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Regular Articles |
From the Laboratory of Organ and System
Pathophysiology,*
Istituto Superiore di Sanità, Rome;
and the Laboratory of Pathophysiology,
Centro
Ricerca Sperimentale, Istituto Regina Elena, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, a chemokine active on DCs and
lymphocytes, and its receptor CCR6 were up-regulated in the CNS
during EAE. These findings suggest that intracerebral recruitment and
maturation of DCs may be crucial in the local stimulation and
maintenance of autoreactive immune responses, and that
therapeutic strategies aimed at manipulating DC migration could be
useful in the treatment of CNS autoimmune disorders.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs have been implicated both in the initiation and maintenance of autoimmune diseases.5 DCs capturing self-antigens in the target organ and migrating to regional lymph nodes are thought to initiate the activation of autoreactive T cells and to support chronic inflammation by sustaining successive waves of priming of naïve T cells. DCs recruited to the target tissue may participate in the maintenance of the inflammatory milieu by local activation of T cells and formation of organized lymphoid structures.6,7
Experimental autoimmune encephalomyelitis (EAE) is a T-cell- and antibody-mediated autoimmune disease of the central nervous system (CNS) widely used to study the pathogenesis of the inflammatory disease multiple sclerosis. EAE can be induced in genetically susceptible animals by active immunization with myelin antigen(s), or by adoptive transfer of myelin-reactive CD4+ T cells.8 Evidence for the involvement of DCs in initiating EAE comes from the observation that DCs efficiently activate encephalitogenic T cells for transfer of EAE,9 and from a recent study showing that DCs pulsed with an encephalitogenic myelin basic protein peptide (Ac1-11) interact with Ac1-11-specific T cell receptor transgenic naïve T cells in the peripheral lymph nodes of recipient mice leading to induction of EAE.10 After migration through the cerebrovascular endothelium, encephalitogenic CD4+ T cells recognize their target antigen on local APCs (reportedly perivascular macrophages and intraparenchymal microglia) and are activated to perform effector functions.11,12 Intracerebral antigen presentation promotes secretion of T helper 1 (Th1) cytokines and the cascade of inflammatory events leading to massive recruitment of macrophages and of additional antigen-specific and nonspecific T cells to the CNS.13
Progression to chronicity in EAE has been associated with the spreading of T cell-mediated autoreactivity to myelin determinants distinct from the initiating ones14,15 and with humoral autoimmunity.16,17 These findings raise two important issues: where the stimulation of new CNS autoreactive T and B cells takes place and whether the CNS harbors or recruits APCs capable of T cell priming locally or after migration to CNS-draining lymph nodes or spleen. Among resident CNS APCs, microglia are less effective than DCs in T cell priming18 and are unlikely to migrate out of the CNS. In the normal CNS of humans and rodents, DCs are strategically located in potential sites of antigen entry such as the choroid plexuses and the meninges, but are not present within the CNS parenchyma.19-22 In a rat model of acute EAE, small numbers of DCs were shown to localize within some spinal cord perivascular inflammatory infiltrates.22 More recently Suter and colleagues23 have demonstrated intracerebral expression of the MHC class II transactivator form I, specific for DCs, and the presence of CD11c+ DCs within CNS perivascular and meningeal inflammatory infiltrates during mouse EAE development. Although these findings suggest that DCs may contribute to intracerebral antigen presentation, it remains to be determined whether DCs are actively recruited to the CNS and play a role in sustaining autoreactive immune responses at the effector site.
The present study provides a detailed immunohistochemical description
of the temporal appearance, spatial distribution, and functional
phenotype of cells expressing DC markers (DEC-205, CD11c, MIDC-8) in
the CNS of SJL mice with proteolipid protein (PLP) 139-151
peptide-induced EAE. It also shows that macrophage inflammatory protein
3 (MIP-3), a chemokine involved in the trafficking of DCs and
lymphocytes to peripheral tissues, and its receptor CCR6 are
up-regulated in the CNS of EAE-affected mice. Collectively, our data
suggest that DC recruitment and maturation within the CNS may be
pivotal in local T cell activation and in maintaining cellular and
humoral autoimmune responses leading to EAE progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female adult SJL mice (Charles River, Calco, Italy) were used at 8 to 12 weeks of age and housed in a controlled environment in accordance with the guidelines of the European Community Council of the Welfare of Experimental Animals (86/609/EEC). All experimental procedures were approved by the Italian Ministry of Health.
