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Are Required for IL-12 But Not Prostaglandin E2 Secretion by Microglia During Antigen Presentation to Th1 Cells1
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Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanità, 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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secretion by
activated Th1 cells. Th1 and, to a lesser extent, Th2 cells also
stimulate the production of PGE2 by microglia. T
cell-mediated induction of PGE2 requires MHC class
II/peptide/TCR interactions but does not depend on CD40 engagement or
on the presence of IFN-. Astrocytes, which preferentially activate
Th2 cells, fail to produce IL-12 and secrete negligible amounts of
PGE2 upon interaction with either Th1 or Th2 cells. These
results suggest that during CNS infection or immunopathology, IL-12
produced by microglia upon Ag-specific interaction with Th1 cells may
further skew the immune response to Th1, whereas the T cell-dependent
production of PGE2 by microglia may represent a negative
feedback mechanism, limiting the propagation of Th1
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK and T
cells (1). Recent studies indicate that IL-12 is important for the induction of protective immune responses against neurotropic viruses (2) and may play a role in virus-induced central nervous system (CNS)3 immunopathology (3). IL-12 has been implicated in the pathogenesis of multiple sclerosis (MS), a putative autoimmune CNS disease, and of experimental allergic encephalomyelitis (EAE), a Th1-mediated animal model of MS (4). The demonstration of IL-12 mRNA-expressing cells in the CNS of animals with EAE (5) and in active MS lesions (6), where the presence of Th1-type cytokines has been documented, suggests that intracerebral production of IL-12 is an important event in the development of Th1 responses contributing to CNS immunopathology. This concept is further supported by the finding that anti-IL-12 treatment inhibits EAE (7), while administration of IL-12 exacerbates the disease (7, 8).
The identification of the cells producing IL-12 and of the signals
regulating IL-12 production within the CNS is important to the
understanding of how T cell responses are regulated during
immune-mediated CNS inflammation. Microglia, the resident brain
macrophages, are believed to function as the principal APC of the CNS
parenchyma (9). Upon CNS inflammation, microglia up-regulate MHC class
II and adhesion/costimulatory molecules (9, 10, 11, 12) and secrete a number of
immunoregulatory mediators, including IL-12. IL-12 p40 mRNA has been
detected in microglia isolated from the CNS of mice with EAE (13) and
in microglia in situ after i.p. injection of LPS (14, 15). Cultured
microglia secrete bioactive IL-12 p75 after combined stimulation with
IFN- and bacterial products (16, 17). Microglia, which process and
present Ag (18, 19, 20, 21) and secrete IL-12, could thus favor the development
of Th1 responses within the CNS. In agreement with this concept, in
vitro activated microglia efficiently restimulate Th1 cells to secrete
cytokines (22). Conversely, astrocytes, the major glial cell population
of the CNS, behave as nonprofessional APC and, despite controversial
results (14, 23), fail to produce IL-12 in response to proinflammatory
stimuli (16, 17), providing an explanation for their capacity to
restimulate preferentially Th2 responses (22).
Another mediator secreted by cultured microglia and astrocytes in response to inflammatory stimuli, such as cytokines or LPS, is PGE2, a product of arachidonic acid metabolism (24, 25, 26). Recent studies indicate that PGE2 exerts important anti-inflammatory and immunoregulatory activities on Th1 responses. PGE2 inhibits cytokine production by Th1 cells (27, 28) and primes naive T cells for production of Th2-type cytokines (IL-4, IL-5, IL-10, and IL-13) (29, 30). In addition, PGE2 can inhibit Th1 responses via its ability to down-regulate IL-12 production by APC (31), including microglia (17), and IL-12R expression (32). PGE2 has been detected in the CNS parenchyma during the recovery phase of EAE (33), and a stable PGE2 analogue inhibits EAE development (34), suggesting an anti-inflammatory role for PGE2 in the CNS. The cellular sources of PGE2 and the signals inducing PGE2 in the inflamed CNS, however, remain to be determined.
