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Institute for Medical Microbiology and Virology, Heinrich Heine University, Duesseldorf, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, GM-CSF, and
NO. In comparison, the DC were more potent stimulators of naive or
allogeneic T cell proliferation. Both DC and CD11c-
microglia/macrophages from inflamed brain primed naive T cells from
DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-
and
IL-2. Resting microglia that had been purified from normal adult brain
generated immature DC upon exposure to GM-CSF, while CD40 ligation
triggered terminal maturation. Consistently, a functional maturation of
brain DC was observed to occur following the onset of encephalitis. In
conclusion, these findings indicate that in addition to inflammatory
macrophage-like brain cells, intraparenchymatical DC exist in
autoimmune and infectious encephalitis. These DC functionally mature
upon disease onset and can differentiate from resident microglia. Their
emergence, maturation, and prolonged activity within the brain might
contribute to the chronicity of intracerebral Th1
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(4, 5, 6, 7). Despite such limitations imposed on the immune
system, vigorous inflammatory responses can rapidly develop within the
brain, resulting in the recruitment of naive bystander T cells
(8). Experimental autoimmune encephalomyelitis (EAE)3 is a murine model of autoimmune disease directed against CNS Ags. Disease is initiated in animals of susceptible strains by sensitization or the adoptive transfer of neural Ag, e.g., myelin basic protein-specific CD4+ T cells leading to multiple inflammatory, often demyelinating, lesions in spinal cord and brain (reviewed in Ref. 9). A prototypic model of infectious CNS inflammation is murine toxoplasmic encephalitis (TE), which is induced by experimental infection with the intracellular parasite Toxoplasma gondii. While TE is transient in mice that are genetically resistant to the pathogen (e.g., BALB/c), susceptible mouse strains (e.g., C57BL/6) develop progressive lethal encephalitis during chronic infection (reviewed in Ref. 10). Common to EAE and TE is a type 1 CD4+ T cell-mediated immunopathology, although it is still unclear how the intracerebral T cell response is polarized. In EAE, brain microglia that function as resident APC are thought to skew the cellular response, as they have been shown to preferentially activate Th1 cells (11). Besides microglia, other potential APC are brain macrophages and astrocytes (reviewed in Refs. 12 and 13) as, of course, are the dendritic cells (DC), which have been recently identified in the brains of BALB/c mice during chronic latent TE (14). These brain DC seem to be related phenotypically to macrophages/microglia.
Since myeloid DC progenitors are present in the newborn mouse brain (15) and mature upon challenge by T. gondii of glial cells in vitro (14), the emergence of brain DC in TE raises the question of whether these DC appear and expand as a direct consequence of the presence of the pathogen or as a result of the local inflammatory reaction. In the present study we address the hypothesis that CNS inflammation in general is associated with an intracerebral presence of functional DC. EAE as a noninfectious inflammatory CNS disease and progressive TE as a severe infectious encephalitis were examined for the presence and location of brain DC. Isolated DC were compared with coisolated microglia/brain macrophages with respect to their morphology, T cell stimulatory activity, and cytokine secretion profiles. Furthermore, we delineate a Toxoplasma-independent differentiation pathway that leads from resting microglia to functionally mature DC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 mice were bred at the Tierversuchsanlage,
University of Duesseldorf, from breeding stock supplied by Biological
Research Laboratories (Fuellinsdorf, Switzerland). SJL/NBom mice were
obtained from M&B (Ry, Denmark). Mice transgenic for TCR that recognize
OVA323–339 peptide in the context of
I-Ad (DO11.10 TCR/ transgenic mice) on a
BALB/c background (16) were provided by Dr. M. Kopf (Basel
Institute of Immunology, Basel, Switzerland). EAE was elicited in
8-wk-old female SJL mice according to the method of Miller and Karpus
(17) by s.c. immunization with 180 µg of myelin basic
protein 89–101 peptide (VHFFKNIVTPRTP; synthesized and HPLC purified
by the Center for Biological and Medical Research, University of
Duesseldorf) in CFA containing 200 µg of Mycobacterium
tuberculosis H37Ra. In addition, mice were injected i.p. with 400
ng of pertussis toxin (Alexis, Lausen, Switzerland) on days 0 and 2.
