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*
Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanità, Rome, Italy;
Institute of General Pathology, Catholic University, Rome, Italy; and
Roche Milano Ricerche, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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microglia express MHC class II molecules, CD40, and ICAM-1 and
efficiently present OVA 323-339, leading to T cell proliferation and
production of IL-2 and IFN- by Th1 and of IL-4 by Th2 cells.
IFN--treated astrocytes, which express MHC class II and ICAM-1,
present OVA 323-339 less efficiently to Th1 cells but are as efficient
as microglia in inducing IL-4 secretion by Th2 cells. However,
astrocytes are much less potent than microglia in presenting naturally
processed OVA peptide to either T cell subset, indicating inefficient
Ag processing. The capacity of astrocytes and microglia to stimulate
Th1 and Th2 cells depends on their MHC class II expression and does not
involve ICAM-1, B7-1, or B7-2 molecules. However, CD40-CD40L
interactions contribute to Th1 activation by microglia. These data
suggest that microglia may play a role in the activation of Th1 and Th2
cells, whereas astrocytes would restimulate mainly Th2 responses in the
presence of appropriate peptides. This differential capacity of brain
APC to restimulate Th1 and Th2 responses may contribute to the
reactivation and regulation of local inflammatory processes during
infectious and autoimmune diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes have been subdivided into two distinct subpopulations
that differ in their cytokine profile and effector functions. Th1
lymphocytes, characterized by IL-2, IFN-, and TNF-ß secretion,
induce cell-mediated immune responses, whereas Th2 lymphocytes
selectively produce IL-4, IL-5, IL-10, and IL-13 and promote humoral
immune responses (7, 8). Th1-dominated responses are often associated
with inflammation and tissue destruction that lead to organ-specific
autoimmune diseases (5, 9, 10). Autoreactive CD4+ T
lymphocytes exhibiting a Th1 phenotype are thought to play a role in
the pathogenesis of MS (11) and experimental allergic
encephalomyelitis, an animal model of CNS autoimmune disease (12, 13).
Conversely, several cytokines produced by Th2 lymphocytes, such as
IL-4, IL-10, and IL-13, have anti-inflammatory activities.
Activation of Th2 lymphocytes may inhibit CNS inflammation and limit
the noxious effects of Th1-mediated immunity, as observed in
experimental allergic encephalomyelitis (14, 15). IL-12 promotes the
differentiation of naive T cells to Th1 and is a potent costimulus for
the activation of differentiated Th1 cells (16, 17), whereas IL-4
drives the development of Th2 responses. In addition, the development
of Th1 and Th2 lymphocytes from a common undifferentiated precursor
depends on several other factors, including the avidity of TCR-ligand
interaction and costimulatory signals delivered by the APC
(7).
Within the CNS parenchyma, microglia, a type of highly differentiated
and quiescent tissue macrophage, constitutively express MHC class II
molecules (18, 19, 20). MHC class II expression is greatly increased on
reactive microglia in response to neuronal damage, infection, or
inflammation (21, 22). IFN- and/or LPS stimulate cultured microglia
to express MHC class II as well as adhesion/costimulatory molecules and
to produce bioactive IL-12 (23, 24, 25, 26, 27). In vitro, activated microglia are
able to prime alloreactive T cell responses (24, 28) and to stimulate T
cell lines to proliferate and secrete cytokines (23, 29, 30).
Adhesion/costimulatory molecules, such as ICAM-1, CD40, CD80, and CD86,
are also expressed by reactive microglia in inflammatory MS lesions
(25, 31, 32, 33), suggesting a role for these APC in T cell activation
in vivo.
Astrocytes, the major CNS glial cell type, have also been proposed to
have a role as APC. While MHC class II expression on astrocytes in situ
remains controversial (34, 35, 36, 37, 38, 39), astrocytes stimulated in vitro by
IFN- and TNF-
express MHC class I and II molecules as well as
ICAM-1, vascular cell adhesion molecule-1, and LFA-3 (24, 40, 41, 42, 43).
Conflicting data have been reported on the capacity of cultured
astrocytes to activate T cell responses. Depending on the source of
astrocytes and the type of responding T cell, astrocytes have been
shown to act as stimulators or inhibitors of the proliferation of
primed T cells (29, 41, 43, 44, 45, 46) and to be able or unable to stimulate
the proliferation of naive T cells (24, 28, 46, 47). Conflicting data
also exist on the type of T cell costimulatory signals (e.g., B7 and
IL-12) expressed or inducible in astrocytes (25, 26, 27, 47, 48, 49).
T cell activation within the CNS is still poorly understood and no information is yet available on the efficiency of CNS APC to restimulate Th1 and Th2 responses. In this study, we have determined the capacity of microglia and astrocytes from BALB/c mouse forebrains to process and present Ag to OVA-specific TCR transgenic Th1 and Th2 cells, leading to T cell proliferation and cytokine secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were purchased from Charles River (Calco, Italy).