EAE Induction
The peptide corresponding to amino acids 139 to 151 of mouse PLP24 was purchased from Primm, Milano, Italy. Female SJL mice were injected subcutaneously in the flank with 0.2 mg of PLP 139-151 peptide in complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI) on days 0 and 7, and with pertussis toxin (0.2 µg; Sigma Chemical Co., St. Louis, MO) intraperitoneally on days 0, 1, 7, and 8.25 Control mice were injected with phosphate-buffered saline (PBS) in CFA and pertussis toxin, according to the same schedule. Mice were weighted and examined daily for clinical signs of EAE, which was scored on the following scale: grade 0, no abnormality; grade 1, reduced tail tonus or slightly clumsy gait; grade 2, tail atony, moderately clumsy gait, impaired righting ability, or any combination of these signs; grade 3, additional hind limb weakness; grade 4, hind limb paralysis and fore limb weakness; grade 5, tetraplegia or moribund state. Mice were sacrificed for immunohistochemical and molecular studies at different times after disease induction: during preclinical EAE (day 10 or 13 after immunization; for this time point, we selected mice that already exhibited marked weight loss); during acute EAE (day 14 to 16 after immunization); during the early remission phase (day 20 to 22 after immunization, 24 hours after disappearance of clinical signs); 48 hours after the onset of relapses that followed an almost complete remission phase (day 25 to 30 after immunization); during chronic EAE (day 30 to 32 after immunization). At least two animals were examined for each time point.
Immunohistochemistry
For specimen collection, mice were anesthetized with xylazine
chloride hydrate and ketamine and perfused intracardiacally with
PBS followed by cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer
(pH 7.4). Brains and spinal cords were removed, kept overnight in 4%
paraformaldehyde at 4°C, passed in 15% and 30% sucrose, frozen in
dry ice-chilled isopentane, entirely cut in serial sections with
cryostat, and stored at -20°C. For immunohistochemistry, 10
µm-thick sections were air-dried, passed in 70%, 95%, and 100%
ethanol, and then dried again. After rehydration with PBS and
pre-incubation with 10% normal rabbit or goat serum, sections were
incubated at 4°C overnight with monoclonal or polyclonal antibodies
optimally diluted in PBS/1% BSA. To identify DCs, three widely used
markers for mouse DCs were used: hamster monoclonal antibody (mAb)
N418, which binds the CD11c integrin;26
rat mAb NLDC-145,
which binds the multilectin receptor DEC-205;27
and rat
mAb MIDC-8, which binds a still unidentified antigen within
intracellular granules of mature, interdigitating DCs in
secondary lymphoid organs28,29
(all from Serotec, Oxford,
UK). Anti-CD4 (RM4–5; PharMingen, San Diego, CA), anti-CD8 (53-6-7;
PharMingen), anti-CD45R/B220 (RA3–6B2; PharMingen) and
anti-CD11b/Mac-1 (5C6; Serotec) rat mAbs were used to identify
CD4+ T cells, CD8+ T cells,
B cells, and macrophages/microglia, respectively. To investigate
the distribution of molecules which are typically expressed on APCs, we
used mouse mAb anti-rat/mouse MHC class II
(I-Ak,s) (MRC OX-6; Serotec), rat mAb anti-mouse
CD40 (3/23; PharMingen) and rat mAb anti-mouse CD86 (B7-2) (GL1;
PharMingen). Sections were also stained with a goat polyclonal
antiserum recognizing rat/mouse MIP-3 (R&D Systems, Minneapolis,
MN). After extensive washings with PBS, sections were incubated with
the corresponding biotinylated secondary antibodies (rabbit
anti-goat IgG, rabbit anti-rat IgG, goat anti-hamster IgG, goat
anti-mouse IgG, all from Vector Laboratories, Burlingame, CA) and
avidin:biotinylated peroxidase complex (ABC), using the ABC Vectastain
Elite kit (Vector Laboratories), according to the manufacturer’s
instructions. Staining reactions were performed with 3,3
diaminobenzidine (DAB; Sigma) as substrate. DEC-205, MIDC-8, and CD86
immunostainings were also performed using the amplification procedure
described by Adams,30
with minor modifications. After
incubation with ABC, sections were incubated with 0.01%
H2O2 and 28 mmol/L
biotinylated tyramine, prepared as described,30
for 10
minutes at room temperature. After extensive washings, sections were
incubated with ABC for 20 minutes, PBS for 30 minutes, and DAB for
another 5 minutes. To eliminate endogenous peroxidase activity,
sections were incubated with 0.3%
H2O2 in PBS, for 20
minutes, before secondary antibody addition. For negative controls, the
primary antibody was omitted from the diluent. Sections were
counterstained with hematoxylin and viewed with a Zeiss Axiophot
photomicroscope (Oberkochen, Germany).
