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*
Institute for Medical Microbiology and Virology, Heinrich-Heine University, Duesseldorf, Germany; and
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. These brain DC were mature, as indicated by
high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They
triggered Ag-specific and primary allogeneic T cell responses at very
low APC/T cell ratios. Among mononuclear cells from encephalitic brain,
DC were the main producers of IL-12. Evidence for a parasite-dependent
development of DC from CNS progenitors was obtained in vitro: after
inoculation of primary brain cell culture with T.
gondii, IL-12-secreting dendriform cells emerged, and DC marker
genes were expressed. Different stimuli elicited the generation and
maturation of brain DC: neutralization of parasite-induced GM-CSF
prevented outgrowth of dendriform cells and concomitant release of
IL-12. IL-12 production was up-regulated by external IFN-
but was
stopped by inhibiting parasite replication. Consistently, DC isolated
from GM-CSF-treated brain cell culture were activated to secrete IL-12
by exposure to parasite lysate. In sum, these results demonstrate
T. gondii-induced expansion and functional maturation of
DC in the CNS and, thus, highlight a mechanism that may contribute to
the chronicity of the host response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD1d in addition to the pan-DC marker CD11c.
In contrast, myeloid-related DC generate from bone marrow cells or
blood monocytes in the presence of GM-CSF (12, 13) and
selectively bear the 33D1 surface Ag and share CD11b and F4/80 markers
with cells of the monocyte/macrophage lineage (3, 4, 5).
Although immature DC efficiently capture and process Ag but fail to
stimulate T cells due to their weak expression of interaction
ligands, mature DC are excellent APC expressing MHC class II and T
cell costimulatory molecules at high levels and strongly producing
IL-12. Functional maturation of DC is triggered by inflammatory or
microbial stimuli such as IL-1, TNF-, and LPS or by CD40 ligation
and is associated with increased cellular mobility (reviewed in Ref.
14).
DC are rare but comparatively ubiquitous cells. Besides their classical
sites in lymphoid and skin tissues, DC are located in the interstitial
connective tissue of many nonlymphoid organs including liver, kidney,
and heart (reviewed in Ref. 15), often exhibiting an
immature or Ag-processing phenotype. So far, DC have not been detected
within the brain. Presumptive local APC are astrocytes and
microglia/CNS macrophages, all of which require IFN- for this
function (reviewed in Ref. 16). However, evidence for the
existence of an intracerebral DC was provided by a recent study that
attests an immature DC phenotype to myeloid dendriform cells grown in
mouse brain primary culture with GM-CSF (17). Accordingly,
such brain cell-derived DC can present soluble Ag independently of
IFN- and exhibit a unique, Kv1.3-dominated profile in voltage-gated
K+ currents similar to that of splenic DC
(17, 18, 19).
To examine the possibility that DC are present in the CNS, murine toxoplasmosis was chosen as the model system in which an intracerebral T cell response occurs and determines the outcome of brain infection (20). In mice resistant to infection with Toxoplasma gondii, parasite challenge of the brain causes transient encephalitis, leaving the persistent parasite stage in cysts scattered in a histologically unobstrusive CNS parenchyma (21). In this inapparent phase of chronic infection, the brain continues to harbor elevated T cell numbers (22). In the present study, we demonstrate the expansion and concomitant activation of brain DC during toxoplasmic encephalitis (TE) and reveal a pathway by which T. gondii triggers the generation and maturation of DC from CNS progenitors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and C57BL/10 mice were used at 12–16 wk of age for in situ and ex vivo analyses. Newborn animals of either sex were used for the preparation of brain cell cultures. Mice were bred under specific pathogen-free conditions from breeding stock supplied by Biological Research Laboratories (Füllinsdorf, Switzerland) and The Jackson Laboratory (Bar Harbor, ME), respectively.
Parasites and infection
The DX strain of T. gondii (intraspecies group II) was used. For infection of mice, cysts were harvested from the brains of chronically infected animals. Minced brain tissue was homogenized by forced extrusion through 18-, 20-, and 22-gauge needles. Cysts were purified by centrifugation through Ficoll-Paque (Pharmacia, Uppsala, Sweden) and then with washes in PBS. All experiments of infection in vivo were performed with BALB/c mice. Mice were injected i.p. with three cysts, and infection was serologically verified 2–3 wk postinfection (p.i.). For inoculation of brain cell cultures, bradyzoites were prepared as described (23) by mild trypsinization of cell culture cysts. Parasite lysate containing 10 mg/ml protein was prepared from tachyzoites (strain BK) as detailed in (24). Feeder cells and toxoplasms were routinely tested for mycoplasma contamination.
