How to drain without lymphatics? Dendritic cells migrate from the... fluid to the B-cell follicles of cervical lymph nodes

PHAGOCYTES
How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal
fluid to the B-cell follicles of cervical lymph nodes
Eric Hatterer, Nathalie Davoust, Marianne Didier-Bazes, Carine Vuaillat, Christophe Malcus, Marie-Franc¸oise Belin, and Serge Nataf
The lack of draining lymphatic vessels in
the central nervous system (CNS) contributes to the so-called “CNS immune privilege.” However, despite such a unique
anatomic feature, dendritic cells (DCs)
are able to migrate from the CNS to cervical lymph nodes through a yet unknown
pathway. In this report, labeled bone marrow–derived myeloid DCs were injected
stereotaxically into the cerebrospinal fluid
(CSF) or brain parenchyma of normal
rats. We found that DCs injected within
brain parenchyma migrate little from their
site of injection and do not reach cervical
lymph nodes. In contrast, intra-CSF–
injected DCs either reach cervical lymph
nodes or, for a minority of them, infiltrate
the subventricular zone, where neural
stem cells reside. Surprisingly, DCs that
reach cervical lymph nodes preferentially
target B-cell follicles rather than T-cell–
rich areas. This report sheds a new light
on the specific role exerted by CSFinfiltrating DCs in the control of CNStargeted immune responses. (Blood. 2006;
107:806-812)
© 2006 by The American Society of Hematology
Introduction
Under normal conditions, the transport of immune cells from blood
to the central nervous system (CNS) is restricted by 2 physical
barriers: the blood-brain barrier formed by CNS parenchymal
microvessels and the blood cerebrospinal fluid (CSF) barrier
formed by the choroid plexuses. Also, the circulation of immune
cells from brain to lymphoid organs is hampered by the lack of
CNS-draining lymphatic vessels. Nevertheless, immune responses
may develop in the CNS, and cervical lymph nodes are considered
as major sites of antigen presentation during neuroinflammatory
diseases.1,2 Indeed, antigens are drained from the CNS to cervical
lymph nodes along the axons of craniofacial peripheral nerves.3,4
Also, it was reported that dendritic cells (DCs) are able to migrate
out of the CNS and, in turn, to elicit a CNS-targeted immune
response.5,6 However, it is not clear whether DCs circulating out of
the CNS actually migrate from brain parenchyma or from the CSF
compartment. This point is of importance because DCs are absent
from normal CNS parenchyma,7 but they can be detected in CSF
and in compartments associated with CSF circulation or production, including meninges and choroid plexuses.8-10 Moreover, under
neuroinflammatory conditions, DCs accumulate in the CSF11,12 as
well as in perivascular spaces,13,14 anatomic compartments draining
into the CSF. These findings, along with others, suggest that the
CSF may be a major transport route for DCs circulating in the CNS
and migrating either from CSF to CNS parenchyma or from CSF to
the lymphoid organs.11,12,15,16
In the present study, we tracked bone marrow–derived myeloid
DCs injected stereotaxically into the CSF or brain parenchyma of
rats under normal conditions.
Materials and methods
From the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM)
U433, Institut Fe´de´ratif de Recherche (IFR) 19, Faculte´ de Me´decine Laennec,
Universite´ Claude Bernard, Lyon, France.
Recherche sur la Sclerose en Plaques (ARSEP) (N.D. and C.V.).
Submitted January 12, 2005; accepted September 9, 2005. Prepublished
online as Blood First Edition Paper, October 4, 2005; DOI 10.1182/blood-200501-0154.
Supported by grants from INSERM, the Rhoˆne-Alpes region, and the Faculte´
de Me´decine Lyon-Nord (S.N.) and by fellowships from the Ministry of
Research and Technologies (MRT) (E.H.) and from the Association Pour la
806
Animals
Animal care and procedures were conducted according to the guidelines
approved by the French Ethical Committee (decree 87-848) and the
European Community directive 86-609-EEC and meet the Neuroscience
Society guidelines. The study protocol was approved by the ethical
committee of Faculte´ de Me´decine Laennec, Lyon, France. Eight- to
10-week-old female Sprague Dawley rats were obtained from Harlan
(Gannat, France).