About 70% of the animals showed symptoms that corresponded to grades
0.5–2 (beginning tail paresis and additional hind limb paresis,
respectively) in EAE scoring (17). Mice were analyzed at
the peak of disease. Control animals treated with CFA and pertussis
toxin were simultaneously analyzed. Female BALB/c or C57BL/6 mice were
infected at 8–12 wk of age with the DX strain of T. gondii
by i.p. injection with three cysts prepared as previously described
(14). Infection was serologically verified 2–3 wk later.
Unless otherwise stated, T. gondii-infected mice were
analyzed between days 28 and 42 postinfection when TE was
histologically apparent.
Bromodeoxyuridine (BrdU) labeling
For pulse-labeling of proliferating cells in vivo, mice were injected i.p. with 1.2 mg of BrdU (Roche, Mannheim, Germany) 12 h before preparation of brain cells.
Immunohistochemistry
Mice were transcardially perfused with ice-cold PBS. The brains
were excised, mounted in OCT compound (Sakura, Zoeterwoude, The
Netherlands), snap-frozen in 2-methylbutane, and stored at -70°C.
Frozen sections, 5 µm thick, were stained with anti-CD11c mAb
N418 (Endogen, Cambridge, MA), anti-TCR mAb H57-597 (PharMingen,
San Diego, CA), or hamster isotype control mAb G235-2356 (PharMingen)
as detailed previously (14). Ab binding was detected using
biotinylated goat anti-hamster IgG (The Jackson Laboratory, Bar
Harbor, ME) and peroxidase-conjugated avidin-biotin complex (Vector
Elite ABC kit, Vector, Burlingame, CA). Sections were developed using
the 3,3'-diaminobenzidine substrate kit (Vector) and were
counterstained with hematoxylin.
Preparation of microglia and DC populations
Brains were removed from perfused mice, minced, passed through an 18-gauge needle, and digested by incubation with 250 µg/ml collagenase/dispase and 250 µg/ml DNase I (Roche) at 37°C for 45 min each. The resulting cell suspensions were fractionated on 30/60% Percoll gradients at 1000 x g for 25 min (18), and brain mononuclear cells (BMNC) were collected from the interface. Due to the low number of BMNC obtained, cells from 3–12 mice/experiment were pooled. For isolation of DC or microglial cells, BMNC were depleted of T cells by treatment with anti-Thy1.2 mAb 30-H12 (PharMingen) and complement (low tox rabbit complement; Cedarlane, Hornby, Canada). Then, CD11c+ cells were magnetically sorted by positive selection using N418-coated beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instruction, resulting in a >90% pure population as controlled by flow cytometry. Spleen DC were similarly isolated from normal splenocytes. DC-free microglia/brain macrophages were prepared from the residual CD11c- BMNC by positive selection using M1/70.15-coated magnetic beads (Miltenyi). In general, the resultant population contained >96% CD11b+ and <0.5% CD11c+ cells. For assays, all MACS-enriched cells were resuspended in phenol red-free DMEM with 10% FCS (containing <10 pg/ml LPS as determined by Limulus amebocyte lysate assay), 2 mM L-glutamine, and 50 µM 2-ME.
In vitro differentiation and activation of brain DC
Microglia prepared from normal adult BALB/c mice were plated at 2 x 105 cells/ml into low adherence six-well plates (Costar, Cambridge, MA) with medium containing 50 ng/ml murine GM-CSF (provided by Dr. F. Seiler, Behringwerke, Marburg, Germany) and cultivated for 4–6 days. Microglia were also harvested from GM-CSF-supplemented primary culture of newborn mouse brain (19). CD11c+ cells were then immunomagnetically isolated, washed, and recultured for a further 24 h with GM-CSF. During that time, 10 µg/ml soluble CD40L and cross-linking Ab (Alexis) or 10 µg/ml LPS (Escherichia coli O55:B5; Difco, Detroit, MI) were added to some cultures to trigger DC terminal maturation.