For double-immunohistochemical stainings with anti-CD11b and anti-CD11c mAbs, after an initial blocking with normal sera, sections were incubated with the two mAbs and then in sequence with goat biotinylated anti-hamster IgG (Vector Laboratories), ABC-alkaline phosphatase (Vector laboratories), and fuchsin (DAKO, Glostrup, Denmark) as chromogen (red color). After washing, sections were incubated with avidin-biotin (Vector Laboratories), rabbit anti-rat IgG, ABC-peroxidase, and DAB as chromogen (brown color).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Mice were anesthetized as above and perfused intracardiacally with
cold PBS. Total RNAs were extracted from the CNS, spleen, and small
intestine of control (n = 2), presymptomatic
(n = 2), EAE-affected (n
= 5), and remitting (n = 2) SJL mice using the
SV Total RNA Isolation System (Promega, Madison, WI), according to the
manufacturer’s instructions. One µg of purified RNA was
reverse-transcribed using the Reverse Transcription System (Promega),
with 15 U of avian myeloblastosis virus reverse transcriptase. For
detection of murine MIP-3 transcripts,31
one-tenth of
this reaction was added to 40 µl of PCR mix and amplified for 36
cycles (30 seconds at 94°C, 30 seconds at 60°C, 45 seconds at
72°C), with 2.5 U of Taq polymerase (Promega), using the
following primers: 5'-TACAGACGCCTCTTCCTTCC-3' (sense) and
5'-TCTTGACTCTTAGGCTGAGG-3' (antisense) (size of the amplified product
of MIP-3 cDNA was 174 bp). For detection of murine CCR6
transcripts,31
equal amounts of cDNA were subjected to PCR
amplification for 36 cycles (15 seconds at 94°C, 15 seconds at
59°C, 45 seconds at 72°C), using a forward primer specific to the
untranslated 5' region (5'-CTGCAGTTCGAAGTCATC-3') and a reverse primer
specific to the CCR6 open reading frame (5'-GTCATCACCACCATAATGTTG-3')
(sizes of the amplified products of CCR6 cDNA were 330 and 420 bp). As
control, transcripts of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase were also amplified for 25
cycles (30 seconds at 94°C, 30 seconds at 60°C, 45 seconds at
72°C), using the following primers: sense 5'-CCATGGAGAAGGCCGGGG-3',
antisense 5'-CAAAGTTGTCATGGATGACC-3' (size of the amplified product of
glyceraldehyde-3-phosphate dehydrogenase cDNA was 194 bp). Negative
controls lacking template RNA or RT were included in each experiment.
The PCR products were fractionated on a 2% agarose gel and visualized
by ethidium bromide staining. The data shown were obtained in
individual, representative animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal Mouse CNS: Localization of DCs in the Choroid Plexuses and Meninges
In agreement with previous studies on DC localization in the
normal rat and human CNS,19-22
the murine DC markers
DEC-205 and CD11c were found to be expressed by process-bearing cells
in the choroid plexus (fourth ventricle) and in the meninges of
untreated SJL mice (Figure 1, A–C)
, but
not in the spinal cord, brain stem, or cerebellar parenchyma. Meningeal
and choroid plexus DCs had the phenotype of immature, tissue resident
DCs as they did not bind the mAb MIDC-8, reacting with an intracellular
granule antigen of mature DCs28,29
(not shown). In the CNS
of control mice, CD11b, a marker for
macrophages/microglia32
and myeloid DCs,33
stained round cells, most likely macrophages, in the meninges and
choroid plexuses and only few intraparenchymal microglia (not shown).