One-day-old mice were used for the preparation of brain cell cultures,
whereas 2- to 3-mo-old female mice were used for the preparation of
spleen cells. DO11.10 TCR transgenic mice on BALB/c background (50)
were kindly provided by Dr. D. Y. Loh (Hoffmann-La Roche, Nutley,
NJ). In these transgenic mice, 95 to 100% of the CD4+
T cells are Vß8.1.2+ (51) and express a TCR-ß
specific for OVA peptide 323–339 bound to I-Ad.
Cell cultures
Primary mixed glial cultures were established from the forebrains of 1-day-old BALB/c mice, following previously published procedures (48). Forebrains were carefully freed of meninges, chopped into 0.25-mm sections, and dissociated by a mild trypsinization procedure and gentle mechanical disruption with a Pasteur pipette. The cells were seeded into poly-L-lysine (10 µg/ml)-coated 175-cm2 flasks at the density of 4 x 104 cells/cm2 and grown at 37°C in a 92% air-8% CO2 humidified atmosphere in DMEM (HyClone, Cramlington, U.K.) containing 0.45-µm filtered, 10% FCS Myoclone (Life Technologies, Gaithersburg, MD), 2 mM glutamine (BioWhittaker, Verviers, Belgium), penicillin (100 U/ml), and streptomycin (100 µg/ml). The medium was replaced after 24 h and then every 3 days when preparing astrocyte cultures, or only once after 4 days when preparing microglial cultures. After about 10 days in vitro, microglial cells were detached from the astroglial monolayer by rapid (15–30 s) and gentle manual shaking of the culture flasks; the supernatants were collected and centrifuged, and the cells were reseeded on plastic surfaces in the same medium as above. After 1 h, the medium was replaced to remove nonadherent cells.
For the preparation of purified astrocyte cultures, 10-day-old primary cultures were vigorously shaken to detach microglia and oligodendrocytes growing on top of the astrocytic layer. The remaining adherent cells were detached with trypsin (0.25%)/EDTA and the resulting cell suspension was left at room temperature in uncoated flasks to allow adherence of microglia to the plastic surface. After 20 to 30 min, the nonadherent or loosely adherent cells were collected after mild shaking of the flasks and the adhesion step was performed once more. The supernatants containing the nonadherent cells were collected and centrifuged; the cells were resuspended in fresh medium as above and reseeded on poly-L-lysine-coated plastic surfaces.
Spleen cells were prepared from normal BALB/c mice, irradiated with 3000 rad, and seeded in 96-well flat-bottom culture plates in RPMI 1640 (Life Technologies) supplemented with 50 µM 2-ME, 2 mM L-glutamine, 50 µg/ml gentamicin, and 10% FCS (Sigma, St. Louis, MO).
T cell lines
CD4+ cells were positively selected from inguinal and mesenteric lymph nodes of naive DO11.10 TCR transgenic mice by anti-CD4-coated magnetic microbeads (Miltenyi-Biotec GmbH, Bergish, Germany). CD4+ T cells (2 x 105 cells/well) were cultured with OVA peptide 323-339 (0.3 µM), synthesized as previously described (52), and mitomycin C-treated BALB/c splenocytes (5 x 106 cells/well) as APC in a total volume of 2 ml in 24-well plates, in the presence of either 0.1 ng/ml recombinant mouse IL-12 (Hoffmann-La Roche, Nutley, NJ) and 10 µg/ml anti-mouse IL-4 (11B11; American Type Culture Collection (ATCC), Rockville, MD), or 20 ng/ml mouse recombinant IL-4 (Hoffmann-La Roche AG, Basel, Switzerland) and 10 µg/ml anti-mouse IL-12 mAb (10F6; Hoffmann-La Roche) to obtain Th1 or Th2 cell lines, respectively. Cells were cultured in RPMI supplemented with 50 µM 2-ME, 2 mM L-glutamine, 50 µg/ml gentamicin, and 10% FCS (Sigma). After 3 days in vitro, T cells were expanded and grown in complete medium containing 10 ng/ml of recombinant human IL-2 (Hoffmann-La Roche AG).
Ag presentation
Varying numbers of microglia and astrocytes were seeded in
96-well flat-bottom tissue culture plates. After 16 h, the culture
medium was replaced with fresh medium (DMEM/10% FCS) without or with
cytokines or LPS, and cells were incubated at 37°C for additional 24
to 48 h. Mouse recombinant cytokines used in this study were:
IFN- (sp. act. 1 x 107 U/mg), TNF- (sp.
act. 4 x 107-4 x 109 U/mg), and
IL-1ß (sp. act. 4.5 x 109 U/mg), all from Genzyme,
Cambridge, MA. LPS (from Escherichia coli, serotype 026:B6)
was purchased from Sigma. Immediately before the addition of T cells
from transgenic mice, astrocytes and microglia were gently washed three
times with DMEM/10% FCS to completely remove stimulating agents.