Immunohistochemistry
Brains from infected or control mice were mounted in OCT compound (Miles Scientific, Naperville, IL), snap-frozen in prechilled 2-methylbutane, and stored at -70°C. Cryostat sections (5 µm) were thaw-mounted on poly-L-lysin-coated microscope slides and fixed in ice-cold acetone for 10 min. Sections were incubated for 30 min with 0.3% H2O2/0.2 M NaN3 to quench endogenous peroxidase activity before blocking with 10% goat serum (Vector Laboratories, Burlingame, CA) in HBSS. Sections were then stained for 1 h with anti-CD11c mAb N418 (Endogen, Cambridge, MA), DC-specific mAb 33D1 (Biotrend, Cologne, Germany), or hamster and rat isotype control Ab (PharMingen, San Diego, CA). After washing, sections were incubated with biotinylated rabbit anti-rat IgG (Vector) or goat anti-hamster IgG (The Jackson Laboratory). Subsequent incubation of the slides with peroxidase-conjugated avidin-biotin complex (Vectastain Elite ABC Kit; Vector) was performed according to the manufacturer’s protocol. Sections were developed with 3,3'-diamino-benzidine (DAB) using the DAB substrate kit (Vector), slightly counterstained with hematoxylin, dehydrated, and mounted.
Preparation of brain mononuclear cells (BMNC)
Mice were transcardially perfused with 2 x 20 ml ice-cold PBS while being kept under deep Metofane (Janssen, Neuss, Germany) anesthesia. The brains were excised, minced in DMEM with 10% FCS, and passed through an 18-gauge needle. After tissue digestion with 0.3 U/ml collagenase D and 0.2 mg/ml DNase I (Boehringer Mannheim, Mannheim, Germany) at 37°C for 45 min each, cells were washed, resuspended in cold medium, and passed through a 70-µm sieve. Then, cells were centrifuged on a 30%/60% Percoll gradient (one gradient/brain) at 1000 x g for 25 min as described (25). Cells in the interphase and pellet were collected and washed, and gradients containing red blood cells in the pellet were discarded. As determined by flow cytometry, BMNC were enriched in the interphase. Due to the low numbers of BMNC obtained, per experiment cells from 3 to 10 mice were pooled.
Isolation of DC and brain macrophages/microglia
Splenocytes or 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 isolated by positive selection using N418-labeled magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. Purity of selected cells was controlled by FACS analysis indicating >90% CD11c+ cells in the positive fraction. Analogously, CD11b+ brain macrophages/microglia were immunomagnetically sorted from CD11c- BMNC by using M1/70.15-labeled beads (Miltenyi Biotec) with <5% contamination by residual CD11c+ cells. All cells were resuspended in phenol red-free DMEM supplemented with 10% FCS, glutamine, and 50 µM 2-ME.
Flow cytometry
CD11c+ BMNC were subjected to
immunofluorescence staining of mouse DC markers by using the following
Abs: hamster anti-CD11c mAb N418 (Endogen), rat anti-DEC-205
mAb NLDC-145 (Dianova, Hamburg, Germany), PE- or FITC-labeled rat
anti-F4/80 mAb CI:A3-1 (Dianova), FITC-labeled rat anti-DC mAb
33D1 (Biotrend), PE-labeled mouse anti-CD8 mAb 53-6.7,
biotinylated rat
anti-I-Ad/I-Ed mAb 2G9,
hamster anti-CD40 mAb HM40-3, biotinylated hamster anti-CD54
mAb 3E2, PE-labeled hamster anti-CD80 mAb 16-10A1, and PE-labeled
rat anti-CD86 mAb GL1 (PharMingen). Species and isotype-matched
control Abs, extravidin-FITC, and FITC-labeled
F(ab')2 fragments of goat anti-rat or
anti-hamster IgG as secondary reagents were from PharMingen or
Dianova.
