, thus containing only GFP+ cells that had
extravasated. To eliminate signal differences between experiments, the
resulting image was thresholded and the area occupied by cells measured,
regardless of fluorescence intensity. Hence, cellular infiltration was calculated as the ratio of the surface covered by GFP+ cells over the parenchymal surface.
For the experiments in which we investigated the role of IL-1R1 in
neutrophil adhesion to the inflamed spinal cord microvasculature, EAEimmunized mice were injected i.v. daily with 100 ml of the IL-1R1 antagonist anakinra (Kineret; Swedish Orphan Biovitrum). Anakinra was
injected at a concentration of 20 mg/ml in saline for a dose of 0.1 mg/g body
weight, starting the day before MOG35–55 immunization until 14 d.p.i.
Purification of blood neutrophils
Purification of bone marrow neutrophils
Neutrophils were also purified from the bone marrow of naive and EAE
mice whose peripheral blood was collected. After ensuring proper euthanasia, femurs and tibias were removed and the bone marrow was flushed
with HBSS using a 25-gauge needle. Erythrocytes were lysed with ammonium chloride and cells were then passed through a 70- to 100-mm nylon
mesh. Cells were washed and neutrophils isolated using two consecutive
Percoll gradients of 64.8 and 61.5%, respectively. They were then washed
in culture medium and their purity was assessed using flow cytometry as
described above. The average neutrophil purity from bone marrow preparations was 97%.
Primary cultures of mouse BBB endothelial cells and
neutrophil transmigration assay
Primary cultures of mouse brain capillary endothelial cells (ECs) were
prepared from 6- to 8-wk-old female C57BL/6 mice. Mouse brain tissue,
free of meninges, was minced, homogenized, and digested in a mixture of
0.7 mg/ml collagenase type II and 39 U/ml DNase I in DMEM for 75 min at
37˚C. Myelin was removed by centrifugation at 1000 3 g for 20 min in
20% BSA-DMEM. The cell pellet was then incubated for another hour at
37˚C with a mixture of 1 mg/ml collagenase-dispase and 39 U/ml DNAse I
in DMEM. Microvascular ECs were separated on a 33% continuous Percoll gradient, collected, and plated on culture dishes coated with 10 mg/ml
collagen type IV and 5 mg/ml gelatin. Mouse brain capillary ECs were
grown in DMEM supplemented with 20% (v/v) FBS, 1 ng/ml basic fibroblast growth factor, 100 mg/ml heparin, 1.4 mM hydrocortisone, and 13
antibiotic-antimycotic solution. Puromycin (10 mg/ml) was added to the
media for the first 2 d of culture. On the third day, media was changed for
fresh culture media containing 4 mg/ml puromycin. Cultures expressed
vascular endothelial–cadherin protein. No immune reactivity for a-smooth
muscle actin, glial fibrillary acidic protein, or neuronal nuclei protein could
be detected, confirming the absence of contaminating smooth muscle cells,
astrocytes, and neurons, respectively.
Primary cultures of mouse BBB-ECs were used to generate an in vitro
model of the BBB. After reaching confluence (typically 4–6 d), mouse
BBB-ECs were seeded onto collagen type IV/gelatin-coated Transwell
permeable inserts (3-mm pore size), at a density of 5 3 104 cells per well in
EC culture media. Brain ECs were allowed to grow for 4 d to form a confluent monolayer. The upper compartment of each well of the 24-well
Transwell was loaded with 200 ml cell suspension containing 1 3 106 purified mouse neutrophils obtained from the blood or bone marrow of naive
and EAE mice at 7 d.p.i. Neutrophil migration was assessed by counting the
absolute number of cells that transmigrated to the lower chamber after 18 h.
Real-time intravital videomicroscopy
Neutrophils were purified from the bone marrow of EAE mice at 10 d.p.i.,
as described above. Neutrophils were stained with the CellTracker green
fluorescent probe (Life Technologies, 1:2000 dilution), washed with HBSS,
resuspended in sterile saline at a final concentration of 1 3 106 cells per
100 ml volume and then systemically injected via the right carotid artery. A
total of three injections were made (each injected during 1 min) at 2-min
intervals in IL-1R1–ko and wild-type (i.e., C57BL/6) mice with EAE at 15
d.p.i. Detailed methods describing the surgical procedure, intravital microscopy using the epi-illumination technique, as well as procedures for
off-line quantitative analysis of neutrophil interactions in spinal cord white
matter postcapillary venules at the lower cervical (C5) level have been
published elsewhere (50, 51). The intravital microscopy imaging was
performed on a custom-made Mikron IVM500 epifluorescent microscope
(Mikron Instruments, San Marcos, CA) equipped with a low light–imaging
camera VE-1000 silicone-intensified target system (DAGE-MTI, Michigan
City, IN). Firmly adherent neutrophils were identified as fluorescent cells that
are stuck to the vessel wall without moving or detaching from the endothelium. Firm neutrophil adhesion was quantified at 10, 30, and 60 min after
the first injection in four different fields of view (at 310 original magnification) containing a sufficient number of postcapillary venules. Importantly,
the fields of view were selected before performing the first injection and
contained approximately the same number of postcapillary venules.
Postmortem brain and spinal cord tissue from MS and NMO
patients
MS patient. An autopsy was performed on a 32-y-old MS female. Postmortem diagnosis was severe inflammatory rebound following cessation of
natalizumab, as confirmed by Dr. Wolfgang Bruck and Dr. Imke Metz
(Göttingen University Medical School, Göttingen, Germany). Because the
patient was off natalizumab for .4 mo, she was not treated with plasma
exchange. JC virus Ab serology was weakly positive, but JC virus PCRs on
CSF and brain tissue were negative (four times). Pathological analyses of
CNS frozen material revealed innumerable inflammatory infiltrates in the
brain, brainstem, cerebellum, and spinal cord. Final pathological diagnosis
was acute and severe exacerbation of MS with multiple actively demyelinating lesions following cessation of natalizumab. Whether this MS patient suffered from an immune reconstitution inflammatory syndrome
remains a possibility, but this was not the diagnosis made by the neuropathologists (W. Br€uck and I. Metz, personal communication).
NMO patients. Formalin-fixed, paraffin-embedded thoracic spinal cord
material from two clinically suspected and autopsy-confirmed NMO
patients were retrieved from the Pathology Department archival material at
the Centre Hospitalier de l’Université de Montréal. Patients were 68 and
50 y old. Disease duration was 5 and 14 y, respectively, and the cause of
death was pneumonia in both cases. Clinical and pathological diagnoses
were confirmed to be opticospinal disease, also known as NMO or Devic’s
disease. Because the year of death/autopsy were 1988 and 1995, anti–
aquaporin 4 Ab status is not available.
Immunostaining of MS and NMO tissue sections
CNS material from one MS patient affected by an acute and severe episode
of relapses (see above) was collected at autopsy and snap-frozen in n-methyl
butane (270˚C). Brain sections (n = 3) were fixed in acetone for 10 min
and transferred to ethanol for 5 min, hydrated in PBS, and blocked with
10% donkey serum at room temperature for 30 min. Sections were then
incubated for 60 min with primary Abs diluted in 3% donkey serum. The
following two primary Abs were used: mouse anti-human elastase (Dako
Canada, 1:300 dilution) and rabbit anti-human fibrinogen (Innovative
Research, 1:500). Next, sections were washed with PBS and 0.05% Tween
20 and incubated with secondary Abs at room temperature for 30 min.