In this study we have addressed the question of whether T cells, which
may be recruited into the CNS after peripheral sensitization to viral
or self Ags, could stimulate CNS APC to produce mediators with a role
in the local regulation of T cell responses. We have used purified
populations of microglia and astrocytes from newborn BALB/c mice and
OVA-specific, I-Ad-restricted Th1 and Th2 cells from TCR
transgenic BALB/c mice as a model to investigate the cellular and
molecular requirements for IL-12 and PGE2 production by
brain APC upon Ag-specific interaction with Th1 and Th2 cells. Our
results indicate that Th1, but not Th2 cells can induce, via IFN-
secretion and cognate interactions, the production of IL-12 by
microglia, thereby favoring the development of Th1 responses. In
addition, Th1 and to a lesser extent Th2 cells can stimulate
PGE2 production by microglia providing a potentially
relevant feedback mechanism for down-regulation of Th1 responses within
the CNS.
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Materials and Methods |
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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 (35)
were provided by Dr. D. Y. Loh, Hoffmann-La Roche (Nutley, NJ). In
these transgenic mice, >95% of the CD4+ T cells are
Vß8.1.2+ (36) and express a TCR-
ß specific for the
OVA323–339 peptide 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 (17). 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 pore size 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 medium described 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 20–30 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 described above and reseeded on poly-L-lysine-coated plastic surfaces. Flow cytometric analysis revealed that microglia and astrocyte cultures contained 92–95% Mac-1+ cells and 95–98% glial fibrillary acidic protein+ cells, respectively (22).
Spleen cells were prepared from normal BALB/c mice, treated with mitomycin C, 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).
In some experiments, microglia (2 x 104 cells/well in 96-well plates) were seeded on plates precoated by overnight incubation at 4°C with 5 µg/ml 14-4-4S anti-IE (mouse IgG, American Type Culture Collection, Manassas, VA), B21.22 anti-I-Ab,d (rat IgG2b, American Type Culture Collection), HM40-3 anti-CD40 (hamster IgM, PharMingen, San Diego, CA), 3/23 anti-CD40 (rat IgG2a, PharMingen), isotype controls (mouse IgG, rat IgG2a, rat IgG2b, and hamster IgM from PharMingen), TSST-1 (Toxin Technologies, Madison, WI), staphylococcal enterotoxin A (SEA), or SEB (Sigma).
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, Bergish, Germany). CD4+ T cells (2 x 105 cells/well) were cultured with OVA323–339 (0.3 µM), synthesized as previously described (37), 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 mouse rIL-12 (Hoffmann-La Roche) and 10 µg/ml anti-mouse IL-4 mAb (11B11, American Type Culture Collection) or 20 ng/ml mouse rIL-4 (Hoffmann-La Roche, 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 of culture, T cells were expanded in complete medium containing 10 ng/ml of human rIL-2 (Hoffmann-La Roche).
Ag presentation
Microglia and astrocytes were seeded at cell densities ranging
from 1 x 103 to 3 x 104 cells/well
in 96-well flat-bottom tissue culture plates. After 16 h, the
culture medium was replaced with fresh medium (DMEM/10% FCS) with or
without mouse rIFN- (100 U/ml; sp. act., 1 x 107
U/mg; Genzyme, Cambridge, MA), and cells were incubated at 37°C for
an additional 24–48 h. Immediately before the addition of T cells from
TCR transgenic mice, astrocytes and microglia were washed three times
with DMEM/10% FCS to completely remove the stimulating agent. Spleen
cells were seeded at densities ranging from 3 x 104
to 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
OVA323–339 or native OVA (Sigma). At the indicated times,
supernatants were collected, centrifuged at 1200 rpm, and stored at
-20°C until used for cytokine or PGE2 determination. In
some experiments the following mAbs (10 µg/ml) were added to cultures
30 min before addition of T cells: B21.22, anti-I-Ab,d
(rat IgG2b, American Type Culture Collection); 14-4-4S,
anti-I-E (mouse IgG, PharMingen); 3E2, anti-CD54
(ICAM-1; hamster IgG, PharMingen); 1G10, anti-CD80 (B7-1; rat
IgG2a, PharMingen); GL1, anti-CD86 (B7-2; rat IgG2a, PharMingen);
37.51, anti-CD28 (hamster IgG, PharMingen); MR1, anti-CD154
(CD40 ligand, gp39; hamster IgG, PharMingen); anti-IFN- (hamster
IgG, Genzyme); XMG1.2, anti-IFN- (rat IgG1, PharMingen);
anti-IL-10 (rat IgG1, Genzyme); and isotype controls (rat IgG2a,
rat IgG2b, and rat IgG1, from PharMingen).