Cells were surface stained according to standard procedures. In samples
without anti-rat secondary reagent, cells had been preincubated
with rat anti-FcR mAb 2.4G2 (PharMingen) to block unspecific
binding of primary Ab. Propidium iodide was added in the final wash to
label dead cells. For flow cytometry using a FACScan (Becton Dickinson,
Heidelberg, Germany), samples were gated on
CD11c+ live cells. Per sample,
104 events were acquired and analyzed using the
Lysys II software.
Ag presentation assays
The APC function of CD11c+ spleen or brain cells was measured in T cell proliferation assay with the CD4+ T cell line LNC.2 (26) specific for purified protein derivative (PPD) of tuberculin. Tests were performed in A/2 microtiter tissue culture plates (Costar, Cambridge, MA) using IMDM with 5% FCS and glutamine. In a total volume of 100 µl, irradiated APC titrated from 104 to 101/well were cocultured with 104 LNC.2 T cells in the presence or absence of 60 µg/ml PPD (Behringwerke, Marburg, Germany). Replicate wells additionally contained 30 µg/ml of anti-I-Ad mAb MKD-6 or control mouse IgG. Test cultures were pulsed with [3H]TdR (7.4 kBq/well) during the last 18 h of 3-day incubation. Results from liquid scintillation counting are given as mean cpm of triplicate test cultures ± SD.
The capacity of isolated DC to stimulate naive T cells was measured in a primary allogeneic MLR. CD11c+ splenocytes and BMNC of BALB/c origin were added in graded doses to 105 C57BL/10 T cells in A/2 microtiter wells (100 µl). The T cells had been prepared by passing spleen cell suspensions through a nylon wool and an Ab-coated T cell isolation column (R&D Systems, Minneapolis, MN) to eliminate surface Ig and FcR-positive cells. The resulting cell preparation contained >99% CD3+ cells. Five days after starting test cultures, the T cell proliferation was measured via [3H]thymidine uptake as described above.
In vitro challenge by T. gondii of brain cells
Primary cultures prepared from newborn mouse brains
(23) were inoculated with T. gondii bradyzoites
(strain DX) at a host-cell-to-parasite ratio of 10/1 when the cell
layer reached confluence. Control cultures were left untreated. As
indicated, 100 U/ml IFN- (Genzyme, Cambridge, MA), 20 µg/ml
sulfadoxin plus 1 µg/ml pyrimethamin (Roche, Rheinach, Switzerland),
10 µg/ml of neutralizing anti-GM-CSF mAb MP1-31G6, or control rat
IgG (Endogen) was added. During 14 days of continued culture, samples
of the supernatant (SN) were taken in 2- or 3-day intervals while one
quarter of the medium was renewed. Samples were stored at -80°C
until being assayed for cytokine content.
From GM-CSF-treated brain cell cultures, loosely adherent DC had been
isolated (17) and were incubated at a density of
106/ml in cytokine-free medium with 100 µg/ml
soluble T. gondii lysate Ag and/or 100 U/ml IFN-. After
48 h, samples of the SN were collected and tested by ELISA for
IL-12.
Measurement of cytokine secretion
Brain cells or splenocytes were seeded into microtiter wells at a density of 2.5 x 105 cells/250 µl in medium with FCS. After 24 h of incubation, samples of the culture SN were collected. Herein, as in samples from brain cell cultures, IL-12 (p40 + p75), IL-10, and GM-CSF were quantified by using specific sandwich ELISA (Genzyme). Recombinant mouse cytokines served as standard. Assays had a minimum sensitivity of 7 (IL-12) or 5 pg/ml (IL-10, GM-CSF). Values represent means ± SD from triplicate SN productions.