Secondary Abs were donkey anti-mouse Alexa-488 (1:500) and donkey
anti-rabbit rhodamine red-X (1:500), both from Jackson ImmunoResearch
Laboratories. Finally, sections were mounted using Gelvatol containing
TO-PRO-3 (Life Technologies, 1:300). Each experiment included a negative control in which the primary Ab was omitted.
Formalin-fixed paraffin-embedded CNS material from two clinically and
pathologically confirmed NMO patients was also used. For immunohistochemistry and fluorescence staining, 3-mm-thick tissue sections (n = 4)
were deparaffinized in three successive changes of toluene and rehydrated
in 100 and 95% ethanol, water, and PBS. Slides were mounted with Permount. All reagents were from Sigma-Aldrich Canada.
For neutrophil defensins (subtypes 1, 2, and 3) and fibrinogen stains, Ag
retrieval was performed with sodium citrate at 95˚C for 30 min, cooled at
room temperature, immersed for 3 min in PBS/Tween 20, and blocked in
10% rabbit serum. Sections were incubated with mouse anti-human neutrophil defensins (Leica Biosystems, 1:150) for 1 h at 37˚C and then
washed in PBS/Tween 20. Secondary Ab (rabbit anti-mouse HRP; from
Dako Canada, 1:150) was incubated at room temperature for 30 min
and the immunoperoxidase reaction was developed using 3,3-diaminobenzidine as a chromogen. For the immunofluorescence stainings, the
mouse anti-human neutrophil defensins (1:150) and rabbit anti-human fibrinogen (1:300) primary Abs were combined in the same incubation solution, following the method described above. In all cases, control
stainings were performed omitting the primary Ab, and no immunopositive
cells could be detected. Staining was visualized using either a Leica
DM6000 microscope with OpenLab software or a Leica SP5 confocal
microscope and analyzed using the Leica LAS AF software.
Statistical analysis
Statistical analyses pertaining to EAE courses were performed using the
nonparametric Mann–Whitney U test. Otherwise, data were analyzed with
Blood from naive and EAE mice was collected as described above and
erythrocytes were lysed with 0.2% NaCl. Osmolarity was restored upon
addition of 1.6% NaCl. Neutrophils were isolated by positive selection
using immunomagnetic anti-Ly6G microbeads (Miltenyi Biotec) according
to the manufacturer’s instructions. Eluted cells were washed, resuspended
in culture medium, and their purity, assessed by flow cytometry, was on
average 98%.
2441
2442
a two-tailed unpaired t test, one-way ANOVA, or two-way repeatedmeasures ANOVA, followed by the Bonferroni posttest. A p value
,0.05 was considered statistically significant, and data are expressed as
means 6 SEM unless otherwise noted. All analyses were performed with
Prism software (GraphPad Software, San Diego, CA).
1G). These data therefore demonstrate that the surgery/imaging
strategy can be implemented in EAE studies in lys-eGFP-ki mice
and permits the collection of data prior to and following disease
onset without compromising the pathophysiological events taking
place at any time during disease development.
Results
BSCB disruption is transient and initiated shortly before EAE
onset
Nonterminal intravital imaging sessions do not influence the
initiation, progression, or severity of EAE in mice
BSCB permeability to low–molecular mass fluorescent tracers
(NaFl, 376 Da; Alexa-594, 760 Da) was evaluated in C57BL/6
(NaFl; n = 28) and lys-eGFP-ki (Alexa-594; n = 30) mice at different time points throughout disease course (Fig. 1A). Animals
were allowed to survive following imaging to associate the level
of permeability with the day of disease onset and thus establish
the temporal dynamics of BSCB disruption. On average for both
mouse lines (n = 58), the integrity of the BSCB was undistinguishable among animals yet unaffected by motor deficits up to
the day before the manifestation of symptoms, as demonstrated by
a marginal permeability 4 d up to 2 d prior to disease onset
(Fig. 1B). In lys-eGFP-ki mice, a small increase in permeability
was detected on the day before onset when compared with other
pre-onset time points (Fig. 1C). The compromised status of the
BSCB reached a maximum during a 3-d period starting at disease
onset, during which we observed tracer leakage in ∼40% of the
blood vessels (Fig. 1A–C). After this peak period, the permeability steadily declined toward baseline values. Six days after
onset, the extent of vascular leakage was about a fifth of the value
observed during the peak period and by day 8 postonset it returned
to pre-onset values (Fig. 1B). Because of the limited number of
transgenic lys-eGFP-ki mice available, we chose to direct our
imaging efforts in those animals around disease onset (n = 30
between 24 and 4 d versus onset; Fig. 1C). Hence, no data were
collected later than 4 d postonset in lys-eGFP-ki mice. No permeability has been observed for either NaFl or Alexa-594 in the
spinal cord of naive C57BL/6 and lys-eGFP-ki mice (data not
shown).
The surgical/imaging strategy we have implemented enables us
to express BSCB permeability data relative to the day of disease
onset, which is not possible with histological tissue preparations
and terminal imaging sessions. Interestingly, linear regression
analysis revealed that the clinical score attributed to individual
animals was not correlated with the extent of tracer leakage
measured (r2 = 0.135, Fig. 1D). However, it was evident that mice
displaying partial or total limb paralysis (EAE score $2) presented high BSCB disruption. Hence, in our hands the EAE score
is only partially related to the extent of BSCB disruption in the
lumbar spinal cord and as such is not the optimal point of reference for reporting BSCB permeability. This slightly differs from
other studies reporting a correlation between clinical severity and
the extent of BSCB disruption, although they used different assays, immunization protocols, or mouse strains (4, 55).
Many active EAE induction protocols, including ours, involve
administration of PTX on the day of immunization and 24 or 48 h
later (0 and 1 or 2 d.p.i.). It has been proposed that the toxin
momentarily alters tight junction architecture via vasoactive amine
sensitization, resulting in transient opening of the BBB in susceptible mouse strains (56–58). Therefore, we assessed whether
breaches in the BSCB were detectable early in the course of the
pathology, that is, 24 h following each i.v. PTX injection in immunized animals. None of the eight mice imaged at 1 or 3 d.p.i.
(n = 4 mice/day, disease incidence = 100%) displayed fluorescent
tracer accumulation outside blood vessels (data not shown). Additional control experiments were conducted in mice that received
a combination of adjuvants (without MOG) to ascertain that the
In MOG-induced chronic EAE in C57BL/6 mice, disease progression follows a predictable monophasic pattern once animals
start exhibiting clinical deficits. The most unpredictable parameter
remains the onset of disease, which can occur anytime between 8
and 12 d.p.i. This poses considerable uncertainty in the investigation of pathological events taking place prior to disease onset,
because it is difficult to establish correlation between observations
made before onset and subsequent changes in behavior or disease
course for instance. To alleviate the uncertainty pertaining to the
day of EAE onset, we implemented an intravital imaging strategy in
which mice are imaged once and allowed to survive until termination of the experimental protocol. This enables collecting data
about cellular infiltration and status of the BSCB in asymptomatic
animals and relating them to the exact timing of disease onset.
Chronic implants have been described and would be the optimal
strategy to put forward to remedy the situation, because they allow
repetitive imaging of the same animal in a longitudinal fashion (52,
53). However, in our hands they are not suitable in the study of
autoimmune diseases because they require animals to be administered with immunosuppressant or anti-inflammatory drugs to
limit fibrosis over the surface of the spinal cord, a critical step to
achieve acceptable success rates and in-depth high-resolution
imaging.