Flow cytometry
Freshly prepared BMNC or cells isolated from brain cell culture
were stained using the following Abs: biotinylated anti-CD11c mAb
N418 (Endogen) or HL3 (PharMingen), FITC-labeled anti-F4/80 mAb
CI:A3-1 and FITC-labeled anti-DEC-205 mAb NLDC-145 (Dianova,
Hamburg, Germany), and PE- or FITC-labeled anti-CD11b mAb M1/70.15,
FITC-labeled anti-CD8 mAb 53-6.7, and PE- or FITC-labeled
anti-CD45 mAb 30-F11 (PharMingen). Species- and isotype-matched
control Ab and FITC- or PE-conjugated streptavidin as secondary reagent
were obtained from PharMingen. To block unspecific binding of
primary Ab, cells were preincubated with anti-FcII/IIIR mAb
2.4G2 (PharMingen). Double staining of surface markers was performed
according to standard procedures (18). Propidium iodide
was added in the final wash to label dead cells, which were excluded
from the analyses. BrdU incorporated into cellular DNA was
detected by using the BrdU flow kit (PharMingen). After surface
staining, cells were fixed and permeabilized, and DNA was denatured as
detailed in the kit protocol. Cells were then incubated with
FITC-labeled anti-BrdU mAb 3D4 (PharMingen). All samples were
immediately measured on a FACSCalibur flow cytometer,
104 events were acquired, and data were analyzed
with CellQuest software (Becton Dickinson, Heidelberg, Germany).
Phase contrast and electron microscopy
Freshly isolated CD11c+ and CD11c- microglia/brain macrophages were incubated in culture medium for 6 h at 37°C to allow reconstitution of cell morphology. Light microscopy was performed on a Zeiss photomicroscope III (Zeiss, Jena, Germany). For standard electron microscopy, cells were fixed in Karnovsky’s solution, postfixed in 2% osmium tetroxide, dehydrated in ethanol, and embedded in Durcupan ACM (Fluka, Neu-Ulm, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a Zeiss EM 109 transmission electron microscope.
Mixed leukocyte reaction
CD11c+ splenocytes and BMNC, CD11c-/CD11b+ BMNC, or in vitro differentiated brain DC were tested as stimulators of allogeneic T cell responses by MLR. T cells were prepared from spleens of C57BL/6 mice by passing splenocytes through nylon wool and an Ab-coated T cell enrichment (R&D Systems, Minneapolis, MN) column. In A/2 microtiter wells containing 100 µl of IMDM with 5% FCS, glutamine, and antibiotics, 1.5 x 105 purified T cells (>99% CD3+) were mixed with titrated numbers of gamma-irradiated APC of BALB/c origin. On day 3 test cultures were fed by adding 50 µl of medium. T cell proliferation was measured via [3H]TdR incorporation during the last 18 h of a 5-day incubation.
Priming and activation of TCR transgenic T cells
To analyze T cell priming by brain APC, naive T cells purified
from DO11.10 mice were exposed in primary stimulation culture to sorted
CD11c+ or
CD11c-/CD11b+ BMNC from
chronically T. gondii-infected BALB/c mice.