Reagents
Murine GM-CSF, human Flt3-L, murine IL-4, and human TGF-␤ were
obtained from PeproTech (Tebu). Mouse monoclonal antibodies recognizing rat MHC class II molecules (OX6 antibody), CD11b/CD11c (OX42
antibody), ␣E2 integrin or CD103 (OX62 antibody), CD80 (B7-1, clone
3H5), CD86 (B7-2, clone 24F) or CD54 (ICAM-1, clone 1A29) were
purchased from Becton Dickinson Biosciences (Meylan, France). Mouse
monoclonal antibody recognizing CD11c (clone 8A2) was purchased from
Serotec (Oxford, United Kingdom). For flow cytometry experiments,
FITC-labeled rat-adsorbed goat anti–mouse antibody (Serotec) was used as
a secondary antibody and mouse anti–human CD3 antibody (Beckman
Coulter, Marseille, France) was used as a control primary antibody. For
immunocytochemistry, a fluorescein-conjugated goat anti–mouse antibody
(Alexa Fluor 488; Molecular Probes, Leiden, The Netherlands) was used as
a secondary antibody.
Generation of rat bone marrow–derived DCs
Female Sprague Dawley, Dark Agouti, or Lewis rats were killed, and bone
marrow was flushed from femurs and tibias using 10 mL DMEM in a
The online version of this article contains a data supplement.
Reprints: Serge Nataf, INSERM U433, Faculte´ de Me´decine Laennec, 07 rue
Guillaume Paradin, 69372 Lyon, France; e-mail: [email protected]
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
10-mL syringe with a 26-gauge needle. Bone marrow cells were then
resuspended and passed trough a cell strainer (70-␮m pore). After 1 wash in
phosphate-buffered saline (PBS), cells were resuspended in 10% DMSO,
20% FCS and stored in liquid nitrogen until use. When needed, frozen vials
of cells (10-15 ⫻ 106/vial) were thawed, cells were washed once in DMEM
(Gibco) then cultured in 25 cm2 culture plates at a density of 106 cells/mL in
IMDM (Gibco, Karlsruhe, Germany) supplemented with 15% FCS (Fetal
Clone II; Perbio Science, Bonn, Germany) and antibiotics (penicillin/
streptomycin; Invitrogen, Cergy Pontoise, France). Myeloid rat dendritic
cells were then generated as previously described,17,18 with slight modifications. Briefly, bone marrow cultures were grown for 7 days at 37°C in 5%
CO2 in the presence of murine GM-CSF (10 ng/mL) and human Flt-3 ligand
(10 ng/mL). By the end of this period, clusters of nonadherent cells had
formed that were removed, dispersed, and replated in fresh media consisting of DMEM supplemented with 10% FCS (BioWest, Nuaille´, France),
penicillin/streptomycin (Invitrogen), murine GM-CSF (10 ng/mL), and
murine IL-4 (10 ng/mL). After 3 days, large numbers of free-floating cells
harboring irregular cell surfaces could be observed, as well as a small
population of plastic-adherent macrophages and stromal cells. In some
experiments, 1-␮m diameter fluorescent microspheres (Molecular Probes)
at a dilution of 0.01% solid were added to the culture medium for an
additional 24-hour period. Following this culture procedure, free-floating
cells were harvested, washed once in PBS, and used for cytologic
examination, fluorescence-activated cell sorting (FACS) analysis, or in vivo
experiments.
EFFLUX OF DENDRITIC CELLS FROM CEREBROSPINAL FLUID
807
Tucson, AZ), and contrasted with uranyl acetate and lead citrate. Observations were made on a JEOL 1200EX transmission electron microscope
(Jeol, Tokyo, Japan) equipped with a MegaView II high resolution
transmission electron microscope (TEM) camera and an Analysis Soft
Imaging system (Eloı¨se SARL, Roissy, France).