Spleen cells were seeded at densities ranging from 3 x
104 to 1 x 106 cells/well. TCR transgenic
Th1 and Th2 cells were collected from 6- or 7-day-old cultures and
added (5 x 104) in RPMI/10% FCS (supplemented as
described above) to wells containing the different APC in the presence
of different doses of OVA 323-339 peptide (0.001–0.3 µM) (52) or
native OVA (grade V, from Sigma) (0.01–30 µM). For inhibition of Ag
presentation, the following mAbs (10 µg/ml) were added to the
cultures 30 min before addition of T cells: B21.22,
anti-I-Ab,d (ATCC); 14-4-4S,
anti-I-E (ATCC); YN1/1.7.4, anti-ICAM-1 (ATCC);
1G10, anti-B7-1 (PharMingen, San Diego, CA); 37.51, anti-CD28
(PharMingen); 2D10, anti-B7-2 (53); MR1, anti-CD40 ligand
(PharMingen); hamster IgG (PharMingen); rat IgG2a (PharMingen); rat
IgG2b (ATCC). For analysis of T cell-derived cytokines, supernatants
from triplicate cultures were harvested after 24 or 48 h,
centrifuged at 1200 rpm and stored at -20°C until used for cytokine
determination. For proliferation assays, microglia, astrocytes, and
spleen cells were irradiated (3000 R) immediately before addition of T
cells and Ag. Proliferation was measured by incorporation of 0.5 to 1
µCi/well [3H]thymidine (Amersham Italia, Milan, Italy;
sp. act. 25 Ci/mmol) during the last 16 h of a 48-h incubation
period. Radioactivity was detected using a Packard Topcount microplate
scintillation counter (Packard Instrument, Meriden, CT).
Cytokine assays
IL-2, IFN-, and IL-4 were quantified by a two-site sandwich
ELISA as previously described (54). All mAbs, except anti-IFN-,
were purchased from PharMingen. The anti-IFN- mAb used for
capture was AN.18.17.24 (55) and the mAb used for detection was
peroxidase-conjugated XMG1.2 (56). Cytokines were quantified from three
to four titration points using standard curves generated by purified
recombinant mouse cytokines (IFN- and IL-4 from Hoffmann-La Roche
AG, and IL-2 from PharMingen) and results expressed as cytokine
concentration in ng/ml. Detection limits for all cytokines were in the
range of 7 to 15 pg/ml.
Intracellular staining for IFN- and IL-4 production
Polarized Th1 and Th2 cells were restimulated with PMA (50
ng/ml) and ionomycin (0.75 µg/ml) for 4 h at 37°C, with 10
µg/ml of brefeldin A (Sandoz, Basel, Switzerland) added for the last
2 h. After fixing for 20 min at room temperature in 2%
paraformaldehyde, the cells were stained for intracytoplasmic IFN-
and IL-4 using the method described by Openshaw et al. (57). All
incubations were performed at room temperature in PBS containing 5%
FCS, 0.5% saponin (Sigma), and 0.1% sodium azide. After washing and
10 min of preincubation in PBS/FCS/saponin, cells were incubated with
FITC rat anti-mouse IFN- (XMG1.2; PharMingen) and phycoerythrin
(PE) rat anti-mouse IL-4 (11B11; PharMingen) or with FITC- and
PE-labeled rat IgG1 isotype controls (R3-34; PharMingen). After 30
min, cells were washed twice with PBS/FCS/saponin and then with PBS/5%
FCS to allow membrane closure. Cell membranes were then stained with
Cy-Chrome-labeled anti-CD4 (L3T4; PharMingen) for 15 min. Analysis
was performed with a FACScan flow cytometer (Becton Dickinson, Mountain
View, CA) equipped with CELLQUEST software. In all, 30,000 events were
acquired.
Flow cytometry
Sixteen hours after isolation from primary cultures, microglia
and astrocyte cultures (5 x 105 cells/35-mm
diameter culture dishes) received fresh culture medium or medium
containing cytokines or LPS, as above. After 24 to 48 h of
incubation, cells were washed with DMEM, preincubated on ice for 30 min
with DMEM containing 30% goat serum, and then incubated on ice for
another 30 min with FITC- or PE-conjugated mAbs in DMEM/30% goat serum
to inhibit binding to FcR. The following fluorochrome-conjugated mAbs
(all from PharMingen) were used at 1:100 to 1:200 dilution: PE rat
anti-mouse Mac-1/CD11b (M1/70, IgG2b), FITC rat anti-mouse B7-2
(GL1, IgG2a), FITC rat anti-mouse CD40 (3/23, IgG2a), FITC mouse
anti-mouse I-Ad (39-10-8, IgG3), FITC mouse
anti-mouse I-E (14-4-4S, IgG2a). The background
fluorescence was evaluated by staining the cells with isotype control
Abs: PE rat IgG2b, FITC rat IgG2a, FITC mouse IgG2a, and FITC mouse
IgG3 (all from PharMingen). For CD54 (ICAM-1) and B7-1 stainings, cells
were incubated for 30 min with rat anti-mouse ICAM-1 mAb (IgG2a;
Serotec, Oxford, U.K.), rat anti-mouse B7-1 (1G10, IgG2a;
PharMingen) or isotype control Ab (rat IgG2a; PharMingen) and then for
an additional 30 min with FITC goat F(ab')2 anti-rat
IgG (Organon Teknika-Cappell, Durham, NC). At the end of the
incubations, cells were washed three times with DMEM. Microglial cells
were detached from the culture dishes using a cell scraper, whereas
astrocytes were detached by gentle pipetting after incubating at 4°C
for 5 to 10 min in PBS/0.02% EDTA. Cells were centrifuged and
resuspended in 0.3 ml PBS/0.5% paraformaldehyde. For detection of the
astrocyte-specific intermediate filament protein glial fibrillary
acidic protein (GFAP), microglia and astrocytes were detached from the
culture dishes as above and fixed with 4% paraformaldehyde in PBS for
5 min at room temperature. Cells were then permeabilized with Triton
(0.1% in PBS) for 5 min and incubated with mouse anti-GFAP mAb
(IgG, 1:200) (BioGenex Laboratories, San Ramon, CA) or control isotype
Ab followed by FITC goat F(ab')2 anti-mouse IgG
(1:200). Cells were analyzed on a FACScan flow cytometer, and 3000 to
5000 events were acquired.