CD11c+ normal splenocytes that had been
immunomagnetically depleted of CD45R+ cells by
negative selection using B220-coated microbeads (Miltenyi) served as
reference APC. Naive T cells were purified from the spleens of 8-wk-old
DO11.10 mice via nylon wool and a T cell enrichment column and
subsequent sorting for CD62Lhigh cells using
MEL14-coated magnetic beads (Miltenyi). In a total volume of 1 ml
(48-well plate), 5 x 105 DO11.10 T cells
were coincubated with 5 x 104 irradiated
APC as described above, but in medium containing 0.3 or 1 µM
OVA323–339 peptide (ISQAVHAAHAEINEAGR;
synthesized and HPLC purified by the Center for Biological and Medical
Research, Duesseldorf, Germany). On day 3 cultures were fed with 1
ng/ml IL-2. Subsequently, proliferating T cells were allowed to expand
by transfer to 24-well plates. On day 6 T cell blasts were washed, and
2 x 105 cells/200 µl were plated using
medium without IL-2 in microtiter wells precoated with anti-CD3
mAb (1 µg/ml). Supernatants (SN) from restimulated T cells were
collected at 24 h and were stored at -70°C until tested for
cytokine content.
Cytokine assays and measurement of NO production
Freshly prepared brain cells were seeded into microtiter wells
at a density of 2 x 105/200 µl in DMEM
with 10% FCS. After 24 and 72 h of incubation, samples of the SN
were collected. In SN from 24-h culture of brain cells or T cells,
IL-1, IL-10, IL-12p40, IL-12p70, TNF-, GM-CSF, IFN-, IL-4, and
IL-2 were quantified using specific sandwich ELISAs (Endogen,
PharMingen, and R&D Systems) with recombinant mouse cytokines as
standard. Assays had a minimum sensitivity of 5 (IL-10, IL-12p40,
GM-CSF), 10 (IL-1, IL-2), 20 (IL-4, IL-12p70, TNF-), and 50
(IFN-) pg/ml, respectively. In 3-day SN from brain cell cultures,
the concentration of nitrite indicating cellular NO production was
determined photometrically with the Griess reagent and an
NaNO2 standard (20). The detection
limit of this assay was 2.5 nM
NO2-.
Statistics
The significance of differences was examined by Student t test, using Prism software (GraphPad, San Diego, CA), and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microgliosis is a sign of CNS inflammation (21). For
a quantitative approach mononuclear cells were prepared from perfused
normal and inflamed brain, and the percentage of
CD11b+ cells was determined by flow cytometry. As
calculated from the BMNC number in pooled cell isolates, on the
average, 3 x 105
CD11b+ mononuclear cells were recovered per brain
of normal adult mice (Table I
). In EAE
and TE as experimental models of brain inflammation, the total yield of
BMNC increased, which is partially due to T cells infiltrating the CNS
(data not shown). In parallel, the number of
CD11b+ cells increased, correlating with the
severity of disease (Table I). Although the origin of the expanding
cells remains unclear, evidence that a proliferation of
CD11b+ cells contributes to this increase was
obtained by pulse-labeling in vivo with the thymidine analogue BrdU;
while mononuclear cells from control brains showed no substantial DNA
synthesis, those from encephalitic brains contained approximately 10%
BrdU-labeled cells, which, by double staining, were identified as
microglia and T cells. About 3% of all CD11b+
BMNC had incorporated the BrdU label (Fig. 1A). In comparison, the
proportion of BrdU-positive cells among CD3+
brain cells was 6–7% (Fig. 1B). The detection of cycling
microglia in EAE and TE confirms a previous observation in
graft-vs-host disease (22). Collectively, these results
indicate a proliferative reaction in a subset of microglia/macrophages
from encephalitic brains.
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C).