Before performing permeability or cellular infiltration studies,
we validated that the surgery and imaging session did not affect the
initiation, development, progression, or severity of the disease. The
premise was to image animals at selected time points in the course
of EAE and compare different parameters to establish whether
indeed the surgery/imaging exerted an influence on any of them.
Prior to immunization, female C57BL/6 (n = 26) mice between 8
and 10 wk of age were randomly assigned to one of six groups
according to the time at which they would be imaged. Imaging
sessions took place at days 1, 3, 7, 10, 14, and 17 following immunization. To avoid bias, animals were imaged regardless of
their presenting EAE symptoms or not. All mice developed EAE
(100% incidence), so based upon their individual EAE course they
were attributed either the group “Pre onset” (n = 16) or “Post
onset” (n = 10), depending on whether they were imaged before or
after first manifesting EAE symptoms, respectively. The mean and
median EAE scores were similar between both groups at all times
throughout the entire duration of the experiment (Supplemental
Fig. 1A, 1B), as were the mean days of onset (Supplemental Fig.
1C) and peak of disease (Supplemental Fig. 1D). Hence, the
surgical protocol and imaging session do not influence the day
when animals first display symptoms or reach their highest clinical
score. To evaluate the total severity of EAE, the area under the
curve and maximal clinical score attained were used as summary
statistics to compare individual animals (54). Both parameters
were identical between the two groups (Supplemental Fig. 1E,
1F), indicating that conducting a single imaging session does
not affect the extent at which animals develop EAE. Moreover,
comparison of the EAE course between C57BL/6 mice and heterozygous lys-eGFP-ki mice in the C57BL/6 background in separate experiments revealed no differences in terms of days of onset
and peak of disease or the severity of EAE (Supplemental Fig.
2443
vascular leakage measured in EAE animals was not caused by the
adjuvants themselves. For this purpose, 16 animals were randomly
assigned to one of four groups (n = 4/group) receiving different
components of the emulsion. Mice were either injected with PTX
alone, PBS plus CFA, PTX plus CFA (categorized as control
groups), or PTX plus CFA plus MOG (i.e., the EAE group). By
virtue of the results presented in Fig. 1B, imaging sessions took
place as mice from the EAE group first displayed clinical symptoms (at ∼10 d.p.i.; disease incidence was 100%) to compare data
acquired in a similar time frame. None of the animals from the
three control groups exhibited symptoms at any time during the
protocol (data not shown) nor did they present evidence of BSCB
permeability (Supplemental Fig. 2A). In sharp contrast, in all four
mice from the EAE group we could measure a significant loss of
BSCB integrity. To definitively rule out the possibility that PTX
induces BSCB disruption, and to study the behavior of myeloid
cells in response to PTX treatment, we examined BSBC leakage in
lys-eGFP-ki mice. For this purpose, both saline-treated (n = 2) and
PTX-treated (n = 4) mice were imaged at 6 h postinjection by
means of two-photon intravital microscopy and then allowed to
FIGURE 1. In vivo permeability of the BSCB to low molecular mass fluorescent tracers shortly precedes EAE onset and coincides with the infiltration of
LysM+ cells. (A) Representative projections of intravital images acquired in the lumbar spinal cord of C57BL/6 mice using NaFl (green, top) and in lyseGFP-ki mice using Alexa-594 (red, bottom). Scale bars, 50 mm. (B) Quantification of BSCB leakage in both mouse strains pooled together (n = 58). An
increase in permeability is apparent 1 d prior to disease onset. Maximal leakage occurs during a 3-d period starting at disease onset, after which it declines
over a few days. (C) Quantification of BSCB leakage and GFP+ infiltration in lys-eGFP-ki mice (n = 30). Infiltration takes place 1 d prior to disease onset,
concomitantly with BSCB disruption. (D) The extent of BSCB permeability is not correlated with the clinical EAE score. However, animals displaying
moderate or heavy signs of paralysis (score $2) have a much more leaky BSCB than animals that do not (score ,2). Data are pooled from both mouse
strains (n = 58). (E) Confocal image of a spinal cord from an EAE lys-eGFP-ki mouse. GFP+ cells were found in the lumen of blood vessels, between the
endothelial and parenchymal basement membranes (pan-laminin staining in red), migrating across the parenchymal basement membrane (arrows), as well
as in the spinal cord parenchyma. Leukocyte infiltration causes a distention of the perivascular space (*). Scale bar, 10 mm. (F) Individual images from an
intravital time-lapse video showing GFP+ cells (white arrows) leaving the vasculature (Alexa -594, red) and entering the perivascular space in a mouse one
day prior to EAE onset. Scale bar, 25 mm. All data are represented as means 6 SEM. **p , 0.01 by two-tailed unpaired t test (D).
2444
Myeloid cell infiltration in the spinal cord precedes EAE onset
and is concomitant with BSCB disruption
Because lys-eGFP-ki mice are reporters of mature granulomyelomonocytic cells (60), we investigated the temporal dynamics of their infiltration in the spinal cord during the course of
acute EAE. Accumulation of GFP+ myeloid cells outside blood
vessels was not observed in asymptomatic animals until the eve of
disease onset (Fig. 1A, 1C, 1E). Before that, we could only visualize cells circulating in the bloodstream with occasional
instances of perivascular macrophages or extravasated cells, as
previously reported in the normal spinal cord of lys-eGFP-ki mice
(61). However, a considerable shift in the infiltration pattern was
evident concomitantly with an increase in BSCB permeability 1 d
prior to onset (n = 4, Fig. 1A, 1C). Interestingly, a large number of
GFP+ cells were bordering the outer surface or in the close vicinity
of blood vessels inside the CNS, suggesting that they were in the
process of transmigration across the BSCB or had recently entered
the perivascular space or spinal cord parenchyma (Fig. 1A, onset
21). We are inclined to think that all of these possibilities are
likely to be true for the following reasons: 1) GFP+ myeloid cells
were seen crossing the spinal cord endothelium in vivo, as demonstrated by a series of images from time-lapse movies (Fig. 1F);
and 2) GFP+ myeloid cells were found in the lumen of blood
vessels, between the endothelial and parenchymal basement
membranes, and in the spinal cord parenchyma upon inspection of
spinal cord sections by laser-scanning confocal microscopy
(Fig. 1E). Notably, some GFP+ cells were observed in the process
of migrating across the parenchymal basement membrane, most
likely toward the parenchyma (see arrow-pointed cells in Fig. 1E).
Animals imaged on the day of clinical onset presented extensive
infiltration as demonstrated by the high CNS coverage we measured, reaching a maximal value 1 d following onset (n = 6,
Fig. 1C). To our surprise, the presence of GFP+ myeloid cells in
the spinal cord parenchyma was not as long-lived as we expected.
Indeed, their surface coverage started declining 2 d after the appearance of symptoms (n = 4) and by 4 d postonset was a third of
the maximum value measured on the day after onset (Fig. 1C).
The temporal course of GFP+ cell infiltration shared many similarities with that of BSCB disruption. Notably, both were detectable shortly before the appearance of motor deficits and reached
a maximum at disease onset for a 3-d period, after which they
declined with a slightly different dynamics. The most noticeable
difference was that BSCB breakdown is considerable on day 2
postonset whereas cellular infiltration is already diminished by
that time (Fig. 1C). This infiltration pattern is reminiscent of
a short-lived myeloid population, which together with their high
number and early course of action relative to BSCB disruption led
us to hypothesize that GFP+ infiltrates are mainly composed of
neutrophils. This hypothesis is supported by earlier studies that
showed that neutrophil infiltration increases during EAE onset,
remains high at the peak of disease, and dramatically declines
thereafter (18, 62).