Statistical analysis
To quantitate the differences between microglia and astrocytes
in their ability to stimulate Th1 and Th2 cytokine secretion, data were
analyzed by Friedman nonparametric ANOVA (58), considering the
experiments as blocks and the "cell type" by "cell density"
combinations as repeated measures within experiments. The main effect
of "cell type" and "cell density" and their interaction were
assessed by the orthogonal partitioning of the overall Friedman
2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As the main goal of this study was to evaluate the efficiency of
microglia and astrocytes in the restimulation of TCR transgenic T
cells, we ascertained that all experiments were conducted using
microglia and astrocyte cultures from BALB/c mouse forebrains devoid of
any cross-contamination. Culture purity was assessed by surface
staining with anti-Mac-1/CD11b, a marker for macrophages/microglia
(59), and by intracellular staining with a mAb specific for the
astrocytic intermediate filament protein GFAP (60). Flow cytometric
analysis revealed that microglia and astrocyte cultures contained 92 to
95% Mac-1+ cells and 95 to 98% GFAP+
cells, respectively (Fig. 1
).
Mac-1+ cells and GFAP+ cells were virtually
absent in astrocyte and microglia cultures, respectively. Examination
of the cultures by the fluorescence microscope after triple staining
with anti-Mac-1, anti-GFAP mAb, and Hoechst 33258 fluorochrome
(to label cell nuclei) confirmed that less than 1% of the total
population expressed GFAP or Mac-1 in microglia or astrocyte cultures,
respectively (not shown).
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Th1 and Th2 cell lines were generated from mice transgenic for the
DO11.10 TCR, which recognizes the OVA epitope 323-339 bound to
I-Ad molecules. CD4+ lymph node T cells
from TCR transgenic mice were cultured with mitomycin C-treated spleen
cells from normal BALB/c mice and OVA peptide 323-339 in the presence
of IL-12 and anti-IL-4 or IL-4 and anti-IL-12 mAb to promote
the development of Th1 or Th2 cells, respectively. As shown in Figure 2, the cell lines obtained exhibit a
clear Th1 or Th2 profile, as detected by intracellular staining for
IFN- and IL-4 production. In both cell lines, >80% of total cells
express the transgenic TCR, as determined by staining with the
clonotype-specific mAb KJ1-26 (not shown).
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To establish optimal in vitro conditions that enable microglia and
astrocytes to present Ag, cultures were treated for 24 to 48 h
with various agents (IFN-, LPS, TNF-, IL-1ß) that induce
expression of MHC class II and adhesion/costimulatory molecules such as
CD54 (ICAM-1), CD40, CD80 (B7-1), or CD86 (B7-2). Expression of these
molecules was evaluated by flow cytometric analysis, as shown in Figure 3.
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(100 U/ml) induces expression of MHC
class II I-A and I-E molecules, and enhances ICAM-1 expression, but has
no effect on B7-1 or B7-2. The anti-B7-1 and anti-B7-2 mAbs
used in this study stain the immortalized microglial cell line BV-2
(not shown), as previously reported (48). IFN- also up-regulates
CD40 expression on microglia. In astrocyte cultures, TNF- (10, 50
ng/ml) increases ICAM-1 expression and potentiates the inducing effect
of IFN- on MHC class II and ICAM-1. IL-1ß (100 U/ml) also enhances
ICAM-1 on astrocytes, but does not affect IFN--induced MHC class II
expression (not shown). None of the surface molecules examined is
induced on microglia by TNF- or IL-1ß (not shown). As expected,
astrocytes do not respond to LPS (0.1, 1 µg/ml) (not shown), whereas
LPS-treated microglia show enhanced expression of CD40 and ICAM-1, but
not of MHC class II, B7-1, or B7-2 molecules. As previously reported
(48), B7-2 is induced on mouse microglia, but not on astrocytes, by
simultaneous treatment with IFN- and LPS. This combined treatment
also enhances the microglial expression of ICAM-1 and CD40, but not of
MHC class II and B7-1. Neither CD40 nor B7 molecules are induced on
astrocytes upon 72 h of treatment with IFN-, TNF-, IL-1ß,
IFN-/TNF-, or IFN-/IL-1ß (not shown). Microglia present more efficiently than astrocytes the OVA peptide 323-339 to Th1 but not Th2 cells
We next tested microglia and astrocytes for their ability to
present OVA 323-339 and to induce cytokine secretion by Th1 and Th2
cells, either constitutively or after appropriate stimulation, as
determined above. Polarized CD4+ TCR transgenic cells
were cultured with microglia or astrocytes in the presence of OVA
323-339. After 24 h, IL-2, IFN-, and IL-4 were quantified in
culture supernatants by specific ELISA. In preliminary experiments,
optimal T cell cytokine secretion was induced by 0.3 µM OVA 323-339
(not shown), and this dose was used in the following experiments.