Allogeneic mixed leukocyte reaction
For T-cell preparations, cell suspensions obtained from the cervical lymph
nodes of Lewis rats were passed through a cell strainer (70-␮m pore) and
negative magnetic selection (Milteny Biotec, Paris, France) was performed
using OX6 (anti–MHC class II molecules), OX33 (CD45RA expressed on
B cells), or OX42 (anti–CD11b/c antibody) primary antibodies. Bone
marrow–derived DCs, obtained from Sprague Dawley rats as described in
“Generation of rat bone marrow–derived DCs,” were pulsed for 24 hours
with LPS (100 ng/mL; Sigma-Aldrich, Deisenhofen, Germany) then
cocultured in graded doses with 2 ⫻ 105 T cells in a 96-well round-bottom
plate. Cells were plated in triplicate in a total volume of 200 ␮L/well with
DC/T cell ratios from 1:20 to 1:160. After 72-hour culture, cells were pulsed
with 2 ␮Ci/well (0.074 MBq/well) 3H-thymidine (Amersham Biosciences,
Uppsala, Sweden) for 18 hours then harvested on fiberglass fibers.
Incorporated thymidine was quantified in a direct beta counter (Matrix 96;
Packard, Groningen, The Netherlands), and results were expressed as the
mean counts per minute (cpm) ⫾ SD of triplicate cultures.
Cell labeling
Cytologic analysis
After fixing in acetone, cells were rinsed 3 times in PBS then incubated for
30 minutes at room temperature with a blocking solution containing 4%
bovine serum albumin and 10% normal goat serum. Cells were then
incubated overnight at 4°C with mouse monoclonal antibody OX42, OX6,
anti-CD80, anti-CD86, or anti-CD54 diluted 1:100 to 1:400 in blocking
solution. After several washes in PBS, cells were incubated for 50 minutes
in blocking solution containing a fluorescein-conjugated goat anti–mouse
antibody (dilution 1:100) then rinsed in PBS and mounted using an aqueous
preparation (Fluoroprep; BioMe´rieux, Marcy l’Etoile, France). In experiments in which fluorescent microspheres had been added to the culture
medium, harvested cells were fixed in 4% paraformaldehyde before being
cytospun and processed for immunocytologic analysis as described above
in this paragraph. In these cases, confocal laser scanning microscopy (LSM
META Zeiss; Carl Zeiss, Jena, Germany) was performed to discriminate
between internalization of particles and attachment to the cell membrane.
Otherwise, images were recorded and analyzed by a computer-assisted
system consisting of a specific image analysis software (analySIS auto; Soft
Imaging System, Mu¨nster, Germany).
FACS analysis
In each experiment, 0.2 ⫻ 106 to 0.5 ⫻ 106 cells were incubated on ice for
30 minutes with mouse OX6 antibody (anti–MHC class II molecules),
OX62 antibody (anti–integrin ␣E2 or CD103), anti-CD11c, anti-CD80,
anti-CD86, or control mouse anti–human CD3 antibody diluted 1:50 in PBS
containing 2% FCS. After one wash in PBS-2%FCS, cells were then stained
with FITC-conjugated rat-adsorbed goat anti–mouse IgG, washed twice in
PBS, measured in a EPICS XL flow cytometer (Beckman Coulter), using
CellQuest software (Becton Dickinson) for analysis.
Electron microscopy
Bone marrow–derived DCs were fixed for 30 minutes in 2% glutaraldehyde–
0.1 M NaCacodylate pH 7.4. They were then washed 3 times in 0.1 M
Nacacodylate/sucrose, pH 7.4, for 15 minutes and fixed afterward with 1%
OsO4-0.15 M NaCacodylate pH 7.4 for 30 minutes. After dehydration in an
ascending gradient of ethanol, 5 minutes for each step, 30%, 50%, 70%, and
95%, impregnation steps and embedding were performed in Epon, finally
polymerized at 60°C for 48 hours. Sixty to 80-nm sections were obtained
using an ultramicrotome RMC-MTX (Research. Manufacturing Company,
For injection experiments, DCs were labeled using CFSE (carboxyfluorescein diacetate succinimidyl ester; Molecular Probes) or fluorescent microspheres. Briefly, for CFSE labeling, cells were washed once in PBS and
incubated for 5 minutes at 37°C in 1 ␮M CFSE. Then, 250 ␮L FCS was
added, and cells were further incubated for 5 minutes at 37°C before being
washed in PBS and resuspended at a dilution of 3 ⫻ 104 cells/␮L in phenol
red–free DMEM. Alternatively, cells were incubated for 24 hours with
fluorescent microspheres as described in “Generation of rat bone marrow–
derived DCs,” and then rinsed twice in PBS and resuspended at a dilution of
3 ⫻ 104 cells/␮L in phenol red–free DMEM.