Flow cytometric analysis of blood from lys-eGFP mice shortly
before EAE onset (8 d.p.i.) confirmed that neutrophils are significantly more numerous than M1 and M2 monocytes, representing ∼40% of all CD45+ leukocytes (Fig. 2A). Neutrophils
constituted ∼75% of all GFP+ cells in the blood at this time point
(Fig. 2B), and the mean GFP fluorescence intensity in neutrophils
was at least 3-fold higher than in either monocyte subsets (Fig. 2C,
2D). Examination of leukocytes in the spinal cord of lys-eGFP
mice revealed infiltration from Ly6G+ neutrophils at similar levels
than F4/80lo and F4/80hi macrophages and CD3+ T lymphocytes
both before and on the day of EAE onset (Fig. 2E, 2G). Interestingly, infiltrated neutrophils from mice not yet displaying
symptoms were forming a high proportion of GFP+ cells (Fig. 2F),
consistent with data obtained from the blood (Fig. 2B). Once mice
started exhibiting locomotor deficits, however, macrophage subsets and neutrophils were indistinguishable in terms of their
contribution to the GFP+ population (Fig. 2H). Of note, no macrophages expressing M2 markers were detected at any time point
in the spinal cord, and T cells did not express GFP in the spinal
cord and blood (Fig. 2F, 2H and data not shown).
In addition to flow cytometry experiments, immunohistochemical stainings were performed on lumbar spinal cord sections to
confirm that neutrophils are indeed present in the CNS shortly
before disease onset in EAE mice. As mentioned in Materials and
Methods, none of the Abs we tested allowed satisfactory discrimination of infiltrating macrophages from resident microglia,
and hence only immunostainings for neutrophils and T cells were
quantified (Fig. 2I, 2J). At 7 d.p.i., Ly6G+ neutrophils were significantly more numerous than CD3+ T cells, whereas both cell
types were present at similar levels at 14 and 21 d.p.i. Taken together, these results support other studies pointing toward an early
involvement of neutrophils in CNS pathophysiology (15, 16, 18,
27), as they infiltrate the spinal cord parenchyma during the preclinical stage of EAE, consistent with our intravital imaging data
(Fig. 1A–C). This suggests an intricate relationship between
neutrophils and events taking place early in the course of the
disease. By virtue of the results from our intravital studies
(Fig. 1A–C), we hypothesized that BSCB disruption is such an
event deserving further investigation.
Neutrophils are eliminated from the circulation of EAE mice
administered the anti-Ly6G 1A8 mAb
To test this hypothesis, we performed depletion experiments in
EAE mice and characterized BSCB integrity in animals devoid
of circulating neutrophils. To selectively deplete the neutrophil
population, we administered anti-Ly6G mAb (clone 1A8) prior to
EAE onset. The efficiency of the treatment in eliminating neutrophils and sparing monocytes was verified in whole blood using
survive until day 2. Animals were then killed by transcardiac
perfusion and their spinal cords processed, immunostained, and
imaged by confocal microscopy. No permeability was observed
for Alexa-594 in the spinal cord of saline-treated and PTX-treated
lys-eGFP-ki mice at 6 h postinjection (Supplemental Fig. 2B).
However, intravital imaging revealed the presence of GFP+ cells
firmly adhered to the endothelium and sometimes crawling along
spinal cord microvessels after PTX treatment, corroborating an
earlier immunohistochemical study by Richard et al. (59). The
only occasional GFP+ cells that were observed in control animals
had a spindle-shaped morphology and were located in the perivascular space, reminiscent of perivascular macrophages.
Importantly, the myeloid cells that adhered to the spinal cord
endothelium of PTX-treated mice did not penetrate into the parenchyma and appeared to be anatomically restricted to the
meningeal vessels (Supplemental Fig. 2C, 2D). Collectively, our
data rule out the contribution of adjuvants in disrupting the BSCB
shortly following immunization or disease onset in EAE animals.
As far as PTX is concerned, this corroborates findings from other
groups who also failed to detect an effect of the toxin in disrupting
the BSCB (9, 11). Note, however, that PTX clearly induces
changes at the level of the spinal cord endothelium, changes that
seem to be related to the recruitment and adhesion of immune
cells. Therefore, the permeability observed is elicited by the
combination of adjuvants and the inflammatory process induced
by myelin fragments as opposed to an action of the adjuvants
alone.
2445
flow cytometric analysis 24 h following the first Ab injection. Cells
were first gated according to their scattering properties and then
doublets were discarded using the forward and side light scatter
parameters (Fig. 3A, left). After selecting live CD45+ cells
(i.e., live leukocytes; Fig. 3A, middle), neutrophils and monocyte
subsets were identified according to their lack of B220 expression
FIGURE 2. Neutrophils infiltrate the spinal cord during preclinical EAE, constituting most GFP+ cells in lys-eGFP-ki mice blood and spinal cord during
the preonset period. (A–D) Flow cytometric analysis of lys-eGFP mouse blood at 8 d.p.i. (n = 4), which is before disease onset. Cells were gated so that
doublets were excluded and only live CD45-expressing leukocytes were considered. Individual populations were identified based on their expression (or
lack thereof) of GFP as well as CD11b, 7/4, Ly6G, and Ly6C markers. Neutrophils are more numerous than M1 and M2 monocytes relative to live
leukocytes (A) and form the dominant population expressing GFP (B) during the preclinical stage of EAE. Histogram and quantification of the mean GFP
fluorescence intensity show that GFP is expressed at a higher level in neutrophils compared with either monocyte subsets (C and D). (E–H) Flow cytometric
analysis of lys-eGFP mouse spinal cord before and on the day of EAE onset (n = 3 mice/time point). Live CD45+ cells were gated as described above, and
microglia (CD45dim) were distinguished from infiltrating blood-derived leukocytes (CD45hi) based on their levels of CD45 expression. The phenotype of
neutrophils, macrophages (Mf; which includes both hematogenous and perivascular macrophages), and T cells was confirmed with Ly6G, F4/80, and CD3,
respectively, in addition to CD11b, 7/4, and Ly6C. Note that monocytes that have recently emigrated from the bloodstream into the spinal cord perivascular
spaces express F4/80 on their surface, but the level is lower than on perivascular and spinal cord–infiltrated macrophages (F4/80lo versus F4/80hi). All
macrophages detected were Ly6Chi, which suggests that no M2 macrophages were present. (E) Neutrophils, macrophages, and T cells infiltrate the lower
spinal cord (T9–S4) at similar levels before EAE onset. (F) Neutrophils appear to constitute the dominant population expressing GFP at this time point,
similar to what has been observed in the blood (B). (G) Neutrophils, macrophages, and T cells infiltrate the spinal cord at similar levels, which are higher
than in mice in the preclinical stage of the disease. (H) Neutrophils and macrophages contribute equally to the GFP+ cellular population. (I) Immunohistochemical stainings of C57BL/6 mouse spinal cords at 7 (left), 14 (right), and 21 (not shown) d.p.i. (n = 3/time point). Scale bar, 200 mm. (J)
Quantification reveals that neutrophils have infiltrated the spinal cord at 7 d.p.i., as opposed to T cells. At 14 and 21 d.p.i., both cell types are present at
similar levels. All data are expressed as means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 with one-way ANOVA followed by Bonferroni posttest (A–
H) or two-tailed unpaired t test (J).