As shown in Figure 4, in the presence of
0.3 µM OVA 323-339, high numbers (
3 x 104/well)
of untreated microglia induce production of IL-2 and IFN- by Th1
cells but little or no IL-4 by Th2 cells. Although I-Ad
molecules are undetectable on unstimulated microglia by flow cytometry
(see Fig. 3
), in high cell density cultures low amounts of
I-Ad or other adhesion/costimulatory molecules could be
sufficient to drive Th1 activation. During the 24-h coculture period,
microglial I-Ad expression could also be stimulated by
IFN-, which is produced by Th1 but not Th2 cells. IFN--treated
microglia, which are MHC class II+, CD40+, and
strongly ICAM-1+, are much more potent than unstimulated
microglia in presenting OVA 323-339 and efficiently activate both T
cell subsets. At any cell density tested, IFN--treated microglia
induce the secretion of large amounts of IL-2 and IFN- by Th1 cells
and of IL-4 by Th2 cells. Stimulation of Th1 and Th2 cytokine
production is dose dependent, maximal stimulation being obtained with
3 x 104 cells/well. However, at the highest cell
density (1 x 105), plateau or even reduced values of
cytokines are sometimes observed. In the presence of
IFN-/LPS-treated microglia, which express high levels of B7-2, CD40,
and ICAM-1 but are MHC class II negative, T cell cytokine secretion is
similar to or lower than that elicited by unstimulated microglia.
Cytokine secretion also is not induced by LPS-treated microglia (not
shown). IFN-/TNF--treated microglia are as efficient as
IFN--treated microglia in inducing cytokine secretion by Th1 and Th2
cells, whereas TNF--treated microglia do not induce T cell
activation at levels higher than those of unstimulated microglia (not
shown). When activated by microglia, Th1 cells do not secrete IL-4, and
Th2 cells secrete no IFN- and little or no IL-2 (Fig. 4), confirming
the polarization of the TCR transgenic T cells used. No cytokine
production has ever been detected in the absence of OVA peptide or by
microglia and T cells cultured alone (not shown).
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).
Astrocytes pretreated with IFN- present OVA peptide and stimulate
secretion of IL-2 and IFN- by Th1 cells as well as of IL-4 by Th2
cells. IFN-/TNF--treated astrocytes are as effective as
IFN--treated astrocytes in stimulating cytokine secretion by either
T cell subset, even if the combined cytokine treatment induces higher
expression of MHC class II and ICAM-1 molecules (see Fig. 3). The
stimulating effect of cytokine-activated astrocytes is dose dependent,
being maximal at 3 x 104 cells/well. As expected,
TNF--, IL-1ß-, or LPS-treated astrocytes, which are MHC class II
negative, are as effective as unstimulated astrocytes. Treatment of
astrocytes with IFN- plus LPS or with IFN- plus IL-1ß induces T
cell activation similar to that induced by IFN- treatment (not
shown). As already observed with microglia, Th1 and Th2 cells secrete
no IL-4 and no IFN- or very little IL-2, respectively, when
activated by astrocytes. No cytokine secretion is observed in the
absence of OVA peptide or of T cells (not shown).
When T cell activation by microglia and astrocytes is compared, it is
evident that IFN--treated microglia are more efficient than
IFN--treated astrocytes in restimulating Th1 cells. The differences
between Th1 cytokine production induced by activated microglia and
astrocytes are statistically significant at all APC numbers tested
(p < 0.001). It is noteworthy that secretion
of IL-2 and IFN- induced by 1 x 105 astrocytes is
similar to that induced by 3 x 103 microglia,
indicating that microglia are about 30-fold more efficient than
astrocytes in stimulating Th1 cytokine secretion. In contrast, the two
APC restimulate Th2 cells with a similar efficiency, although in some
experiments, at the lowest cell densities tested (<1 x
104/well), microglia induce slightly higher IL-4 secretion
than astrocytes.
Involvement of MHC class II and adhesion/costimulatory molecules in
the restimulation of Th1 and Th2 cells by IFN--activated
microglia and astrocytes
To assess the contribution of MHC class II and
adhesion/costimulatory molecules in the restimulation of cytokine
secretion by Th1 and Th2 cells, IFN--stimulated microglia or
astrocytes (3 x 104/well) were cultured with OVA
323-339 and T cells in the presence of specific neutralizing Abs (10
µg/ml) or the same amount of their corresponding isotype controls. As
shown in Figure 5,
anti-I-Ab,d, but not anti-I-E mAb, inhibits almost
completely the capacity of IFN--stimulated microglia and astrocytes
to induce IFN- and IL-4 production by I-Ad-restricted,
TCR transgenic Th1 and Th2 cells, respectively. In contrast,
neutralizing mAbs against B7-1, B7-2, and CD28, the counterreceptor for
B7 molecules on T cells, have no effect on the capacity of microglia
and astrocytes to induce cytokine secretion from either T cell subset.