Within the BrdU-positive gate approximately 60% of brain cells
expressed the pan-DC surface marker CD11c (Fig. 1D). These
data identify DC as a major population within the proliferating BMNC
population during the onset of CNS inflammation. In EAE and TE, CD11b+ BMNC that coexpress CD11c appear at inflammatory sites and exhibit a comparable DC phenotype
To test whether CNS inflammation is generally associated with
local expansion of DC, BMNC from autoimmune and severe infectious
encephalitis were analyzed for the presence of DC. In brain cells from
SJL mice during EAE as well as in those from C57BL/6 mice during
progressive lethal TE, DC were identified within the
CD11b+ cell pool (Fig. 2A). While only minute numbers
were detected among CD11b+ mononuclear cells from
normal brain, CD11b+ cells from encephalitic
brain contained significant numbers (up to 30%) of cells that proved
similar to myeloid-related DC. Double stainings showed that
CD11c+ brain cells in EAE lacked CD8 and
DEC-205, but coexpressed CD11b and F4/80 (Fig. 2B). By
phenotype, the CD11c+ BMNC in EAE thus correspond
to those in TE (14) and further resemble normal mouse bone
marrow-derived DC (23). In agreement with previous studies
(24, 25, 26), CD45 was expressed at a low level on mononuclear
cells from normal brain. By contrast, CD45 was markedly up-regulated on
encephalitic BMNC (Fig. 2C). In an attempt to define levels
of surface CD45 expression in CD11b+ brain cell
populations, BMNC from inflamed brain (progressive TE, wk 6
postinfection) were depleted of T cells and immunomagnetically sorted
for CD11c+ vs
CD11c-/CD11b+ cells. Fig. 2D shows that the purified brain DC expressed CD45 at high
and intermediate levels, while all CD11c-
microglia/brain macrophages exhibited a CD45high
phenotype.
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A–C). Brain DC were found at
perivascular and intraparenchymatic sites of inflammation. A comparable
pattern in tissue distribution of brain DC was observed in progressive
(Fig. 3D) and chronic latent TE (14). In
immunized SJL mice, CD11c+ brain cells were
detected only when EAE was apparent. Local expansion of DC possibly
starts with few cells dispersed in parenchymatic areas lacking
substantial leukocyte infiltrates (Fig. 3E). In TE, a
correlation between the number of brain DC and the severity of disease
was evident. By comparison, significantly more brain DC were isolated
in progressive than in chronic latent disease (Table I). In BMNC from
T. gondii-resistant BALB/c mice after recovery from TE and
in BMNC from immunized, but asymptomatic, or mock-immunized SJL mice,
few DC could be identified only by flow cytometry.
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Given the parallel existence of dendritic and macrophage-like
brain cells during CNS inflammation, both cell types were compared in
shape and function with regard to their putative roles in local
immunity. Freshly prepared CD11c+ and
CD11c-/CD11b+ mononuclear
cells from encephalitic brain were incubated in medium with 10% FCS
for 24 h to allow reconstitution of cell morphology. During this
time, most CD11c+ brain cells remained floating
and exhibited features of DC such as spinous, typically not branched,
processes. Electron microscopic examination revealed irregularly shaped
nuclei, numerous mitochondria, and few electron-dense granules and
phagocytic structures (Fig. 4, A and B). Thereby, CD11c+
brain cells closely resemble bone marrow-derived DC (23).
As a rule, the brain DC population contained some mitotic cells.
Coisolated CD11c-/CD11b+
BMNC became adherent, with extending pseudopodia and branched
cytoplasmic protrusions. Ultrastructural characteristics included
phagosomes, prominent vacuoles and mitochondria, and abundant rough
endoplasmatic reticulum (Fig. 4, C and D).
Comparative measurement of cytokine secretion ex vivo (Fig. 5) identified encephalitic brain DC to
release predominantly IL-12p70. CD11c-
inflammatory microglia/brain macrophages, by contrast, proved superior
in secretion of TNF- and GM-CSF and production of NO
(p < 0.005). IL-1 and IL-10 were released
by both cell populations; the higher levels at
CD11c- microglia/macrophages were not
significant (p > 0.05). These results
characterize brain DC as inflammatory cells specialized in IL-12
secretion, whereas
CD11c-/CD11b+ BMNC, by
producing TNF- and NO, resemble inflammatory macrophages.