Stereotaxic injections of labeled DCs
Stereotaxic injections of labeled DCs were performed in 21 female
Sprague-Dawley normal rats. All injections were performed using phenol
red–free DMEM as a vehicle. For intra-CSF injections in normal rats,
3 ⫻ 105 DCs loaded with fluorescent microspheres (n ⫽ 5) or labeled with
CFSE (n ⫽ 6) were diluted in 10 ␮L vehicle and injected in the left lateral
ventricle. Briefly, each rat was deeply anesthetized by pentobarbital
injection and placed in a stereotaxic frame, and its head was tilted slightly
by raising the tooth bar to 5 mm. Solution (10 ␮L) was then slowly injected
in the left lateral ventricle (stereotaxic coordinates: 1.4 mm lateral to the
bregma and 4.5 mm down from the surface of the skull) over a period of 3
minutes, using a Hamilton syringe. The animal remained in the stereotaxic
frame with the needle in place for 1 minute thereafter, and the needle was
then slowly removed over a period of 2 minutes. Alternatively, DCs loaded
with fluorescent microspheres (n ⫽ 3) or labeled with CFSE (n ⫽ 5) were
injected into the corpus callosum (stereotaxic coordinates: 1.4 mm lateral to
the bregma and 3.7 mm down from the surface of the skull), otherwise
following the same protocol. Following the intra-CSF injections of DCs,
rats were killed on day 1 (n ⫽ 2), 3 (n ⫽ 5), or 8 (n ⫽ 4) after injection.
Following the intraparenchymal injections of DCs, rats were killed on day 3
(n ⫽ 4) or 8 (n ⫽ 4) after injection. Control experiments were performed in
which rats received an intra-CSF injection of vehicle alone (n ⫽ 2).
Histologic analysis
On day 1, 3, or 8 after stereotaxic injection of DCs, animals were
anesthetized by halothane inhalation and killed by intracardiac perfusion
with 250 mL 4% paraformaldehyde in 100 mM pH 7.4 phosphate buffer.
Then brains, cervical lymph nodes, and axillary lymph nodes were
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HATTERER et al
dissected out, immersed overnight in fixative at 4°C, and kept in PBS
containing 30% sucrose at 4°C until use. When needed, tissues were then
frozen in dry ice, and the blocks were embedded in polyethylene glycol and
cut in 14-␮m thick sections with a cryostat.
Results
Generation and characterization of rat
bone marrow–derived DCs
Rat myeloid DCs were generated, as described in “Materials and
methods,” by the sequential treatment of whole bone marrow cultures
with Flt-3 ligand ⫹ GM-CSF for 7 days, then GM-CSF ⫹ IL-4 for 3
days. Free-floating cells harvested after 10 days of culture displayed a
round irregular morphology (Figure 1A) or harbored dendrites of
various lengths (Figure 1E). Immunocytoflurorescence analysis showed
that all cells exhibited strong MHC class II staining (Figures 1B,F) with
50% to 60% of them showing MHC class II⫹ dendrites (Figure 1F).