Both B7-1 and B7-2 mAbs inhibit the proliferation of TCR transgenic
naive T cells stimulated by BALB/c dendritic cells (F. Ria and F.
Aloisi, unpublished observations). These results are consistent with
the lack of expression of B7-1 and B7-2 molecules on IFN--activated
BALB/c mouse microglia and astrocytes, as detected by flow cytometry
(Fig. 3). Despite high expression of ICAM-1 on both microglia and
astrocytes, anti-ICAM-1 mAb does not affect Th1 and Th2 cytokine
secretion induced by either cell type. A modest (about 35%), but
statistically significant reduction of IFN- secretion by Th1 cells,
but not of IL-4 by Th2 cells, is observed when Th1 cells are activated
by microglia in the presence of a mAb against CD40L, the
counterreceptor for CD40 on T cells. This is consistent with the
expression of CD40 on IFN--stimulated microglia (Fig. 3). Anti-CD40L
mAb has no effect on Th1 or Th2 activation induced by IFN--treated
astrocytes. The above results indicate that the presence of MHC class
II on CNS APC is critical for the stimulation of differentiated Th1 and
Th2 cells, whereas CD40-CD40L interactions may play a role in the
restimulation of Th1 cells by microglia.
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Having established that microglia and astrocytes are able to
present OVA 323-339 to Th1 and Th2 cells and that IFN- is the best
stimulus for these APC to induce cytokine secretion from both T cell
subsets, we next assessed their relative capacity to process
Ag.
First, we analyzed the proliferation of TCR transgenic Th1 and Th2
cells in response to native OVA presented by CNS APC or by spleen
cells. In the presence of 3 µM native OVA, no T cell proliferation is
induced by unstimulated microglia or astrocytes (Fig. 6). IFN--treated microglia stimulate
the proliferation of Th1 and Th2 cells at levels similar to those
induced by 30- to 100-fold higher numbers of spleen cells. In contrast,
astrocytes present native OVA very inefficiently to stimulate
proliferation of either Th1 or Th2 cells. In some experiments, little
proliferation of Th1 cells is only observed in response to OVA
presented by 3 x 104 astrocytes. The decreased
proliferative T cell responses with increasing microglia density could
be due to noxious mediators released by activated microglia and/or
could result from activation-induced T cell death (30, 61).
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). Also, astrocytes become able to
stimulate T cell proliferation in the presence of OVA 323–339 peptide.
Optimal proliferative responses of Th1 and Th2 cells are induced by
3 x 104 IFN--treated astrocytes and are similar to
or higher than those induced by microglia. However, at lower cell
densities (<1 x 104), astrocytes are less efficient
than microglia in stimulating proliferation of both Th1 and Th2 cells.
The proliferative response is abrogated by addition of
anti-I-Ad mAb to cultures, and no T cell proliferation
occurs in the absence of added Ag or APC (not shown). In conclusion,
both microglia and astrocytes can present OVA peptide and induce
proliferation of Th1 and Th2 cells, whereas only microglia are able to
process and present native OVA leading to activation of both T
cell subsets.
When examining T cell cytokine secretion, IFN--treated microglia are
the most efficient APC in processing OVA, leading to activation of both
Th1 and Th2 cells (Fig. 7). Compared with
spleen cells, 30- to 300-fold less microglia are required to induce the
same level of IL-2, IFN-, or IL-4 production. Consistent with data
for proliferative assays, IFN--treated astrocytes process OVA very
inefficiently because a very modest production of IFN-, but not of
IL-2, by Th1 cells and of IL-4 by Th2 cells is observed only at the
highest cell density tested (1 x 105 cells). In the
presence of OVA 323–339 (0.3 µM), unstimulated microglia and spleen
cells induce similar Th1 activation, whereas IFN--treated microglia
are again the most efficient APC, being 10- to 100-fold more potent
than astrocytes and spleen cells in inducing IL-2 and IFN-
production (Fig. 7). In agreement with the results reported in Figure 4, the differences between Th1 cytokine production induced by activated
microglia and astrocytes are statistically significant at all APC doses
tested (p < 0.001). Conversely,
IFN--treated microglia and astrocytes are equally able to present
OVA 323–339 to Th2 cells and induce levels of IL-4 secretion that are
similar to those induced by 10- to 30-fold higher numbers of spleen
cells.