2446
Neutrophil depletion delays the manifestation of EAE,
attenuates clinical symptoms, and prevents early BSCB
disruption
Neutrophil depletion has been performed in autoimmune contexts,
where it was shown that the effector phase of relapsing–remitting
EAE was suppressed in SJL mice that received the anti–Gr-1 mAb
(clone RB6-8C5) directed against both Ly6C and Ly6G (32). Interestingly, Carlson et al. (16) demonstrated that Evans blue dye
extravasation was prevented in neutrophil-depleted SJL mice
during acute symptomatic and relapse episodes, in addition to
a restoration of clinical symptoms following cessation of the anti–
Gr-1 injections. We thus verified that neutrophil depletion using
the 1A8 mAb resulted in similar effects on the EAE course of
C57BL/6 animals. For that purpose, the treatment was initiated
prior to the onset of clinical symptoms and continued every other
day until the peak of the disease (Fig. 4A). Similar to the abovementioned studies, the manifestation of motor deficits was delayed
in mice receiving the 1A8 mAb in comparison with the isotype
control, as demonstrated by differences in the mean daily scores
and number of days with symptoms (Fig. 4A, 4B). Moreover, mice
depleted in neutrophils were either protected against EAE or
exhibited attenuated motor deficits, as the maximal clinical score
they reached during the protocols was significantly less than in
their control littermates, which all developed EAE (Fig. 4B,
middle). Finally, the total severity of the disease during the 30 d
of observation was lower in the anti-Ly6G group (Fig. 4B, right).
Therefore, our data corroborate findings by other groups who
observed a delayed and attenuated manifestation of neurologic and
motor deficits in EAE mice depleted of Gr-1+ cells, among which
the neutrophil population is the most abundant (16, 32).
The delayed onset of clinical symptoms elicited by the absence
of neutrophils suggests an intricate relationship with the immunological events taking place early in the course of EAE, among
which BSCB disruption and immune cell infiltration are key elements (63). Therefore, we performed intravital imaging experiments around disease onset in neutrophil-depleted mice to establish
whether the altered EAE courses observed were associated with
changes in BSCB integrity. Considering the time frame during
which we measured significant leakage of low–molecular mass
fluorescent tracers in previous experiments (Fig. 1), imaging
sessions were conducted when animals from the isotype control
group were displaying acute clinical symptoms (i.e., on days 0 and
2 postonset). In mice that were depleted in neutrophils and subsequently imaged, we observed no (in six of eight) or negligible
(in two of eight) amounts of vascular tracer outside a few blood
vessels (Fig. 4C). In sharp contrast, every animal from the control
group displayed evident vascular leakage, with the fraction of
leaky vessels from this group being 7-fold larger than in the antiLy6G group for the same time frame during which imaging took
place (Fig. 4D). Collectively, our data point toward a plausible
involvement of neutrophils in creating breaches in the BSCB early
in the course of EAE, after which they are suited to enter the CNS
environment and promote the entry and perhaps polarization of
T cells, monocytes, and APCs, thus helping the initiation of deleterious inflammatory events resulting in lesion formation and
neurologic deficits.
Neutrophils acquire a higher capacity to transmigrate across
murine endothelial cells during the process of EAE
We next chose to investigate whether the induction of EAE could
affect the capacity of neutrophils to migrate across the BBB, using
our previously established in vitro model of leukocyte transmigration. We found that highly purified neutrophils isolated either
from the blood or the bone marrow of presymptomatic EAE
animals migrate more efficiently in vitro across mouse CNS microvascular EC monolayers than do those collected from nonimmunized animals (Fig. 4E). These data demonstrate that the
transmigration of neutrophils through brain microvascular ECs is
significantly enhanced early during the course of EAE, and that
this process can occur without the contribution of other leukocyte
subsets.
Firm adhesion of neutrophils to the inflamed spinal cord
microvasculature is regulated by IL-1R1 during EAE
Previous studies have suggested an important role of IL-1R1 in
neutrophil recruitment at sites of inflammation (64, 65), as well as
in the pathogenesis of EAE, MS, and NMO (66–69). Thus, we
determined the expression pattern of IL-1R1 in the mouse spinal
cord. We performed immunofluorescence staining on spinal cord
tissue taken from naive C57BL/6 and IL-1R1–ko (negative control) mice using a polyclonal anti–IL-1R1 Ab. IL-1R1 staining
was always found in close proximity to blood vessel basement
membranes and observed on their luminal side where endothelial
cells are found (Fig. 5A). Importantly, no immunofluorescence
signal was detected in the spinal cord of IL-1R1–ko mice
(Fig. 5B), confirming the specificity of the Ab used and suggesting
that endothelial cells are the major source of IL-1R1 in the normal
spinal cord.
Because of the reported importance of IL-1R1 in neutrophil
recruitment in various inflammatory conditions, and because en-
combined with their differential expression of 7/4 (Ly6B.2;
Fig. 3A, right). The addition of the B220 marker enabled normalization of cell counts to the B cell population (Fig. 3B), which
is not affected by the depletion treatment (44). We confirmed the
phenotype of every gated cell population with the CD11b, Ly6C,
and Ly6G markers. Because the depleting Ab was directed against
Ly6G, this marker was not used in the gating strategy but rather to
confirm that Ly6G+ cells were indeed eliminated from the blood.
The B cell population was quantified for a 1-min-long acquisition,
and B cell counts from the three groups (saline, control IgG, and
anti-Ly6G) were identical (Fig. 3B). In contrast, quantification of
the total live CD45+ leukocytes population revealed that animals
injected with the anti-Ly6G mAb had significantly fewer leukocytes in their blood than did the controls (n = 5/group, Fig. 3C),
thus suggesting that the Ab effectively eliminated cells instead of
blocking receptors at their surface, thereby preventing their detection. Gating cells based on the expression of the 7/4 Ag
confirmed that neutrophils were completely absent from the
circulation in anti-Ly6G–treated mice (Fig. 3D). In contrast,
neutrophil counts were similar among the isotype control and
saline groups. Quantification of the Ly6Chi and Ly6Clo monocyte
subsets supported the premise that the anti-Ly6G treatment did not
affect any of them, indicating that 1A8 specifically depletes circulating neutrophils and spares the monocyte populations
(Fig. 3E, 3F), in agreement with previous reports (43, 48). As
a point of interest, the administration route was a critical factor for
keeping mice healthy in depletion experiments. Indeed, previous
independent experiments revealed that i.v. injection of the 1A8 Ab
on day 7 postimmunization was not tolerated by EAE animals, as
4 of 14 died in the first 3 h following the initial injection (data not
shown). In contrast, the i.p. route caused no problems whatsoever
regarding the survival or well being of the animals, and hence for
every depletion experiments whose results are presented in this
study, Abs were administered i.p. Importantly, the depletion of
neutrophils is long-lasting and very effective, with undetectable
numbers of blood neutrophils for at least 3 d after bolus injection
of the anti-Ly6G mAb (Fig. 3G).
2447
dothelial cells are the principal source of IL-1R1 in the mouse
spinal cord, we next reasoned that systemically infused neutrophils
would be incapable of migrating across the BSCB in mice lacking IL-1R1 during EAE. Using intravital epifluorescence videomicroscopy, we therefore quantified the number of fluorescently
labeled neutrophils permanently adhering to spinal cord postcapillary venules at different time points after neutrophil infusion in
both IL-1R1–ko and control (C57BL/6) mice (Fig. 5C–E). As
shown in Fig. 5C, neutrophils were found to undergo firm and
sustained adhesion to the spinal cord endothelium of C57BL/6
mice with EAE. In contrast, almost no neutrophils could firmly
adhere to the microvasculature of IL-1R1–ko mice, as evidenced
by a .90% drop compared with numbers seen in the C57BL/6
recipient group. Taken together, these results suggest that endothelial IL-1R1 is critical for firm adhesion of neutrophils to spinal
cord microvascular walls during EAE.