Analysis by transmission electron microscopy allowed us to distinguish
round irregular cells with a rich endosomal compartment (Figure 1C)
from process-bearing cells with a less-developed endosomal compartment (Figure 1G). When performing a phagocytic assay with fluores-
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
cent microspheres, we found that (1) greater than 95% of cells ingested
substantial amounts of microspheres while expressing MHC class II
molecules (Figures 1D,H) and (2) cells showing the more immature DC
morphology (ie, a round irregular cell body without dendrites) displayed
the highest phagocytic activity (Figure 1D). Finally, by FACS analysis,
we observed that cells uniformly expressed the rat dendritic cell markers
CD11c and OX62,17,19,20 as well as OX42 (CD11b/c) and low-tointermediate levels of MHC class II molecules (OX6), CD80, CD86,
and CD54 (Figure 1I; data not shown). Altogether, these results indicate
that cells, generated under our experimental protocol, consisted of
immature myeloid DCs. Accordingly, when cells were stimulated with
LPS for 24 hours, they all acquired the morphologic, phenotypical, and
functional features of fully mature DCs (Figure 1J and Figure S1; see the
Supplemental Figure link at the top of the online article, at the Blood
website). In particular, LPS-stimulated DCs exhibited dendrites bearing
MHC class II molecules (Figure S1), and the levels of membranous
OX6 and CD86 antigens, as assessed by FACS analysis, were dramatically up-regulated when compared with unstimulated DCs (Figures
1I,J). Similar findings were observed when analyzing CD80 and CD54
antigens by immunocytofluorescence (data not shown). Finally, confirming their mature phenotype, LPS-stimulated DCs elicited a dosedependent allogeneic T-cell response (Figure S1).
Figure 1. Generation of rat bone marrow–derived immature myeloid DCs. Rat bone marrow cultures were sequentially treated with Flt3-ligand ⫹ GM-CSF for 7 days and
then GM-CSF ⫹ IL-4 for 3 days. Nonadherent cells were then harvested and characterized by using hematoxylin-eosin staining (A,E), immunocytofluorescence (B,F,D,H),
electron microscopy (C,G), or FACS analysis (I). (A,D) Cells stained with hematoxylin-eosin show a round irregular morphology (A) or bear multiple dendrites (E). (B,F) Round
irregular cells (B) as well as process-bearing cells (F) display strong immunostaining against MHC class II molecules. (C,G) Electron microscopy allows round irregular cells
with numerous phagosomes and phagolysosomes (C) to be distinguished from process-bearing cells showing a less developed endosomal compartment (G). (D,H) The
phagocytic activity of bone marrow–derived DCs was evaluated by adding, in the culture medium, fluorescent latex microspheres of 1-␮m diameter for 24 hours. Using confocal
microscopy, fluorescent microspheres (red) are observed in the cytoplasm of MHC class II⫹ round irregular DCs (D) or MHC class II⫹ (green) process-bearing DCs (H). The
inset in panel D shows a high magnification view of MHC class II⫹ endocytic vesicles (green) having internalized fluorescent microspheres (red). (I) FACS analysis shows that
cells uniformly express OX62, CD11c, and OX42, indicating they are myeloid dendritic cells. They also show low-to-intermediate levels of MHC class II molecules, CD80, and
CD86, indicating they are immature DCs. (J) When stimulated with LPS, DCs acquire phenotypic features of mature DCs because they express high membranous levels of
CD11c, MHC class II, and CD86 molecules as compared with control staining (gray curve). For results of FACS analysis, in each quadrant the percentage of cells is shown
displaying fluorescence intensity above the background level obtained with a control antibody (gray curve), the mean fluorescence intensity (MFI), and the factor of MFI
increase as compared with control MFI. Scale bars: 8 ␮m (A,E), 4 ␮m (B,F), 2 ␮m (C,G), 2 ␮m (D), 1.5 ␮m (inset in D), and 2 ␮m (H). Data shown are representative of at least
3 experiments.
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
EFFLUX OF DENDRITIC CELLS FROM CEREBROSPINAL FLUID
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brain parenchyma) (n ⫽ 4). Animals were killed for CNS histologic analysis on day 1, 3, or 8 after injection. At all time points
studied, DCs injected in the left lateral ventricle could be found in
the CSF compartment. In particular, groups of labeled cells were
frequently found on the apical surface of the choroid plexus
homolateral to the injection (Figure 2A-B). Also, numerous cells
were detected bilaterally in the recesses of the fourth ventricle,
which communicate with the subarachnoid spaces, and in the brain
cortical meninges (Figure 2C-D; data not shown). Altogether, these
observations suggest that cells had passively followed the CSF
flow through the third and fourth ventricle before getting access to
the outer surface of the brain, where CSF circulates in the
subarachnoid spaces.