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-treated microglia in the presence of as low as 0.03 µM native
OVA and increases with protein doses up to 30 µM. In contrast, 3
x 104 IFN--treated astrocytes induce some cytokine
secretion from Th1 cells only at OVA concentrations equal to or higher
than 10 µM (Fig. 8). Even after a
longer incubation (48 instead of 24 h) of T cells with astrocytes
and native OVA, cytokine production remained very low (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that rodent and human microglia activated
by IFN-, LPS, or phagocytosis can process and present Ag to unprimed
and primed T cells, leading to their proliferation (23, 24, 28, 29). In
a recent ex vivo study, Ford et al. (30) showed that microglia from the
adult rat brain stimulate production of IFN- and TNF- by
long-term Ag-specific T cell lines but fail to induce either IL-2
production or T cell proliferation. Our results provide further
insights into microglial APC function by showing that microglia can
effectively restimulate both Th1 and Th2 cells. Microglia efficiently
present synthetic OVA peptide as well as peptide derived from
processing of native OVA to both T cell subsets, leading to T cell
proliferation and to secretion of IL-2 and IFN- by Th1 cells and of
IL-4 by Th2 cells. In our culture conditions, unstimulated microglia
constitutively express ICAM-1, but little or undetectable MHC class II,
B7, or CD40, and are able to induce some degree of Th1 and little Th2
cytokine secretion, suggesting that low levels of MHC class II or other
costimulatory signals are sufficient to restimulate T cell effector
functions. Pretreatment of microglia with IFN-, which up-regulates
MHC class II, ICAM-1, and CD40 but neither B7-1 nor B7-2, greatly
enhances the capacity of this cell type to stimulate Th1 and Th2
cytokine production and to induce proliferation of both T cell subsets.
Interestingly, optimal T cell proliferation is induced by low microglia
numbers that induce little or no T cell cytokine secretion, whereas at
higher cell densities (>3 x 103) microglia do
not support T cell proliferation. This suggests that products of
activated microglia may negatively affect T cell survival/proliferation
or that T cells undergo apoptosis in response to Ag presentation by
microglia, as shown in two recent reports (30, 62). We are currently
evaluating these two possibilities. These findings also suggest that in
vivo restimulation of Th1 and Th2 cells by microglia may result in
either T cell death or in further expansion of the differentiated T
cell subsets depending on the number of activated microglia and on the
strength of the T cell-activating stimuli.
Optimal Th1 proliferation and cytokine production usually require
costimulatory signals (e.g., B7 and IL-12) that synergize with each
other (16). In our experimental system, MHC class II expression by
microglia is critical to elicit T cell cytokine secretion, whereas
adhesion/costimulatory molecules, such as ICAM-1, B7-1, and B7-2 do not
appear to be involved. Blockade of CD40L on Th1 cells partially reduces
IFN- production, suggesting that CD40-CD40L interactions contribute
to Th1 activation by microglia. Similarly to what was observed in
macrophages and dendritic cells (63, 64), ligation of CD40 on microglia
may induce IL-12 secretion, which in turn promotes cytokine production
by Th1 cells (16, 65). This possibility is currently under
investigation. The observation that IFN-/LPS-stimulated microglia
(that express high levels of ICAM-1, CD40, and B7-2 but are MHC class
II negative) are unable to induce T cell activation further supports a
major role for MHC class II, rather than adhesion/costimulatory
molecules, in the activation of differentiated Th1 and Th2 cells.
However, it is possible that IFN-/LPS-activated microglia secrete
products that have adverse effects on T cells and may therefore inhibit
the positive effects of other costimulatory signals.
Astrocytes are also able to restimulate Th1 and Th2 cells and,
similarly to microglia, IFN- is the most effective stimulus that
enables astrocytes to induce T cell proliferation and cytokine
secretion. Compared with microglia, IFN--activated astrocytes are
less powerful activators of Th1 responses, but are similarly effective
in presenting antigenic peptide to Th2 cells leading to IL-4 secretion.
The present study also shows that astrocytes have a much lower capacity
to process Ag than microglia, as they stimulate some Th1 and Th2
cytokine production only in the presence of very high doses of native
OVA. Inefficient Ag processing by astrocytes is confirmed by
proliferation studies. Although high numbers of astrocytes (1 x
104) induce higher T cell proliferation than microglia to
OVA 323–339, at lower cell densities microglia are clearly more
efficient than astrocytes in stimulating the proliferation of both Th1
and Th2 cells. The present data are in agreement with the work of
Matsumoto, Ohmori, and Fujiwara (29), who reported that microglia, but
not astrocytes, are able to present native myelin basic protein and to
stimulate T cell proliferation. They are, however, in contrast with
several previous reports showing that astrocytes are able to process
and present protein Ag (including OVA) to T cell lines, leading to
their proliferation (41, 46, 47, 62). A likely explanation for these
conflicting data is the degree of contamination of astrocyte cultures
by microglia. In fact, as shown herein, even very small amounts of
microglia (1–3 x 102 cells/well) are sufficient to
trigger T cell proliferation. The presence of peptides in the protein
preparations used as Ag in previous experiments should also be
considered.
As for microglia, astrocyte-induced cytokine secretion by TCR
transgenic Th1 and Th2 cells is totally dependent on MHC class II
expression and does not appear to involve ICAM-1, which is highly
expressed on these cells. We also show that astrocytes cannot be
induced to express B7-1 (CD80) or B7-2 (CD86), as already reported (25, 33, 48), or CD40, and that these molecules do not contribute to either
Th1 or Th2 activation by astrocytes. In contrast, Nikcevich et al. (47)
have recently reported that B7-1 and B7-2 are induced by IFN- on
mouse astrocytes and are involved in the activation of naive T cells as
well as differentiated Th1 cells from the same TCR transgenic mice used
in the present study. Neither mouse strain differences nor the type of
inducing stimuli can explain these conflicting results, which could be
mainly due to contaminating microglia in astrocyte cultures.