No CD4+, CD8+, or
B220+ cells were detected (not shown).
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During preclinical EAE (day 10 after immunization),
DEC-205+ and CD11c+ cells
with thin cytoplasmic processes were detected not only in the meninges
and choroid plexus but also in discrete areas of the spinal cord white
matter, predominantly beneath the pial surface (Figure 1, D–F)
. In
most sections, the distribution of DEC-205+ and
CD11c+ cells gave the clear impression that these
were in the process of migrating from the meninges into the spinal
cord. At this stage, no MIDC-8+ mature DCs were
detected in the CNS (not shown). Double stainings with anti-CD11b and
anti-CD11c mAbs revealed no overlapping of the two immunoreactivities,
excluding that CD11c expression was induced on microglia (Figure 1G)
.
Using adjacent spinal cord sections, we found that process-bearing
cells expressing MHC class II molecules were present in the meninges
and in the same areas as DEC-205+ and
CD11c+ cells (Figure 1H)
. At 10 day after
immunization, no CD4+,
CD8+, or B cells infiltrated the CNS. Moreover,
neither CD11b+ macrophages nor activated
microglia were detected within the CNS parenchyma (not shown),
rendering unlikely that these cell types contributed to early
intracerebral expression of MHC class II molecules. No
DEC-205+ or CD11c+ cells
were present in the spinal cord of PBS/CFA-injected mice.
Acute EAE: Presence of Mature DCs in the CNS Inflammatory Cell Infiltrates
In the CNS of mice with acute EAE (grade 3 to 4), immune
infiltrates were present in perivascular, meningeal, and submeningeal
locations. CD11b+ macrophages and
CD4+ T cells constituted the predominant
infiltrating leukocytes and penetrated extensively into the CNS,
particularly in the spinal cord (Figure 2, A and B)
. Very rare, if any,
CD8+ T cells and B220+ B
cells were detected in acute EAE lesions (not shown).
DEC-205+ cells with characteristic dendritic
shape localized within the CNS infiltrates and only few were scattered
in the spinal cord parenchyma (Figure 2, C–E)
. CD11c did not seem to
be a useful marker for DCs in EAE lesions as it was expressed on most
of the infiltrating cells, likely macrophages (not shown). Notably,
scattered cells within the infiltrates were labeled intracellularly by
MIDC-8 mAb (Figure 2, F and G)
, suggesting that DCs rapidly acquire a
mature phenotype in the inflamed CNS.
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). In
experiments in which the staining protocol did not include the
amplification step with biotinylated tyramine,30
CD86 mAb
selectively stained few infiltrating process-bearing cells, most likely
DCs (Figure 2I)
. This finding is consistent with the fact that mature
DCs express CD86 at higher levels than other blood-derived or tissue
APCs.2 Very rare DEC-205+ or MIDC-8+ cells were detected within the residual inflammatory infiltrates persisting in the perivascular and meningeal locations during the early remission phase of EAE and none were present at late remission stages (not shown).
Chronic EAE: Persistence of DCs in the Inflammatory Cell Infiltrates
Immunohistochemical analysis of the CNS of mice developing chronic
EAE (grade 2 to 3) was performed at 
2 weeks after the onset of
clinical disease. Compared with acutely affected mice, mice developing
chronic EAE had smaller and less invasive immune infiltrates in the
spinal cord, brain stem, and cerebellum, whereas microglia activation
was still evident, particularly in the spinal cord (Figure 3A)
. The meningeal and perivascular
infiltrates comprised CD4+ T cells as well as
CD8+ T cells and B cells (Figure 3, B–D)
. Within
the infiltrates, many DEC-205+ and
MIDC-8+ cells were detected (Figure 3, E and F)
.
Intriguingly, anti-DEC-205 (Figure 3G)
, but not MIDC-8 (not shown), mAb
also stained numerous process-bearing cells in discrete areas of the
spinal cord white matter.