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To assess CD11c+ brain DC and
CD11c-/CD11b+ BMNC for
their capacities to stimulate an alloreactive T cell response, cells
coisolated from encephalitic brain were used in MLR. Titrated numbers
of both APC types were cocultured, each with a fixed number of
allogeneic T cells. Fig. 6A
shows that DC derived from inflamed brain were potent stimulators,
already inducing T cell proliferation at a ratio of 1 cell per 100 T
cells. CD11c-/CD11b+
inflammatory BMNC proved about 4-fold less efficient. Similarly,
CD11c+ cells from normal adult BMNC were
significantly superior in T cell stimulation (p
< 0.05) compared with residual
CD11c-/CD11b+ brain cells
(Fig. 6B), thus indicating that potent stimulator cells were
enriched. The level of T cell response induced by brain DC was
comparable to that induced by spleen DC as reference stimulator cells
and is consistent with previous studies that suggest that brain
microglia comprise cells active in allo-MLR (26, 27).
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. T
cells primed with brain DC as well as those primed with
CD11c-/CD11b+ BMNC
secreted high levels of IFN- and IL-2, minimal amounts of IL-4, and
no IL-10 (Fig. 7). Taking into account
that brain DC are superior in stimulating naive T cell proliferation,
they can be considered the principal inducers of Th1 responses in
inflamed CNS tissue.
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Since microglia from adult mouse brain or brain cell primary
culture share functional and phenotypic characteristics with immature
DC (15, 26), we tested whether mature DC can differentiate
from microglia. Resting microglia were prepared from adult mouse brain
and depleted of CD11c+ cells, resulting in
96–99% F4/80+/CD45low
cells as controlled by flow cytometry (Fig. 8A). To stimulate
differentiation to DC, isolated microglia were maintained with GM-CSF
for 6 days. While a subpopulation of cells rapidly adhered by
exhibiting a ramified morphology, a further population of veiled or
dendriform cells remained in suspension, often forming clusters (Fig. 8, B and C). At the concentration used to promote
differentiation of brain DC, GM-CSF did not induce cellular
proliferation, as assessed by [3H]TdR
incorporation (not shown). After 5 days of incubation with GM-CSF,
approximately 30% of cells expressed CD11c (Fig. 8D)
compared with <0.5% of cells in the initial population. Testing by
MLR of sorted CD11c+ cells revealed their
allostimulatory capacity, although these microglia-born APC were
significantly less potent than reference splenic DC. Engagement of CD40
by overnight incubation with a CD40 agonist, however, resulted in full
competence to trigger maximum proliferation of alloreactive T cells
(Fig. 8E). Similar results were obtained when using
microglia derived from primary brain cell culture, although their basic
stimulatory capacity was minimal compared with that of ex vivo
microglia (Fig. 8F). Measurement of IL-12 secretion
confirmed the activation of CD11c+ microglia via
CD40 ligation (Fig. 8G). These findings demonstrate a
GM-CSF-dependent differentiation of microglia to DC and their
functional maturation in response to CD40 signaling.
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Based on our observation that resting microglia can acquire a DC
phenotype and develop into functionally mature DC, we tested whether
brain DC isolated during all stages of transient CNS inflammation vary
in their maturational stage. Since CD11c is expressed on both immature
and mature DC, sorting of CD11c+ cells as a final
step in the isolation procedure yields highly enriched DC regardless of
their individual maturational stage. The relative proportions of fully
matured DC among CD11c+ BMNC isolated at
different time points during transient TE were estimated by their
allostimulatory capacities in MLR. Compared with cells from normal
brains, DC prepared from the brains of BALB/c mice at the onset of TE
(day 14 postinfection) showed a significantly
(p < 0.005) elevated capacity to stimulate
alloreactive T cell proliferation (Fig. 9). Maximum levels in T cell stimulation
were measured on brain DC prepared during ongoing CNS inflammation (day
24 or 36 postinfection) or later when encephalitis in those mice
resistant to infection with T. gondii was resolved (day 77
postinfection). These results indicate that a maturation of brain DC
occurs in periods of apparent CNS inflammation and that functionally
mature DC continue to be present in the brain after termination of a
local inflammatory process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is produced and prime naive T cells entering the
inflamed brain. The present data demonstrate in autoimmune and
infectious encephalitis that CNS inflammation is associated with the