The present report is the first one clearly establishing differences
between microglia and astrocytes in their Ag-processing capacity and in
their efficiency to restimulate distinct T cell subsets. It is likely
that the phagocytic properties of microglia, which are functionally
similar to macrophages, contribute to the more efficient Ag processing.
The higher efficiency of microglia in restimulating Th1 cytokine
secretion compared with astrocytes is consistent with the finding that
microglia, but not astrocytes, secrete IL-12 (26, 27).
IFN--activated microglia are also more potent than spleen cells in
Ag processing and in the restimulation of Th1 and Th2 cytokine
production. To further define the relative position of astrocytes and
microglia in a hierarchy of APC, future studies will compare the
function of brain and lymphoid (dendritic cells, B cells) APC.
The present data suggest that microglia, when activated in vivo by
IFN- and, possibly, by other stimuli that enhance MHC class II
expression (e.g., factors released during neuronal damage) (21, 22),
may act as powerful APC for the restimulation of peripherally activated
Th1 and Th2 cells that have entered the CNS as a consequence of a viral
infection, or in inflammatory CNS diseases such as MS (5, 6). In MS,
MHC class II, ICAM-1, B7, and CD40 molecules are expressed on activated
microglia present within or around white matter inflammatory lesions
(25, 31, 32, 33), suggesting that these cells can effectively present Ag to
infiltrating T lymphocytes. Th1 responses are thought to be involved in
the generation of MS lesions (5, 15), although both Th1 and Th2
cytokines have been detected in the brain parenchyma and cerebrospinal
fluid at different stages of the disease, indicating disregulation of
both cellular and humoral immune responses (31, 66). Since Th2
responses could inhibit inflammation and limit the noxious effects of
Th1-mediated immunity (7, 9), the capacity of microglia to activate
both T cell subsets may contribute to the pattern of recurrent cerebral
inflammation characteristic of MS (67).
Similarly to other nonprofessional APC (e.g., epidermal keratinocytes,
endothelial cells) (68, 69), cultured astrocytes appear to be very
efficient in Th2 restimulation and are less powerful stimulators of Th1
responses. Despite efficient T cell stimulation demonstrated in vitro,
astrocytes express little or no MHC class II molecules in most CNS
pathologic conditions (38, 70, 71), suggesting that in vivo Ag
presentation by astrocytes may be the exception rather than the rule.
However, intrathecal injection of IFN- in rats induces a delayed
expression of MHC class II molecules on astrocytes (39) and MHC class
II molecules are present on reactive astrocytes in MS lesions (35, 36).
The low efficiency of astrocytes in processing native protein does not
exclude the possibility that astrocytes present Ag at sites of brain
inflammation. In MS lesions, the degradation of myelin by proteolytic
enzymes may lead to the generation of peptide fragments that could be
presented without further processing. Thus, in some circumstances
(e.g., during chronic inflammation), astrocytes could present Ag,
inducing mainly restimulation of Th2 responses. Previous studies have
indicated a role for astrocytes in down-regulating T cell responses
through soluble factors that inhibit Ag presentation by other APC (44, 45) and IL-12 production by microglia (27). This inhibitory activity
and the capacity of astrocytes to activate Th2 responses may represent
important homeostatic mechanisms during recovery from Th1-mediated
inflammation.
In conclusion, our data demonstrate that microglia can act as very efficient APC in the CNS by restimulating both Th1 and Th2 responses. Conversely, astrocytes preferentially restimulate Th2 cells. All together, these data suggest that microglia and astrocytes may play distinct roles in the regulation of immune responses against pathogens and in immune disregulation leading to autoimmune attack of CNS-related components.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francesca Aloisi, Neurophysiology Unit, Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
3 Abbreviations used in this paper: CNS, central nervous system; MS, multiple sclerosis; GFAP, glial fibrillary acidic protein; PE, phycoerythrin.
Received for publication August 4, 1997. Accepted for publication January 9, 1998.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
mRNA in human inflammatory disease. J. Immunol. 147:810.[Abstract]
production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.-treated microglial cells. Eur. J. Immunol. 17:1271.[Medline]
-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells. J. Immunol. 158:614.[Abstract]
and lipopolysaccharide and downregulation by interleukin-10, prostaglandin E2 and cyclic AMP-elevating agents. J. Neuroimmunol. 72:83.[Medline]
interferon. Proc. Natl. Acad. Sci. USA 81:4515.This article has been cited by other articles:
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|
 |
|
  C. van der Putten, E. A. Zuiderwijk-Sick, L. van Straalen, E. D. de Geus, L. A. Boven, I. Kondova, A. P. IJzerman, and J. J. Bajramovic Differential Expression of Adenosine A3 Receptors Controls Adenosine A2A Receptor-Mediated Inhibition of TLR Responses in Microglia J. Immunol., June 15, 2009; 182(12): 7603 - 7612. [Abstract] [Full Text] [PDF]
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|
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|
|
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|
|
|
|
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|
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|
|
|
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 |
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|
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|
|
|
|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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