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). Relapsing EAE: Presence of Mature DCs within the Spinal Cord Parenchyma
For this set of experiments, we analyzed mice with an EAE relapse
that had followed a complete remission. In relapsing EAE (grade 2 to
3), numerous CD4+ T cells were present in the CNS
meninges, perivascular spaces and parenchyma (Figure 4A)
, whereas few
CD11b+ macrophages infiltrated the CNS (not
shown). CD8+ T cells (Figure 4B)
and B cells
(Figure 4C)
also accumulated within the CNS infiltrates.
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. A striking
feature of relapsing EAE was the diffuse infiltration of
DEC-205+ and MIDC-8+ DCs in
the spinal cord and caudal part of the medulla oblongata (Figure 4
; D,
E, G, and H). Intraparenchymal DCs were particularly enriched in areas
of T cell infiltration (compare adjacent sections in panels A and D of
Figure 4
). Moreover, numerous DEC-205+ and
MIDC-8+ cells were disseminated in vast areas of
the spinal cord white matter (Figure 4, G and H)
.
A prominent and diffuse activation of CD11b+
microglia was evident in the CNS of mice undergoing EAE relapses
(Figure 4F)
. In the spinal cord, both the localization and morphology
of CD11b+-activated microglia clearly differed
from those of intraparenchymal DEC-205+ cells
ruling out the possibility that microglia had acquired positivity for
DC markers (compare adjacent sections in panels F and G of Figure 4
).
MHC class II, CD40, and CD86 molecules were expressed on cells of the
inflammatory infiltrates (not shown), some activated microglia, and
intraparenchymal cells with a DC-like morphology (Figure 4, I–M)
.
Up-Regulation of the ß-Chemokine MIP-3 and its Receptor CCR6
in the CNS of EAE-Affected Animals
We then asked whether the CNS of EAE-affected mice would express
chemokines involved in the recruitment of DCs at inflammatory
sites.3
We focused our attention on the ß-chemokine
MIP-3 (also termed liver and activation-regulated chemokine and
Exodus-1), which is expressed constitutively in certain murine tissues
and is up-regulated in inflammation.31,34
CCR6, the
MIP-3-specific receptor, is expressed on murine DCs, T and B
cells,31
and on human immature DCs and memory T
cells.35,36
Analysis of MIP-3 and CCR6 transcripts in
the CNS of control and EAE-affected mice was performed by RT-PCR. Small
intestine and spleen tissues from normal mice served as positive
controls for MIP-3 and CCR6, respectively.31,34
Because
of alternative splicing,31
two amplified CCR6-specific
products were obtained. MIP-3 mRNA was not detectable in the CNS of
control mice (either untreated or injected with PBS/CFA) (Figure 5)
. A very weak band
for CCR6 mRNA was present in the cerebrum only. Both MIP-3 and CCR6
mRNAs became detectable during preclinical EAE (day 13 after
immunization) and were further up-regulated in the acute disease phase
in all CNS areas examined (Figure 5)
. The induction of MIP-3- and
CCR6-specific transcripts in the cerebrum, an area which fails to
develop typical perivascular and intraparenchymal EAE lesions, can be
explained by the presence of abundant inflammatory infiltrates within
the cerebral meninges and ventricular choroid plexuses (our unpublished
observations). During EAE relapses, CCR6 mRNA was up-regulated in the
cerebrum, cerebellum, and spinal cord, whereas expression of MIP-3
mRNA was particularly induced in the spinal cord. During the early
remission phase, transcripts for MIP-3 and CCR6 became again
undetectable, or poorly detectable in the case of cerebral CCR6
transcripts (Figure 5)
.
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in the EAE-affected CNS,
cryostat spinal cord sections were stained with a polyclonal
anti-mouse/rat MIP-3 antibody. No MIP-3 immunoreactivity was
detected in the spinal cord of control and presymptomatic SJL mice (not
shown). During acute EAE, most MIP-3 immunoreactivity co-localized
with the inflammatory cell infiltrates (Figure 6A)
and only few
MIP-3+ cells were scattered in the surrounding
CNS parenchyma. No MIP-3 immunoreactivity was detected in the CNS of
mice undergoing disease remission (not shown). Notably, after EAE
relapses (Figure 6, B and C)
and to a lesser extent in chronic EAE (not
shown), anti-MIP-3 antibody also stained numerous cells regularly
distributed in the spinal cord white matter. Based on their
morphological appearance (Figure 6, B and C)
and localization of the
astrocytic marker glial fibrillary acidic protein in adjacent sections
(not shown), most of the MIP-3+ cells were
identified as astrocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a chemokine implicated in DC and lymphocyte migration.