Finally, we sought to determine whether treatment with the IL1R1 antagonist anakinra could replicate the findings from the
neutrophil depletion studies (Fig. 4), that is, that clinical signs of
EAE are delayed in mice that received the depleting Ab compared
with those treated with the isotype control. Similar to neutrophil
depletion, IL-1R1 blockade through i.v. administration of anakinra
significantly delayed the day of manifestation of clinical signs of
EAE compared with saline treatment (Fig. 5F, 5G). Thus, our
FIGURE 3. Neutrophils are eliminated from the blood of EAE mice after i.p. injection of the anti-Ly6G 1A8 mAb. (A) Flow cytometric analysis of EAE
mouse blood at 8 d.p.i. Cells were first gated according to their scatter characteristics (left), after which doublets were discarded and live CD45+ cells (i.e.,
leukocytes) were selected (middle). Then, B cells, neutrophils, and monocytes were identified based on their expression of distinct markers, including B220
and 7/4 (right). Cellular phenotypes were confirmed with other markers such as CD11b, Ly6C, and Ly6G. (B and C) Quantification of B cells (B) and live
leukocytes (C) at 1 d after neutrophil depletion (n = 5 mice/group). Note that the depletion treatment was initiated at 7 d.p.i. and that cells were counted
during a 1-min acquisition time. The B cell population is unaffected by the anti-Ly6G treatment, as opposed to the overall leukocyte population. This
indicates that the 1A8 mAb effectively depletes cells instead of blocking surface receptors. (D) Neutrophils are eliminated from the circulation after
administration of 1A8 mAb, as opposed to the isotype control and saline treatments. (E and F) Both the Ly6Chi and Ly6Clo monocyte subsets [(E) and (F),
respectively] are unaltered by the 1A8 mAb injection when compared with either control group. Cell counts were normalized to the number of B cells,
which were not affected by the depletion treatment (B). (G) The depletion of neutrophils in the blood is long-lasting, persisting for at least 3 d after the bolus
injection of the depleting anti-Ly6G Ab (100 mg given i.p.). Data are represented as means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 with one-way
ANOVA followed by a Bonferroni posttest.
2448
FIGURE 4. Neutrophil depletion delays EAE onset and severity and prevents early BSCB permeability. (A) Time course from two independent
experiments, showing that initiating the treatment at 7 (left, n = 6/group) or 5 (right, n = 8/group) d.p.i. results in delayed day of onset and reduced severity.
Red arrows refer to days at which mAb injections took place. Disease incidence was 100% in both experiments for the IgG control groups. (B) Summary
statistics for different parameters. Because some neutrophil-depleted animals are protected from EAE, the number of days with symptoms (left) is used as
an alternative to the day of onset, which would be undefined in those animals. Neutrophil-depleted mice display a delayed onset, because they exhibit
symptoms for shorter period of times than do IgG controls. The maximal clinical score is lower in mice devoid of circulating neutrophils (middle), as is the
area under the curve (AUC, arbitrary units; right). Therefore, disease severity is reduced in those animals compared with the IgG controls. Data are pooled
from the two experiments shown in (A) (n = 14/group in total). (C and D) Representative intravital images (C) and quantification (D) of BSCB integrity from
anti-Ly6G–treated (n = 8) and control IgG-treated (n = 4) EAE mice. All animals were imaged during the same time frame, which is when IgG controls
were displaying acute EAE symptoms. The depletion treatment yields a markedly reduced BSCB permeability for the time (Figure legend continues)
results suggest that a key mechanism by which neutrophils are
recruited to the inflamed spinal cord during EAE involves signaling by IL-1R1 in ECs.
The presence of neutrophils correlates with BBB leakage in
acute lesions in MS brain and in the spinal cord of NMO
Discussion
It is well established that activated leukocytes cross the disrupted
BSCB in NMO, MS, and EAE, although the underlying molecular
bases of those pathological events are ill-defined (63). In this study,
we induced EAE in lys-eGFP-ki mice and used two-photon intravital imaging to monitor BSCB leakage and correlate this
process with the infiltration of mature granulomyelomonocytic
cells in the lumbar spinal cord during the course of the disease. We
present evidence to support the notion that neutrophils are involved in the initial steps of the neuroinflammatory response in
EAE. This is demonstrated by the early influx of neutrophils in the
CNS of EAE mice, which precedes the onset of symptoms and
coincides with an increased permeability of the BSCB to low–
molecular mass fluorescent tracers. Consistent with this hypothesis, we found that 1) neutrophil depletion prevented vascular
leakage in the spinal cord of EAE mice, and 2) neutrophils isolated from the bone marrow or peripheral blood of EAE mice
transmigrate more efficiently across the BBB than do naive neutrophils, a phenomenon that occurs without any influence from
other immune cell types. Although sparse, perivascular neutrophils were found in areas of the CNS displaying increased BBB
leakage, in both MS and NMO patients.
To enable disease monitoring and the association of pre-onset
observations to subsequent manifestation of motor deficits, we
devised an experimental paradigm in which mice are allowed to
survive following the single imaging session they undergo. Using
this strategy, we measured an increase in vascular leakage 1 d prior
to disease onset. No leakage of fluorescent tracer outside blood
vessels could be detected prior to that day. Experiments performed
in the rat using low–molecular mass molecules such as mannitol or
radiolabeled ionic tracers revealed a comparable pattern of dif-
fusion, with extravasation of ions from the lumbar spinal cord
vasculature before other CNS regions (71, 72). Similar observations were made in C3H/He mice, in which ascending paralysis
was closely associated with HRP extravasation in the spinal cord,
whereas predominant cerebellar involvement was evident only in
instances of axial rotatory EAE (11). In the EAE SJL mouse
model, it was proposed that the first occurrence of BBB disruption
and rabbit IgG leakage occurred in the cerebellum, and then
spread to the spinal cord (9). Magnetic resonance imaging in the
mouse revealed that BSCB and BBB disruption took place as
animals were already displaying acute motor symptoms (10, 12).
In the brain of EAE mice, Floris et al. (10) further observed
that monocyte infiltration preceded vascular leakage, although
assessments of BBB integrity and cellular infiltration were performed in separate animals that were sacrificed at the end of the
imaging session. Nevertheless, it demonstrated that loss of vascular integrity is closely related to the initiation of the disease.
In recent studies the extent of BSCB disruption was shown to
correlate with the severity of EAE, as measured by fluorometric
analyses of whole homogenized spinal cords (55, 73). In the
present study, we did not find a direct correlation between the
level of BSCB permeability and clinical score, but rather with the
day of disease onset. However, we did observe that the extent of
leakage was significantly higher in animals presenting signs of
paralysis compared with those that did not. Of note, we observed
considerable vascular leakage in vivo between 10 and 30 min
following i.v. injection of the fluorescent tracer, whereas the
authors of the above-mentioned studies transcardially perfused
animals 10 min following the i.p administration of the tracer (55,
73). Hence, this short circulation time combined with the i.p. injection route might have resulted in underestimation of BSCB
disruption in some animals.