Figure 2. Traffic of microsphere-loaded DCs in the CNS of normal rats. DCs
were incubated for 24 hours with fluorescent microspheres, then washed and injected
into the CSF (A-G) or brain parenchyma (H) of normal rats. Photomicrographs show
representative results obtained from analyses of brains on day 3 after injections.
(A-D) Brain sections were examined by light microscopy after hematein-eosin
staining (A,C) or by fluorescent microscopy to detect cells loaded with fluorescent
beads (red) (B,D). Groups of labeled cells are detected in the lateral ventricle,
homolateral to the injection site, on the apical surface of the choroid plexus (A-B;
circle). Injected DCs are detected bilaterally in the recesses of the fourth ventricle
(C-D). (E-G) For the detection of cells loaded with red fluorescent beads, brain
sections were examined by fluorescence microscopy after counterstaining with DAPI
for nuclei visualization (blue). Cells loaded with fluorescent beads are observed in the
subventricular zone homolateral to intravenous injection and particularly within the
germinal zone (E-F; circles). Inset in panel F shows a high magnification view of a cell
located in the germinal zone and harboring numerous intracytoplasmic beads.
Photomicrograph in panel G shows a cell containing fluorescent beads in brain
parenchyma adjacent to the third ventricle. (H) DCs loaded with fluorescent
microspheres and injected into the corpus callosum migrate a short distance from the
injection site, along the adjacent white matter tracts. LV indicates lateral ventricle; cc,
corpus callosum; CP, choroid plexus; Ce, cerebellum; V3, third ventricle. Scale bars:
200 ␮m (C,D,H), 100 ␮m (A,B,E,F), 50 ␮m (G), 2 ␮m (inset in panel F).
Traffic of intra-CSF–injected DCs in the CNS
In a first set of experiments, DCs were incubated with fluorescent
microspheres to track them. Normal rats were then injected with
labeled DCs (3 ⫻ 105 cells) within the left lateral ventricle (n ⫽ 4),
that is, in the CSF compartment or within the corpus callosum (in
Figure 3. Traffic of CFSE-labeled DCs in the CNS of normal rats. Bone
marrow–derived DCs labeled with CFSE were injected into the CSF (A-F) or brain
parenchyma (G-H) of normal rats. For the detection of green CFSE-labeled cells,
brain sections were examined by fluorescence microscopy after counterstaining with
DAPI for nuclei visualization (blue). Photomicrographs show representative results
obtained from analyses of brains on day 3 after injections. (A-B) CFSE-labeled cells
(white arrows) are detected in the germinal zone homolateral to the injection site.
(C-D) CFSE-labeled cells are detected in brain cortical meninges (white dotted line
indicates localization of the glia limitans; circle indicates nuclei of CFSE-labeled cells).
(E-F) A CFSE-labeled cell is observed in the brain parenchyma adjacent to the third
ventricle (circle indicates the nucleus of the CFSE-labeled cell). (G-H) CFSE-labeled DCs
injected into the corpus callosum migrate a short distance from the injection site, along the
adjacent white matter tracts. LV indicates lateral ventricle; cc, corpus callosum; V3, third
ventricle. Scale bars: 100 ␮m (G-H), 50 ␮m (A-B), 30 ␮m (C-F).
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HATTERER et al
Interestingly, some DCs injected in the left lateral ventricle
were found in parenchymal locations, including the subventricular
zone homolateral to the intraventricular injection and, in particular,
the germinal zone, where neural stem cells reside (Figure 2E-F).21
Occasional cells were also detected in the brain parenchyma
adjacent to the third ventricle as well as in frontal cortical locations,
several millimeters away from the site of injection (Figure 2G; data
not shown). Labeled cells located within brain parenchyma were
detected starting day 3 or 8 after injection, indicating they had first
circulated in CSF before infiltrating CNS parenchyma. Thus, our
results show that intra-CSF–injected DCs loaded with microspheres are actually able to infiltrate brain parenchyma.