Based on the present findings, we propose that intracerebral DC
recruitment and maturation are key events in the local activation of
encephalitogenic CD4+ T cells and in the
maintenance of cytotoxic and antibody-mediated autoimmune responses
leading to EAE progression and CNS damage. At a stage immediately preceding EAE clinical signs and appearance of CNS immune infiltrates, cells exhibiting a typical DC morphology and expressing the DC markers DEC-205 and CD11c are found in subpial areas of the spinal cord white matter where the first intraparenchymal MHC class II+ cells are also detected. The present observations are in agreement with studies in other autoimmune pathologies showing that DC infiltration is one of the first abnormalities in the target organ.5 We propose that the early appearance of DCs in the CNS provides an initial pool of intracerebral APCs which efficiently capture CNS antigens and present them to peripherally primed encephalitogenic CD4+ T cells. Matsumoto and colleagues37 first described the presence of Ia+ cells with dendritic morphology in the spinal cord of rats at EAE preclinical stage (day 11 after immunization) but identified them as microglia. Using mice with myelin oligodendrocyte glycoprotein peptide 35-55-induced EAE, Suter and colleagues23 have recently shown that CD11c+ DCs can be detected, together with CD4+ T cells and macrophages, within the spinal cord inflammatory infiltrates shortly before disease onset. These authors have also presented evidence that CD11b+ macrophages are the first hematogenous cell type to be recruited to the CNS. Discrepancies between the study of Suter and colleagues23 and our findings could be due, at least in part, to the use of different mouse EAE models.
During acute EAE, DCs are predominantly distributed within the CNS immune infiltrates and express the mature DC markers MIDC-8 and CD86. We favor the hypothesis that DCs accumulating and maturing in the inflamed CNS play a pivotal role in Th1 effector activation, whereas the more numerous infiltrating macrophages and activated microglia contribute to the amplification and regulation of the immune response. Accordingly, several studies have shown that in the Th1 cytokine-rich microenvironment of EAE lesions the latter cell types are induced to express APC molecules and to secrete pro-inflammatory (interleukin-12, tumor necrosis factor) as well as anti-inflammatory (interleukin-10, transforming growth factor-ß, prostaglandin E2) mediators.11,12,38-40
The persistence of mature DCs in the CNS of mice with chronic and relapsing EAE suggests that DCs are also involved in disease progression. Most intriguingly, we have demonstrated enrichment of mature DCs in the spinal cord parenchyma of mice undergoing EAE relapses. EAE progression is associated with CNS infiltration by CD4+, CD8+, T cells, and B cells, suggesting that DCs may provide the signals necessary for sustaining local activation of these immune cell types. Consistent with this, CNS-infiltrating cells with a DC-like morphology express MHC class II, CD40, and CD86 molecules which are essential for optimal APC function.2 Interaction of CD86 with CD28 on T cells is of critical importance for T cell activation,41 whereas CD40 ligation by CD154 (CD40 ligand) on activated T cells induces DC maturation, cytokine secretion and expression of co-stimulatory molecules.2,42 Encephalitogenic T cells accumulating in the CNS of EAE-affected mice express CD154,43 suggesting that CD40-CD154 interactions are determinants for intracerebral DC maturation. This may be further promoted by cytokines, like granulocyte-macrophage colony-stimulating factor, tumor necrosis factor, and interleukin-1, produced intracerebrally during EAE.44-46 The establishment of DC T and B cell interactions within the CNS parenchyma could sustain the intrathecal B-cell clonal expansion typically associated with multiple sclerosis47 and the production of anti-myelin antibodies detected in EAE and multiple sclerosis lesions.17 Mature intracerebral DCs are also likely to secrete chemokines contributing to the recruitment of additional DCs, CD4+ and CD8+ T cells to the inflamed CNS.48,49
DCs were recently proposed to have a key role in the formation of ectopic lymphoid tissue occurring in target organs in a number of chronic inflammatory diseases.6,7 The presence of organized lymphoid structures in the CNS of mice with adoptively transferred chronic-relapsing EAE50 and in old multiple sclerosis plaques51 suggests that DC might also orchestrate lymphoid neogenesis in the CNS and this in turn may be a key event in the maintenance of an autoimmune response against neural antigens. In addition to a role in sustaining immune responses within the CNS, intracerebral DCs could capture antigen generated during CNS damage and traffic to secondary lymphoid organs to stimulate new autoreactive T cells. This possibility is supported by a recent study showing that intracerebrally injected DCs home to cervical lymph nodes.48