Another important conclusion stemming from our observations
is that the increase in BSCB permeability is transient, peaking 2 d
after disease onset and returning to baseline values ∼6 d postonset.
This is consistent with findings from other groups who also reported a short-term, rather than a permanent, BSCB and BBB
disruption during EAE (10, 72, 74). Interestingly, magnetic resonance imaging studies in MS patients provided evidence that
temporary breakdown of the BBB occurs early in the formation of
new lesions and can be associated with younger age of onset or
more severe disease in relapsing–remitting patients (75, 76). Also
note that we have only used small tracers for permeability studies,
comparable in size to ions (360–760 Da). Therefore, the temporal
changes in BBB integrity that we detected in our study do not
necessarily match alterations of BBB permeability to larger
molecules, such as albumin or dextrans for instance. As Kang
et al. (77) demonstrated, it is erroneous to assume that the BBB
behaves similarly toward small ions and large molecules, supporting the hypothesis of a hierarchical pattern of disruption (4).
Therefore, it is plausible that high–molecular mass vascular
tracers would extravasate according to a different dynamics than
what we measured with low-molecular mass NaFl and Alexa-594.
The triggers responsible for initiating BSCB breakdown in EAE
are yet to be elucidated, although many cellular and molecular
candidates have been shown to influence or modulate its permeability (6). Constituents of the neurovascular unit such as ECs,
period investigated. Scale bar, 50 mm. (E) Neutrophils from EAE mice have a greater propensity to transmigrate than do naive neutrophils. Neutrophils (1 3
106 cells/Transwell) were allowed to migrate for 18 h across a confluent monolayer of primary mouse BBB ECs in a Boyden chamber migration assay. Each
dot represents one Boyden chamber. Neutrophils were isolated from the blood or bone marrow (BM) of naive mice (n = 30) and EAE mice (n = 10) at 7 d.p i.
Data are represented as means 6 SEM. *p , 0.05, **p , 0.01 with a two-tailed Mann–Whitney U test (A and B), a two-tailed unpaired t test (D), or a oneway ANOVA followed by a Bonferroni posttest (E).
In active and chronic MS lesions, the CNS cellular infiltrates are
composed mainly of monocytes/macrophages and T lymphocytes.
However, the presence of neutrophils in the early process of immune cell infiltration in MS is still a matter of debate. To establish
clinical relevance to our findings, we elected to evaluate the
presence of neutrophils in very acute MS lesions and to correlate
the extent of BBB leakage with the presence of neutrophils. Our
data show that in hyperacute MS lesions, the occasional presence of
perivascular elastase+ neutrophils is associated with focal areas
enriched in fibrinogen, confirming that neutrophil infiltration is
associated with focal breaches in the BBB (Fig. 6). This was
confirmed in the spinal cord of NMO patients, an Ab-mediated
disease in which neutrophils are known to play a pivotal role. In
the spinal cord of these patients, defensins+ neutrophils were seen
scattered through the sections, always in close contact with
microvessels (Fig. 7). These neutrophils were often seen in areas
immunopositive for fibrinogen (Fig. 7, lower panels). As Enzmann
et al. (70) reported in acute human stroke lesions, neutrophils were
not seen in CNS parenchyma.
2449
2450
mast cells, and pericytes have received considerable attention
because of their influence on vascular stability (8, 78–80). In the
periphery, cytokines and chemokines released by circulating effector cells such as T cells and neutrophils are also known to affect
CNS barrier homeostasis. Notably, IL-1b has been shown to
modulate BBB permeability in mice and rats and to activate human ECs (7, 81, 82). Interestingly, we found in the present study
that blocking or deleting the endothelial IL-1R1 prevents the adhesion of circulating neutrophils to the spinal cord endothelium
and delays EAE onset. The choroid plexuses were identified as an
alternate route for Th17 cell entry into the CNS via CCR6–CCL20
signaling (83). Interestingly, neutrophils release CCL20 under
inflammatory conditions, and this chemokine is implicated in
a reciprocal activation and recruitment pattern between human
neutrophils and Th17 cells (30, 84). Because of their capacity to
produce and secrete a broad array of molecules and given their
rapid deployment to sites of inflammation, neutrophils have
emerged as likely contributors to the disruption of blood–CNS
barriers induced in EAE (28, 63, 85–88).
Recent studies suggest that myelin-specific encephalitogenic
T cells, upon reactivation in the meninges by dendritic cells, act in
concert with mast cells to promote neutrophil recruitment and
subsequent disruption of the BBB (13, 74). The authors postulated
that neutrophils promote this phenomenon, thereby facilitating
massive leukocyte influx through the compromised BSCB. In
support of this hypothesis, in vitro experiments by Allen et al. (89)
demonstrated that neutrophils acquire a neurotoxic phenotype
upon cerebrovascular transendothelial migration, releasing soluble
factors and proinflammatory cytokines, which could facilitate
such an influx into the CNS. Interestingly, this neurotoxic phenotype of neutrophils was acquired when neutrophils transmigrated across an IL-1–stimulated brain endothelium. Along the
same line, our in vivo data show that blockade of IL-1R1 signaling
with anakinra injected i.v. delays the clinical manifestations of
EAE. Furthermore, we found that activated neutrophils isolated
from EAE mice are unable to firmly adhere to spinal cord
microvessels, and therefore to transmigrate across the BSCB,
when infused into the bloodstream of IL-R1–ko mice. IL-1 is
among the most important differentiation factors for Th17 cells
(67), which are necessary for induction of autoimmune encephalomyelitis (90), and it is also critical for encephalitogenicity of
Th17 cells by regulating GM-CSF production and subsequent
recruitment of effector myeloid cells (91, 92). Work by Segal and
colleagues (16) provides further evidence of the interplay between
T cells and neutrophils in the context of EAE by showing a significant upregulation of neutrophil-attractant chemokines following transfer of myelin-specific CD4+ Th17 cells in naive mice.
Interestingly, these chemokines were present in the CNS of EAE
animals before the clinical manifestation of symptoms. These
findings are consistent with other studies reporting accumulation
of neutrophils in the meninges prior to EAE onset as well as their
prompt entry in the CNS upon immunological challenge, evi-
FIGURE 5. Firm adhesion of neutrophils to the inflamed spinal cord microvasculature is regulated by IL-1R1 during EAE. (A and B) Representative
confocal photomicrographs showing IL-1R1 immunostaining (red) in the spinal cord of a naive C57BL/6 and IL-1R1-ko (negative control) mice. Note the
close proximity between the IL-1R1 signal found at the endothelial cell surface and the pan-laminin immunostaining (green) of blood vessel basement
membranes. Nuclear staining with DAPI is shown in blue. Scale bars, 15 mm. (C and D) Fields-of-view (FOV) images taken from an intravital real-time
video showing neutrophils, stained with the CellTracker green fluorescent probe, adhering to the inflamed BSCB endothelium of an immunized C57BL/6
mouse (C; C57 → C57), but not of an immunized IL-1R1-ko mouse (D; C57 → IL-1R1-ko), at 30 min after cell infusion. Blood vessels are delineated by
dashed red lines. (E) Quantification of firm neutrophil adhesion at 10, 30, and 60 min following systemic infusions of fluorescently stained neutrophils into
C57BL/6 (n = 6) and IL-1R1-ko (n = 6) recipient EAE mice. All data are expressed as means 6 SEM, with the mean representing the average number of
firmly adherent neutrophils per FOV (n = 4 FOV/mouse) using the 310 objective. **p , 0.01 with two-way repeated-measures ANOVA, followed by
a Bonferroni posttest. (F and G) Mice treated with the IL-1R1 antagonist anakinra developed clinical signs of EAE significantly later than those treated with
saline, similar to the response seen in neutrophil-depleted mice (see Fig. 4). Data are represented as means 6 SEM. **p , 0.01, ***p , 0.001 with twoway repeated-measures ANOVA followed by a Bonferroni posttest (F) or a two-tailed Mann–Whitney U test (G).