In contrast to the migratory behavior of DCs injected into the
left lateral ventricle we observed that DCs injected into the corpus
callosum remained mostly confined around the injection site or
migrated only a short distance along the adjacent white matter
tracts (Figure 2H). To ensure that phagocytized microspheres did
not alter the migratory behavior of DCs, a second set of experiments was performed in which CFSE-labeled DCs were injected
into the left lateral ventricle (n ⫽ 6) or corpus callosum (n ⫽ 5) of
normal rats (Figure 3). In these experiments, analysis of CNS
obtained on day 3 (n ⫽ 5) or 8 (n ⫽ 6) after injection gave similar
results to those observed after injections of DCs loaded with
microspheres (Figures 2 and 3). In particular, intra-CSF–injected
DCs could be found in the subventricular zone homolateral to the
injection site (Figure 3A-B), in the meninges (Figure 3C-D), the
brain parenchyma adjacent to the third ventricle (Figure 3E-F) and
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
occasionally in the frontal cortex (data not shown). Also, CFSElabeled DCs injected within corpus callosum migrated little from
their injection site (Figure 3G-H).
Intra-CSF–injected DCs reach the B-cell follicles of cervical
lymph nodes
Histologic examination was performed on cervical lymph nodes
and axillary lymph nodes obtained from animals killed on day 3
(n ⫽ 5) or 8 (n ⫽ 6) after injections of CFSE-labeled DCs into the
left lateral ventricle or corpus callosum (Figure 4). Following
injections in the left lateral ventricle, numerous CFSE-labeled cells
were found in cervical lymph nodes (Figure 4A-B), whereas only
rare labeled cells were detected in axillary lymph nodes (Figure
4C-D). Interestingly, the vast majority of CFSE-labeled cells were
detected within B-cell follicles (Figure 4A, left) and, to a lesser
extent, in the medulla (Figure 4B, right) of cervical lymph nodes. In
accordance with the observed poor mobility of DCs injected within
corpus callosum (Figures 2 and 3), no or only occasional labeled cells
could be detected in cervical or axillary lymph nodes, following
injections of DCs in corpus callosum (Figure 4E-F). To confirm the
preferential distribution of intra-CSF–injected DCs within B-cell follicles of cervical lymph nodes, immunohistofluorescence experiments
were performed using antibodies directed against CD3, OX33 antigen
(CD45RA expressed on B cells), or OX6 antigen (MHC class II
molecules). We observed that CFSE-labeled DCs were mainly localized
in B-cell follicles as compared with T-cell–rich areas (Figure 5A-B).
Figure 4. Cervical lymph node targeting of CSF-injected DCs. Bone marrow–derived DCs were labeled with CFSE then injected into the CSF (A-D) or brain parenchyma
(E-F) of normal rats. Histologic examination of cervical or axillary lymph nodes was performed by fluorescence microscopy after counterstaining with DAPI for nuclei
visualization (blue). Photomicrographs show representative results obtained from analyzing lymph nodes on day 3 after injection. (A-B) In the cervical lymph nodes, numerous
labeled cells are detected within B-cell cortical follicles (A) and in the medulla (B). (C-D) Only occasional labeled cells are detected in axillary lymph nodes and are mainly
localized in the medulla. (E-F) Following injections of DCs into brain parenchyma, no labeled cells are observed in cervical (E) or axillary lymph nodes (F). Scale bars: 200 ␮m.
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Figure 5. Targeting of CSF-injected DCs to B-cell
follicles. To confirm the localization of CFSE-labeled
DCs within B-cell follicles, immunostaining of CD3,
CD45RA (OX33 expressed on B cells), or MHC class II
molecules was performed on sections of cervical lymph
nodes obtained from rats killed on day 3 after injection
(n ⫽ 2). (A-B) Although some CFSE-labeled cells are
present in the T-cell area (A), the majority are located
within B-cell follicles (B). (C) Immunostaining of MHC
class II molecules shows that CFSE-labeled DCs express
MHC class II molecules within B-cell follicles of cervical
lymph nodes. F indicates follicle. Scale bars: 200 ␮m (B),
100 ␮m (A), 50 ␮m (C), 10 ␮m (inset in panel C).
Moreover, colocalization of CFSE labeling with OX6 staining showed
that intra-CSF–injected DCs had maintained the expression of MHC
class II molecules in B-cell follicles (Figure 5C).