local emergence of DC that perform these functions. Concomitant to
microglia proliferation at disease onset, myeloid-related DC appear and
expand at inflammatory sites within CNS tissue and functionally mature
as disease progresses. Generation of DC from microglia, as proved ex
vivo, may represent a brain-intrinsic mechanism by which the organ
acquires full immunoreactivity upon inflammatory challenge and which
may favor the chronicity of cellular responses in the CNS. To date, DC have not been identified in the normal adult brain, whereas outside the blood-brain and meningeal barriers MHC class II+ dendriform cells have been localized in the dura mater, leptomeninges, and choroid plexus (29, 30). An intraparenchymatic presence of DC was indicated by the detection of cells exhibiting MHC class II or DC markers in delayed-type hypersensitivity and EAE lesions (31, 32, 33) and was evidenced on cell isolates in TE (14). Consistently, an intracerebral expression of the DC-specific MHC class II transactivator has recently been observed in mouse EAE (33). By immunohistochemistry in combination with ex vivo phenotyping and testing of brain cells for DC functions, we show here that activated myeloid DC accumulate at perivascular cuffs and parenchymatic foci in the brains of mice suffering from EAE or TE.
It is possible that blood-borne or meningeal DC are recruited by extravasation and via the perivascular space, respectively, to inflammatory sites within the CNS parenchyma. Alternatively, DC can differentiate from monocytes (34) that infiltrate the inflamed brain. Several lines of evidence support a third possibility, a GM-CSF-driven differentiation of brain DC from local, probably microglial, progenitors. It has been shown that the adult brain parenchyma harbors CD11b+ myeloid precursors (35), and immature DC develop in primary culture with GM-CSF (15, 19). GM-CSF, which promotes the development of DC from bone marrow progenitors (23), is produced by glial cells upon exposure to inflammatory or microbial stimuli (36, 37, 38). Consistently, in EAE and TE, expression of GM-CSF is induced early during the onset of disease (39, 40). Intracerebral expression of CSF-1, as a cofactor required by immature brain DC for preserving GM-CSF-dependent APC function (15), is likewise up-regulated early in EAE (41). Furthermore, GM-CSF-triggered development of myeloid DC is accompanied by cellular proliferation (23), which agrees with the findings that most cycling cells from encephalitic brain were CD11c+ and that mitotic cells were enriched in CD11c+, but not in CD11c-/CD11b+, brain cell isolates. Finally, the progressive functional maturation of brain DC after the onset of encephalitis is compatible with a local development of DC. In contrast, an already advanced maturational stage, as would be expected for immigrating DC by virtue of their motility (42), would conflict with the early proliferative activity observed. Accordingly, in EAE single CD11c+ parenchymatic cells were sometimes seen at a distance from T cell infiltrates, which renders a common influx of T cells and DC unlikely.
Evidence for a microglia differentiation pathway leading to brain DC is
provided by the generation of potent allostimulatory
CD11c+ cells from normal resting microglia ex
vivo. This development was independent of IFN-. The alteration of
function and surface phenotype was detected on microglia regardless of
whether they originated from normal adult brain or primary culture,
thus excluding the possibility that the enzymatic extraction of brain
cells might have influenced their functional potential. GM-CSF-induced
expression of the pan-DC marker CD11c was accompanied by the
acquisition of DC-like morphology and T cell-stimulatory activity;
therefore, the cells were designated DC. In recent studies the
GM-CSF-dependent differentiation step has been designated microglia
activation or functional maturation. Exposure of a microglia cell line
or adult microglia to GM-CSF induced up-regulation of CD80/B7.1 and MHC
class II surface expression and of Ag-directed Th1 stimulatory
capacity, respectively (25, 43). However, whether such
GM-CSF-treated microglial cells exhibited DC markers was not analyzed
in these studies. A model integrating the putative differentiation
pathway(s) of brain DC and their relationship to the
microglia/macrophage lineage is shown in Fig. 10. Upon trigger by GM-CSF, e.g., from
reactive astroglia, microglial cells can differentiate via two steps
into functional DC that finally die by apoptosis. Such a concept
obtains support from the even tissue distribution of MHC class
II+ dendritic-like cells in rat EAE in the
prefinal state (32) and the early emergence of
CD11c+ parenchymatic cells (Fig. 3E)
and allows the migration of brain DC from intraparenchymatic sites
also.