2451
As in the case of BSCB permeability, this does not necessarily
rule out disease severity as an indicator of neutrophil infiltration in
the whole spinal cord. In our experimental paradigm, we systematically imaged the same lumbar region of the spinal cord in
mice, thus potentially overlooking other areas of cellular infiltration or leakage. This limitation of our intravital imaging strategy is
supplanted by numerous attributes, including the considerable
temporal resolution and context it enables, as well as the large
sampling volume achieved in the unperturbed CNS of live animals.
Importantly, with standard histological techniques the temporal
evolution of BSCB integrity cannot be precisely determined in
FIGURE 6. The presence of neutrophils correlates with BBB leakage in
acute MS lesions. Confocal images show that fibrinogen (red) extravasation into the brain tissue correlates with the presence of elastase-expressing
neutrophils (green) in the vicinity of CNS vessels. The postmortem tissue
was obtained from an MS patient affected by acute and severe relapse
episodes following cessation of natalizumab therapy. Although not the
final diagnosis made by the two neuropathologists (see Materials and
Methods), we acknowledge the possibility of an immune reconstitution
inflammatory syndrome. Left panels show high magnification views of
insets (A and C); right panels show high magnification views of (B) and
(D). Nuclear counterstaining with TO-PRO-3 is shown in blue. Scale bars,
20 mm.
denced by their conspicuous presence in early inflammatory
infiltrates (17, 18, 27, 74). In keeping with these data, we show
that GFP+ neutrophils are present in the spinal cord of lys-eGFP-ki
mice 1 d prior to the manifestation of clinical symptoms, before
transiently infiltrating the parenchyma. Shortly before onset,
a large number of GFP+ cells are detected in the close vicinity of
blood vessels, as compared with later time points, where widespread infiltration is more evident.
The presence of neutrophils in perivascular infiltrates prior to
disease onset emphasizes their putative involvement in BSCB
disruption, possibly by favoring interactions with cells forming the
BSCB and myelin-specific T cells (16, 74). Our data show that the
presence of neutrophils in the CNS parallels the leakage of vascular tracers over time during the course of the disease, as both
phenomena follow similar temporal patterns. A sharp increase in
tracer extravasation and GFP+ infiltration occurs as animals start
displaying neurologic deficits, irrespective of the clinical severity
of disease at that time. Indeed, the determinant factor influencing
the extent of neutrophil infiltration in the CNS was the time of
onset of motor deficits, suggesting that neutrophil infiltration
was a critical triggering event in the development of the pathology. This reflects the unpredictable nature of EAE pathogenesis, as disease progression over time is not linear and
animals most affected in terms of motor deficits (i.e., higher
scores) have not necessarily been displaying symptoms for
longer than their low-scoring littermates. Hence, the timing between disease onset and evaluation of BSCB integrity or cellular
infiltration is paramount in the optics of establishing correlations
between the manifestation of symptoms and the occurrence of
pathological events.
FIGURE 7. The presence of neutrophils correlates with BSCB leakage
in NMO patients. (A) Immunohistochemistry for elastase shows neutrophil
infiltration (brown cells) in the spinal cord of NMO patients. The tissue
was counterstained using hematoxylin. (B) Confocal images show that the
recruitment of defensins+ neutrophils (green) to the BSCB correlates with
fibrinogen (red) leakage in postmortem tissue obtained from NMO
patients. Insets show an area where neutrophil proximity to endothelium
associates with BSCB disruption. Corresponding high-magnification
images are shown in left panels. Nuclear counterstaining with TO-PRO-3
is shown in blue. Scale bar, 50 mm.
2452
Acknowledgments
We thank Nadia Fortin, Martine Lessard, Nicolas Vallières, Heidi Tardent,
and Gaby Enzmann for invaluable assistance in this work. We are also
grateful to Dr. Denis Soulet (Centre de Recherche du Centre Hospitalier
Universitaire de Québec—Centre Hospitalier Université Laval) for providing access to a laser scanning confocal microscope.
Disclosures
The authors have no financial conflicts of interest.
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Pre onset
Post onset
d
d.p.i.
Onset
f
g
Area Under the Curve
AUC (a.u.)
b
Median EAE scores
Peak
d.p.i.
e
Maximal clinical score
Summary statistics
C57BL/6 strain
lys-GFP strain
Mean day of disease onset
10.1+/- 0.2
10.2 +/- 0.4
Mean day of peak disease
12.8 +/- 0.3
13.8 +/- 0.5
2.6 +/- 0.1
3.1 +/- 0.1
58/58 (100%)
40/42 (95%)
Maximal clinical score
Disease incidence
SUPPLEMENTARY FIGURE 1.
Pre onset
Post onset
EAE score
d.p.i.
d.p.i.
c
Mean EAE scores
Median EAE score
Mean EAE score
a
progression or severity of EAE in C57BL/6 mice. (a) Time points at which imaging (i) was performed are shown above the curves
depicting the daily mean EAE scores from the "Pre Onset" (n = 16) and "Post Onset" (n = 10) groups. Both are similar throughout the
duration of the protocol, as are the median (b) EAE scores. (c-f
c) and peak (d), as well as the total severity of clinical symptoms, measured with the maximal clinical score attained (e) and area under the curve (AUC, arbitrary units) (f). (g) Comparison of the EAE
course between C57BL/6 and lys-eGFP-ki mice. Data represented as mean ± SEM. p > 0.05 with 2-tailed Mann-Whitney test (ns = not
a
b
Saline, t = 6 h
PTX, t = 6 h
Fraction leaky vessels
Alexa 594
LysM+ cells
c
d
Saline, t = 48 h
PTX, t = 48 h
DAPI
eGFP
7/4 A555
Pan-laminin A633
DAPI
eGFP
7/4 A555
Pan-laminin A633
SUPPLEMENTAL FIGURE 2. The administration of PTX does not elicit transmigration of myeloid cells in the spinal cord of
non-immunized mice or BSCB disruption in an EAE context. (a) Individual components of the emulsion cocktail (without MOG35-55)
do not induce BSCB leakage as opposed to the whole cocktail. Animals (n = 4/group) were imaged as mice from the EAE group were
all displaying acute EAE symptoms, i.e. at about 10 d.p.i. Data are represented as mean ± SEM. **: p < 0.01 by 1-way ANOVA followed
by Bonferroni post-test. (b) Representative intravital images from lys-eGFP-ki mice showing that administering PTX to non-immunized mice (i.e. without CFA and MOG35-55) does not result in transmigration of hematogenous granulomyelomonocytic cells into
+
cells to the spinal cord
microvasculature when compared to saline-injected mice. Scale bar: 50 um. (c-d) Representative confocal micrographs of lumbar
spinal segments from lys-eGFP-ki mice treated with either saline (c; n = 2) or PTX (d; n = 4) and killed 48 hours following the injection.
GFP+
the presence of adherent cells only on the luminal side of blood vessel basement membranes (stained with the pan-laminin
+
cells are either

Aubé et al., J Immunol, 2014

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