The present findings raise two important questions: where do intracerebral DCs come from, and how are they recruited to the CNS? During preclinical EAE, the subpial localization of DCs within the spinal cord white matter suggests that they could derive from DCs or DC precursors normally residing in the meninges.21,22 The observation that DCs do not enter the parenchyma of the brain stem and cerebellum at any stage of EAE indicates a unique permissiveness of the spinal cord white matter to DC migration and/or differentiation. Appearance of intracerebral DCs early in the disease process occurs before detectable T cell infiltration into the CNS, suggesting that pro-inflammatory stimuli related to the immune response cause subtle alterations in the vascular endothelia or CNS tissue (eg, induction of adhesion molecules and/or chemokines), leading to selective DC recruitment. Because of the lack of a blood-brain barrier, the meninges are among the CNS compartments more readily affected by peripheral immune stimuli. It should be noted that peripheral immune activation in PLP 139-151 peptide-treated SJL mice is already very prominent at day 10 after immunization.52 Although early DC mobilization is probably not restricted to the CNS, the CNS parenchyma represents a unique site to detect such event, as it normally does not contain a resident DC population.
After EAE development, the presence of DCs within the CNS inflammatory cell infiltrates is consistent with rapid recruitment of DCs or DC precursors from the blood circulation. Conversely, the sparse, intraparenchymal DC localization observed in the spinal cord of mice with chronic and relapsing EAE suggests that DC progenitors residing in the CNS parenchyma may be induced to differentiate into DCs. Monocytes/macrophages share a common myeloid precursor with DCs53 and myeloid precursors continuously enter the CNS to differentiate into perivascular macrophages and microglia.54,55 Chronic neuroinflammation and widespread glial activation could contribute to the establishment of a cytokine (granulocyte-macrophage colony-stimulating factor, interleukin-1, tumor necrosis factor)-rich milieu promoting intracerebral DC development and maturation.53 This hypothesis is currently under investigation.
Chemokines are small proteins that regulate leukocyte migration to
lymphoid organs and nonlymphoid tissues. They are produced by activated
leukocytes themselves as well as by endothelial and parenchymal cells
during inflammation.56
Depending on their maturation
stage, DCs respond to specific chemokines and express the corresponding
subsets of chemokine receptors.35,57,58
MIP-3 has been
proposed to mediate constitutive DC trafficking to certain peripheral
tissues and to be involved in the rapid recruitment of DCs during
inflammation.31,34,35
We have shown that transcripts specific for MIP-3 and its receptor
CCR6 are up-regulated in the CNS during EAE development and that the
spatial distribution of MIP-3 immunoreactivity matches that of DCs,
suggesting that this chemokine may have a role in intracerebral DC
recruitment. Although the localization of MIP-3 in acute EAE lesions
indicates that the first chemotactic signals are provided by
blood-derived cells, the presence of numerous intraparenchymal
MIP-3+ cells, predominantly astrocytes, in the
CNS of mice with chronic and relapsing EAE supports the idea that the
injured CNS becomes the major source of MIP-3. Because CCR6
receptors are expressed on DCs as well as on T and B
lymphocytes,31,35,36
it is likely that intracerebral
MIP-3 production is important for coordinating interactions among
these cell types, resulting in sustained immune activation. In addition
to MIP-3, several other chemokines (eg, MIP-1, MIP-1ß, RANTES,
and MCP-1) are chemotactic for immature murine DCs in
vitro59
and are produced in the CNS during acute and
chronic EAE.60,61
Further studies need to be performed to
understand whether and which chemokine(s) are critically involved in DC
recruitment to the inflamed CNS. Given the presence of numerous DCs
with a mature phenotype in EAE lesions, it will be interesting to
verify whether chemokines acting on mature DCs, such as
MIP-3ß35
and SLC,62
are also produced in
the CNS of EAE-affected mice and contribute to intracerebral DC homing.