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myeloid-related phenotype which argues for a general process causing
the intracerebral presence of DC in CNS inflammation. Immunomagnetic
sorting of T cell-depleted CD11c+ and
CD11c-/CD11b+ mononuclear
cells from encephalitic brain allowed the separation of DC from other
microglia/brain macrophages for comparative analyses ex vivo.
Surprisingly, cells in both populations showed an intermediate to high
expression of CD45; the proportion of CD45high
cells correlated with the severity of disease and the total number of
BMNC isolated. Assuming that low level expression of CD45 stably
characterizes microglia, the fact that only
CD45high cells are present in the
CD11c-/CD11b+ BMNC isolate
from progressive TE might be due to the relative rarity of microglial
cells among the exogenous-derived inflammatory macrophages and the
preceding separation of CD11c+ cells, which,
indeed, contained considerable numbers of cells expressing CD45 at a
low to intermediate level. Another explanation would be an
up-regulation of CD45 on resident microglia under the conditions of
progressive lethal TE. No correlation between the level of CD45
expression and the expression of CD11c was found on encephalitic
BMNC.
With regard to cell morphology and ultrastructure, the brain DC
strikingly resembled bone marrow (23) or liver-derived DC
(44), whereas most inflammatory
CD11c- microglia/brain macrophages exhibited
features of other tissue macrophages. Distinctive immune functions were
the secretion of IL-12p70 and efficient induction of proliferative T
cell responses by brain DC, and the production of TNF-, GM-CSF, and
NO by CD11c-/CD11b+ BMNC,
respectively. Based on these profiles, non-DC
CD11b+ brain cells seem to be predestined as
amplifier and effector cells of CNS inflammation. Among them, microglia
and brain macrophages can alternate in TNF- production
(45). By producing IL-10, inflammatory microglia/brain
macrophages might provide a local counter-regulation of IL-12 effects
(46, 47, 48). While in TE, NO is crucial for parasite
inhibition (49), the role NO plays in CNS autoimmunity,
mediating tissue damage or antagonizing autoaggressive cellular
activity, is still debated (50).
On the other hand, brain DC functionally resemble (by their weak IL-10
production and strong Th1-inducing capacity) bone marrow-derived and
splenic DC, but not liver-derived or Peyer’s patch DC (51, 52), and thus must be considered initiators of Th1-mediated CNS
immunopathology. The Th1-promoting activity of brain DC and their
long-lasting presence even after resolution of a local inflammatory
process suggest a key role in the exacerbation or maintenance of
immune-mediated CNS diseases. As stimulators of naive T cells
infiltrating the inflamed brain, these DC might be crucially involved
in the phenomenon of epitope spreading during chronic T cell-mediated
autoaggression against CNS Ags. Whether microglia are primed in situ to
differentiation into the Th1-inducing DC type by IFN-, as has been
demonstrated on human monocyte-derived DC (53), or by
another, possibly tissue-specific, mechanism remains an issue relevant
for future brain DC-targeted immune intervention.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hans-Georg Fischer, Institute for Medical Microbiology and Virology, Universitaetsstrasse 1, D-40225 Duesseldorf, Germany.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; BMNC, brain mononuclear cells; BrdU, 5-bromo-2'-deoxyuridine; DC, dendritic cell(s); SN, supernatant; TE, toxoplasmic encephalitis; CD40L, CD40 ligand.
Received for publication August 22, 2000. Accepted for publication November 27, 2000.
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