RNA extraction and RT-PCR
Total RNA was extracted by the guanidinium thiocyanate method from brain cells (2 x 106/sample) or T. gondii parasites (107). Reverse transcription of poly(A)+ mRNA was performed using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Alto, CA). Synthesized cDNA was used as template in PCR. Gene-specific primers and internal probes were designed according to published sequences: for DEC-205, 5'-ACTGGAGAACGGGAAGACCAACTGTGAAAA-3' (sense), 5'-TGCTCTGCCATCGGACCATTCG-3' (antisense), and 5'-GTCCTCATAGTCAACCATTTCAATTTTACAAGCAG-3' (probe); for IL-12p40, 5'-CTTCATCTGCAAGTTCTTGGGC-3' (sense), 5'-CGTGCTCATGGCTGGTGCAAAG-3' (antisense), and 5'-TCTGTCTGCAGAGAAGGTCACA-3' (probe); for Kv1.3, 5'-GGCATTGCCATTGTGTCAGTGC-3' (sense), 5'-GAAGCTGGAGGCTCCAGAAGGGG-3' (antisense), and 5'-AGCAGAAGATGACAATGGAGATGAGAATGA-3' (probe); for CD86, 5'-GGGGGATCCATGGGCTTGGCAATCCTTAT-3' (sense), 5'-TCGGGTGACCTTGCTTAGACGTGCAGG-3' (antisense), and 5'-TGAACATTGTGAAGTCGTAGAGTCCAGT-3' (probe); for T. gondii tachyzoite marker SAG1, 5'-GCCGTTGTGCAGCTTTCCGTTCTTC-3' (sense), 5'-ATCCCCCGTCCACCAGCTATCTTCT-3' (antisense), and 5'-GCGCGTCTCACTGCCTTCGGAAACATACTC-3' (probe); and for bradyzoite SAG4, 5'-GGGCGGTGCTCTGTTTCCTTATTTTATTTG-3' (sense), 5'-ACGGGCTCATCCTTGCAGTGGTCTC-3' (antisense), and 5'-ACTTGACTAGGGTTGGGCGGGATGTA-3' (probe). The G3PDH gene expression was detected using primers from Clontech. PCR was conducted by incubation of the reaction mix in 30 cycles of 1 min each at 95/60/72°C. PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining and were visualized with UV.
Homology of PCR products to the predicted transcript sequences was controlled by Southern blot analysis. Gels were blotted to Nytran membrane (Schleicher & Schüll, Dassel, Germany). Then, the membrane was UV cross-linked, and blots were hybridized with the oligonucleotide probes, which had been 3'-end-labeled with digoxigenin using a DIG Oligonucleotide 3'-End Labeling Kit (Boehringer Mannheim). Subsequent immunodetection with alkaline phosphatase and the chemiluminescence substrate CSPD was conducted using the DIG Luminiscent Detection Kit (Boehringer Mannheim).
Statistics
The significance of differences was examined using the Student t test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cellular immunity controls T. gondii infection in the
CNS (20); however, it is still unclear which APC direct
the intracerebral T cell response. In mononuclear cells prepared from
mouse brain, an up-regulation of IL-12p40 and CD86/B7.2 and an
induction of Kv1.3 gene message was observed during the chronic stage
of infection (Fig. 1
). Transcripts
for DEC-205, a surface marker of several DC populations (3, 4, 5, 27) that has also been found on brain endothelial cells
(28), were detected in BMNC from both infected and naive
animals. Although none of these parameters alone compellingly indicates
the presence of DC, their coincidence raised the question whether
DC-like cells exist in the brain during TE. This hypothesis was tested
by immunohistochemical staining of mouse brain for the DC markers CD11c
and 33D1. Neither molecule was expressed in the brains of naive mice
(Fig. 2, A and D)
or during acute infection at day 7 p.i. (data not shown).
However, in the brains of mice infected for 5 wk with T.
gondii, CD11c was detected at inflammatory foci in the brain
parenchyma and perivascular cuffs (Fig. 2, B and
C). Labeling of 33D1 resulted in a congruent pattern, thus
confirming the localization of brain DC in TE (Fig. 2, E and
F). In combination, these data demonstrate the presence of
DC in the T. gondii-infected encephalitic
brain.
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To isolate DC from the brain, BMNC were depleted of T cells and
immunomagnetically sorted for CD11c+ cells. Based
on such a protocol, the intracerebral DC population was estimated
during the course of T. gondii infection. As shown in Fig. 3A, considerable yields of
CD11c+ BMNC were obtained from day 15 p.i.,
with numbers increasing until day 35 p.i. Simultaneously, the mean
proportion of CD11c+ cells within BMNC rose from
<1% up to 10%. At maximum, the number of isolated
CD11c+ cells per brain reached
106 and thus exceeded that of
CD11c+ cells prepared by the same technique from
normal spleen. At 5 mo p.i., the number of total BMNC had dropped near
background level (p > 0.1 compared with normal
brains), whereas the number of CD11c+ brain cells
still remained elevated (p = 0.04). As one
parameter for the course of TE in BALB/c mice, the brain cyst load was
monitored, revealing a peak cyst number in week 4 p.i. (Fig. 3B). The comparable kinetics of the numbers of cysts, BMNC,
and DC in the T. gondii-infected CNS suggest a correlation
between the inflammatory process and the emergence of DC. To obtain
sufficient numbers, in additional experiments,
CD11c+ BMNC were isolated between day 30 and day
43 p.i. unless otherwise stated.