In conclusion, our study strongly suggests that DCs recruited to and maturing within the CNS parenchyma may have a pivotal role both in the onset and progression of EAE. Manipulation of DC recruitment and function within the CNS may represent a new strategy to treat experimental and human neuroinflammatory diseases.
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Acknowledgements |
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Footnotes |
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Supported by Research Project on Multiple Sclerosis and Project "Inflammatory, Oxidative and Autoimmune Mechanisms in CNS Diseases" of the Istituto Superiore di Sanità/Italian Ministry of Health.
Accepted for publication August 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. FEBS Lett 1998, 440:188-194[Medline]
. J Immunol 1999, 162:186-194This article has been cited by other articles:
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  C. Ling, Y. I. Verbny, M. I. Banks, M. Sandor, and Z. Fabry In Situ Activation of Antigen-Specific CD8+ T Cells in the Presence of Antigen in Organotypic Brain Slices J. Immunol., June 15, 2008; 180(12): 8393 - 8399. [Abstract] [Full Text] [PDF]
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R. Penitente, C. Nicolo, P. Van den Elzen, G. Di Sante, C. Agrati, F. Aloisi, E. E. Sercarz, and F. Ria Administration of PLP139-151 Primes T Cells Distinct from Those Spontaneously Responsive In Vitro to This Antigen J. Immunol., May 15, 2008; 180(10): 6611 - 6622. [Abstract] [Full Text] [PDF]
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A.-C. Brehin, J. Mouries, M.-P. Frenkiel, G. Dadaglio, P. Despres, M. Lafon, and T. Couderc Dynamics of Immune Cell Recruitment during West Nile Encephalitis and Identification of a New CD19+B220-BST-2+ Leukocyte Population J. Immunol., May 15, 2008; 180(10): 6760 - 6767. [Abstract] [Full Text] [PDF]
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 I. Ifergan, H. Kebir, M. Bernard, K. Wosik, A. Dodelet-Devillers, R. Cayrol, N. Arbour, and A. Prat The blood-brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells Brain, March 1, 2008; 131(3): 785 - 799. [Abstract] [Full Text] [PDF]
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 D. D. C. Ireland, S. A. Stohlman, D. R. Hinton, R. Atkinson, and C. C. Bergmann Type I Interferons Are Essential in Controlling Neurotropic Coronavirus Infection Irrespective of Functional CD8 T Cells J. Virol., January 1, 2008; 82(1): 300 - 310. [Abstract] [Full Text] [PDF]
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 K. Hirota, H. Yoshitomi, M. Hashimoto, S. Maeda, S. Teradaira, N. Sugimoto, T. Yamaguchi, T. Nomura, H. Ito, T. Nakamura, et al. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model J. Exp. Med., November 26, 2007; 204(12): 2803 - 2812. [Abstract] [Full Text] [PDF]
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Y. Li, N. Chu, A. Hu, B. Gran, A. Rostami, and G.-X. Zhang Increased IL-23p19 expression in multiple sclerosis lesions and its induction in microglia Brain, February 1, 2007; 130(2): 490 - 501. [Abstract] [Full Text] [PDF]
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A. L. Zozulya, E. Reinke, D. C. Baiu, J. Karman, M. Sandor, and Z. Fabry Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1{alpha} Chemokine and Matrix Metalloproteinases J. Immunol., January 1, 2007; 178(1): 520 - 529. [Abstract] [Full Text] [PDF]
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J. Karman, H. H. Chu, D. O. Co, C. M. Seroogy, M. Sandor, and Z. Fabry Dendritic Cells Amplify T Cell-Mediated Immune Responses in the Central Nervous System J. Immunol., December 1, 2006; 177(11): 7750 - 7760. [Abstract] [Full Text] [PDF]
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