Discussion
The prototypic migratory pattern of tissue-resident DCs was
initially established by studying dermal DCs and epidermalresiding Langerhans cells.22-24 In this paradigm, DCs circulating in
the interstitial fluid of the skin are drained by lymphatic vessels and
reach first the outer surface of lymph nodes before gaining access
to T-cell–rich areas. In the skin, as in many other tissues, migration
of immature DCs is accompanied by a process of maturation,
allowing the acquisition of costimulatory molecules and of chemotactic receptors such as CCR7.25,26 By the end of such a process,
mature DCs localize within T-cell–rich areas of draining lymph
nodes and direct the antigen-specific proliferation of T cells that, in
turn, may amplify B-cell responses through the release of TH2-type
cytokines. Our results suggest that such a functional scheme does
not apply to the CNS. The fact that, within cervical lymph nodes,
intra-CSF–injected DCs preferentially target the B-cell follicles
suggests that, under neuroinflammation, specific mechanisms direct the migration of DCs to this location. Interestingly, the
presence of dendritic cells in the germinal centers of lymphoid
organs has been previously acknowledged.27,28 However, the
mechanisms driving DC migration to germinal centers are unclear.
In this regard, it should be noted that a subset of blood-circulating
myeloid DCs has been shown to specifically target splenic B-cell
follicles and to stimulate B-cell proliferation.29 On the basis of this
finding, one may thus hypothesize that the transport of DCs from
the CSF to the B-cell follicles of cervical lymph nodes might occur
through blood. Further studies are required to clarify this point. In
addition, one has to consider that migration of DCs from the CNS
to cervical lymph nodes may be partly conditioned by the
maturation state of the DCs. In this case further studies are needed
to compare the migratory behavior of mature versus immature DCs
when injected into the CNS.
As exogenously delivered DCs may not behave as endogenous
antigen-presenting cells (APCs), we performed preliminary experiments in which fluorescent microspheres were injected into the
CSF of rats with experimental allergic encephalomyelitis. Results
from these preliminary studies suggest that intra-CSF–injected
microspheres accumulate in the meninges and in the cervical
lymph nodes, where again B-cell zones are targeted. This result
further suggests that under inflammatory conditions APCs circulating within the CSF express a peculiar migratory behavior because
they seem to target the B-cell follicles of cervical lymph nodes.
Besides information on the migration of DCs from brain to
lymphoid organs, our data bring new insights into the traffic of
CSF-circulating DCs within the CNS. Thus, the migration of DCs
in the CSF compartment reproduces some aspects of the physiologic CSF circulation, from lateral ventricles to the brain cortical
meninges.30 However, not all intra-CSF–injected DCs follow the
CSF flow as some of them either adhere to the apical surface of the
choroid plexuses or infiltrate the germinal zone, where neural stem
cells reside. The expression of adhesion molecules by choroid
plexus epithelial cells has been reported and may participate in the
physical interactions between choroid plexuses and the injected
DCs.31,32 Similarly, the presence of infiltrating DCs within the
germinal zone of normal rats suggests that chemotactic factors
812
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
HATTERER et al
locally synthesized in the subventricular zone might direct DC
migration through the ependymal layer.
CNS-resident cells, including astrocytes and microglia, are
thought to shape neuroimmune interactions under normal or
inflamed conditions. Our data indicate that, owing to their unique
migratory behavior, CSF-circulating DCs may also play a major
role in CNS immune responses. In this view, deciphering the
molecular mechanisms supporting the targeting of CSF-circulating
DCs to B-cell follicles of cervical lymph nodes may allow new
therapeutic targets for neuroinflammatory diseases to be identified.
Acknowledgments
We thank Gae¨lle Cavillon for technical assistance in flow
cytometry studies and Christine Servet-Delprat and JeanFranc¸ois Ghersi-Egea for their helpful comments. We also thank
Dr Simone Peyrol and the CeCIL (Centre Commun d’Imagerie
Laennec) for the use of their electron microscopy facilities. We
thank Patricia Hulmes (“allenglish,” Lyon) for critical reading
of the manuscript.
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