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DC freshly isolated from infected brain showed the typical
morphology with thin membrane projections. Unlike
CD11c- CD11b+ brain
macrophages/microglial cells, most of CD11c+ BMNC
remained nonadherent during incubation overnight and kept their
dendritic shape. Flow cytometric analysis of
CD11c+ BMNC from chronically infected mice
confirmed the coexpression of the myeloid DC-restricted surface
molecule 33D1 by >90% of isolated cells (Fig. 4). Consistently, a majority of >80%
stained positive for the monocyte/macrophage marker F4/80. In contrast,
the lymphoid-related marker CD8 was lacking, and DEC-205 was only
expressed on 
10% of cells (Fig. 4). By exhibiting this phenotype,
the DC population isolated from T. gondii-infected brain
apparently belongs to the myeloid DC subset.
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indicating that the population of CD11c+ cells
from infected brain consists of terminally differentiated mature
DC. Allostimulatory activity and presentation of foreign Ag
As definitive criterion for being classified as DC, the isolated
brain cells were tested by MLR for their capacity to stimulate
alloreactive naive T cells. Graded numbers of
CD11c+ cells from T. gondii-infected
brain or normal spleen were incubated with a fixed number of allogeneic
T cells. Fig. 5A shows that
<100 brain DC per 105 T cells could already
trigger a substantial response the level of which continuously
increased with rising stimulator cell concentrations. In comparison,
normal spleen DC proved less efficient at low cell numbers but induced
a higher level of T cell proliferation at doses >2500 cells/well. This
differential activity of both DC populations was observed in all
experiments.
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B,
the brain DC were excellent APC inducing maximum T cell proliferation
at a ratio of already 1/100 T cells, whereas the reference DC were
about 10 times less effective. The T cell response was Ag-specific and
dependent on MHC-restricted cellular interaction because blocking with
anti-I-Ad mAb completely abrogated the
proliferation triggered by an optimal number of APC (data not
shown). Brain DC are the predominant IL-12 producers in TE
Production of IL-12 by brain DC ex vivo was monitored during the
course of T. gondii infection and compared with that by
other BMNC. Cytokine release by constant numbers of isolated cells was
quantified by ELISA after 24 h of incubation. As summarized in
Fig. 6A,
CD11c+ BMNC from naive mice secreted little IL-12
equal to the IL-12 release by splenic DC. Compared with this background
level, IL-12 production by brain DC rose upon infection up to a >20
times higher peak value on day 43 p.i. At later time points, the
amounts of IL-12 decreased; however, about 1 year p.i., IL-12 levels
were still significantly (p < 0.001) higher
than those produced by cells from uninfected mice. Considering the
minute number of brain DC months after infection (Fig. 3A),
these findings imply a long-lasting presence of few, but activated, DC
in the chronically infected brain. As is evident from Fig. 6A, brain DC are the most important source of IL-12 among
BMNC because relatively low amounts of IL-12 were produced by the
CD11c- cell population
(p < 0.001). To compare the cytokine profiles
of brain DC and brain-associated macrophages/microglial cells in TE,
CD11b+ cells were enriched from
CD11c- BMNC and were tested against the
CD11c+ cells for secretion of IL-10 and IL-12. As
shown in Fig. 6B, brain DC were confirmed to be the
principal IL-12 producers by releasing 10 times more of this
cytokine than brain macrophages/microglia which, conversely, proved
superior in production of IL-10 (p <
0.001).
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To elucidate brain-specific cellular interactions involved in
parasite-induced development of DC, we used an in vitro system that
mimics CNS infection by T. gondii. After inoculation of
primary brain cell culture with the DX strain of T. gondii,
formation of both parasite cysts and lytic foci starts from day 5
p.i. (29). In parallel, we observed that dendriform cells
emerged and proliferated on the glia monolayer in the absence of
exogenous cytokines (Fig. 7A).
According to their CD11c+
CD11b+ 33D1+
CD8- DEC-205-/+
phenotype (data not shown), these cells correspond to the immature DC
derived from GM-CSF-supplemented primary culture (17).
Additional evidence for the development and functional maturation of DC
upon T. gondii challenge of brain cells was provided by
analysis at the transcriptional level. Expression of IL-12p40 was
induced within 7 days p.i. when a shift in SAG4/SAG1 expression
indicated parasite conversion to the tachyzoite stage. Induction of
Kv1.3, CD86, and DEC-205 gene message was detected in the second week
p.i. (Fig. 7B). Parallel testing by ELISA of SN samples over
the 2-wk interval after inoculation revealed that IL-10, a product of
parasitized microglia (23), is released rapidly (from day
2 p.i.), whereas IL-12, putative indicator of brain DC activation,
is produced later (from day 5 p.i.). IL-12 levels were in the
ng/ml range during week 2 p.i. (Fig. 8A).
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B). In sum, our
findings demonstrate a parasite-triggered, GM-CSF-dependent pathway by
which functional DC develop from CNS progenitors. Activation of these
brain cell-derived DC to produce IL-12 does not require contact with T
cells.
IL-12 secretion upon toxoplasmic challenge of brain cells
correlates with tachyzoite proliferation and is synergistically
enhanced by IFN-
The impact of parasite replication on brain DC development and
function was investigated by treatment of infected primary cultures
with sulfadoxin/pyrimethamin. At the concentrations used to block
T. gondii growth in vitro, both drugs affect neither the
cytokine response of brain cells to LPS nor the development of DC in
GM-CSF-treated primary culture. Administration of
sulfadoxin/pyrimethamin to infected brain cells produced, with a 48-h
delay, no further increase in the IL-12 level of culture SN (Fig. 8C). From day 9 of the 2-wk test period, the level of
accumulative cytokine production was significantly reduced compared
with the IL-12 release by infected but untreated cells
(p < 0.05). Whether the proliferating stage of
T. gondii, the tachyzoite, induces functional maturation of
already DC-committed brain cells was tested by exposure of GM-CSF-grown
brain cells to tachyzoite lysate. As shown in Fig. 9, parasite challenge of these
brain-derived immature DC led to a strong secretion of IL-12,
indicating cellular activation by Toxoplasma products.
Costimulation with additional IFN- accelerated and enhanced the
IL-12 response in parasitized brain cells (Fig. 8C) and by
isolated immature brain DC after incubation with tachyzoite lysate
(Fig. 9). Together with the findings described in the preceding
paragraph, these data specify the different roles of T.
gondii in the development and terminal maturation of brain DC. For
the latter, IFN- provides an efficient costimulus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As indicated by the yield of DC purified from brain tissue, the DC
number in situ reflects the degree of CNS inflammation during all
phases of chronic toxoplasmosis. There are four potential scenarios
that may account for the observed 50- to 100-fold expansion of brain DC
upon onset of brain infection: 1) DC develop from blood monocytes
infiltrating into the inflamed CNS tissue; a monocyte-to-DC
differentiation pathway recently has been shown (13, 35);
2) meningeal DC are recruited in very large numbers to inflammatory
foci beyond the blood-brain barrier; 3) perivascular MHC class
II+ macrophages which function as APC in the
brain (36, 37) proliferate and differentiate to DC and
migrate to inflammatory sites even deep in the parenchyma; and 4) brain
DC develop locally from intracerebral progenitors or resident microglia
due to signals from the T. gondii-infected neural
environment. Prerequisites for the latter, most intriguing possibility
are the existence of a myeloid precursor cell pool in the brain as
demonstrated in the mouse (38) and a parasite-triggered
pathway by which such brain cells expand and differentiate to DC. The
first evidence supporting this hypothesis was obtained in a previous
study that showed the generation of immature DC in GM-CSF-treated brain
cells (17). In addition, secretion of cytokines critical
in DC differentiation, like GM-CSF, IL-1, and TNF-, is induced by
T. gondii in glial cells (23). Accordingly,
GM-CSF expression is induced in the brains of infected mice at day
15 p.i. (39). These findings were complemented with
the present data, which demonstrate that the development and maturation
(in terms of IL-12 production) of brain DC from parasitized glial cells
requires endogenous GM-CSF but can already occur in the absence of T
cells. A final determination of the origin of brain DC and their
possible migration route(s) and destiny remains an issue of further
research, which may be done by utilizing congenic chimera models and
prelabeled cells.
The CD11c+ 33D1+
F4/80+ CD8-
DEC-205-/+ phenotype of the cells isolated from
brain resembles that of their counterparts from brain cell culture
(17). Thus, brain-derived DC correspond to the myeloid
subset of mouse DC and are related to macrophages/microglia but differ
from those by exhibiting the DC-restricted markers CD11c and 33D1. Due
to difficulties in discriminating rat CD11b and CD11c by mAb, brain DC
could have been recognized as inflammatory macrophages in the rat
systems commonly studied (33, 37, 40, 41). The ontogenetic
relationship of both cell populations renders a microglial or monocytic
origin of brain DC to be most likely.
With the definitive detection of brain DC, a novel type of APC is
identified for the CNS. Because the known brain APC, namely astrocytes,
microglial cells, and other CNS macrophages, require IFN- for their
function (for review, see Ref. 16), the presence of DC in
the inflamed brain solves the "hen and egg" dilemma of
intracerebral T cell activation before IFN- is produced. Upon
isolation, brain DC are often found clustered with T lymphocytes (M.
Sahm and H.G. Fischer, unpublished observations), indicating an APC
activity in situ. Similar aggregates have been reported on MHC class
II+ rat microglia in brain graft-vs-host disease
(41). A role for brain DC as amplifiers of T cell
responses in TE is suggested by their strong expression of MHC class
II, CD40, and B7 costimulatory molecules and by their excellent
stimulation of both naive and Ag-primed T cells ex vivo. Unlike MHC
class II+ rat (40) but comparable to
human microglial cells (42), the DC from T.
gondii-infected mouse brain triggered the proliferation of T
cells. In stimulating primary or secondary T cell responses at
suboptimal APC doses, these brain DC proved superior to normal spleen
DC. This is probably due to a higher proportion of fully matured cells
in the brain DC as confirmed by their IL-12 secretion (see Fig. 6A). Both populations were purified via the
ß2-integrin CD11c, which is
expressed as a pan-DC marker also on immature DC (3, 43).
A notable feature of brain DC in TE was their high-level production of
IL-12 ex vivo coupled with a low production of IL-10. Co-isolated
microglia/brain macrophages exhibited a converse profile in secretion
of these cytokines. Evidence from mice suffering from experimental
autoimmune encephalomyelitis or injected with LPS indicates that
microglia-like cells in the inflamed CNS tissue express IL-12p40
(44, 45). Because activated astrocytes can antagonize
microglial IL-12 release (46) and IL-12 production by
brain DC was measured on purified cells, our approach perhaps detects a
functional capability masked in vivo. However, IL-12 was also secreted
by cells developing in parasitized brain cell culture adjacent to
astrocytes (Fig. 8), which would suggest that IL-12 production of brain
DC is an intrinsic function that distinguishes them from CNS
macrophages. The long-lasting presence of activated DC in the infected
brain might contribute to the chronicity of the intracerebral cellular
response in TE.
The parasite itself is involved, via induction of GM-CSF, in the
generation of brain DC and, via soluble tachyzoite compounds, in their
terminal maturation. Whether both effects are triggered by the same or
by different parasite molecules remains unclear. To date, two
cytokine-inducing activities of T. gondii have been
distinguished biochemically and functionally (24, 47).
Although IFN- is not crucial for the IL-12 response of brain DC in
vitro (Fig. 8) or of splenic DC in vivo (48), it
synergistically up-regulates DC activation by T. gondii.
Thereby, the parasite acts in an LPS-like manner according to the
conventional model of DC stimulation (49, 50). Finally,
the dependency on parasite proliferation of IL-12 secretion in
parasitized brain cells implies a feedback loop by which the host
response can adapt to the actual parasite burden during persistent
infection.
In conclusion, the present data prove the existence of functional DC in the T. gondii-infected mouse brain and reveal a mechanism by which CNS tissue can generate DC in response to parasite challenge. This unique potential gives a novel meaning to the traditional concept of the brain as an "immunoprivileged" organ.
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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 Current address: Institute for Medical Microbiology and Virology, Heinrich-Heine University, Dnesseldolf, Germany.
4 Abbreviations used in this paper: DC, dendritic cell; TE, toxoplasmic encephalitis; BMNC, brain mononuclear cells; p.i., postinfection; PPD, purified protein derivative; SN, supernatant.
Received for publication November 3, 1999. Accepted for publication February 14, 2000.
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