Cytokine determination
IFN-, IL-2, IL-4, IL-12 p75, and IL-12 p40 were quantified by
two-site sandwich ELISA, as previously described (17, 22, 38).
Cytokines were quantified from three or four titration points using
standard curves generated by purified recombinant mouse cytokines
(Hoffmann-La Roche), and results are expressed as the cytokine
concentration in picograms or nanograms per milliliter. Detection
limits for all cytokines were in the range of 7–15 pg/ml. In some
experiments, IL-12 p75 and p40 were quantified using commercially
available ELISA (Genzyme), with detection limits of 5 and 10 pg/ml,
respectively. IL-10 was quantified using an ELISA from Genzyme with a
detection limit of 15 pg/ml.
PGE2 determination
PGE2 secreted into the culture medium was measured using a specific RIA, as previously described (24). The amount of PGE2 present in medium containing 10% FCS was measured and subtracted from the value obtained for each sample. The detection limit was 25 pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 when
stimulated with IFN--preatreated microglia and spleen cells, but no
IL-4 or IL-10, whereas IFN--pretreated astrocytes are less efficient
in inducing Th1-type cytokine secretion (Fig. 1
). Pretreatment of microglia and
astrocytes with IFN- is necessary to induce MHC class II expression
and to stimulate the APC function of these cells. IFN--pretreated
microglia and astrocytes are similarly efficient in stimulating Th2
cells to secrete high levels of IL-4, as previously reported (22), and
IL-10, low IL-2, and no IFN-. The amounts of Th1 and Th2 cytokines
induced by CNS APC are similar to or higher than those induced by
spleen cells. These data confirm the polarization of the T cell lines
used and the capacity of microglia and astrocytes to function as APC
for the restimulation of CD4+ Th cells.
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We next examined IL-12 production by microglia, astrocytes, and
spleen cells during Ag presentation to Th1 or Th2 cells. In the
presence of OVA323–339 (0.3 µM), little or no IL-12 p75
and p40 are secreted in cultures containing Th1 or Th2 cells and
unstimulated microglia, as determined by specific ELISA (Fig. 2). Substantial amounts of IL-12 p75 and
p40 are produced in cultures containing IFN--pretreated microglia
and Th1, but not Th2 cells. As observed after stimulation with IFN-
and LPS (17), microglia secrete more IL-12 p40 than IL-12 p75. Maximal
IL-12 production is obtained with 104 microglia/well. At
this cell density, IL-12 p75 secretion by microglia is similar to that
induced during Ag-dependent interaction between 
3 x
105 spleen cells and Th1 cells (Fig. 2). However, microglia
secrete more IL-12 p40 than spleen cells. Th1-driven IL-12 p75 and p40
production by microglia is dependent on the concentration of Ag in
culture, since larger amounts of IL-12 p75 and p40 are secreted with
increasing concentrations of OVA323–339 or native OVA
(Fig. 3). This is consistent with
previous findings that higher Ag concentrations result in greater
cytokine secretion by microglia-activated Th1 cells (22). No IL-12 is
produced by IFN--pretreated microglia in the presence of either T
cells or Ag alone (not shown). Together, these findings indicate that
IL-12 production requires Ag-specific interaction between activated
microglia and Th1 cells. Kinetic analysis of IL-12 p75 secretion in Th1
cell-microglia cultures revealed that the largest increment in IL-12
p75 occurs between 4–8 h, reaching about 70% of the peak values
observed after 24 h (Fig. 4).
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), astrocytes fail to produce IL-12 p75 and p40 upon interaction with
either T cell subset (Fig. 2).
Cell contact via MHC class II/peptide-TCR and CD40-CD154
interactions and secretion of IFN- by Th1 cells are required for
Th1-induced IL-12 secretion by microglia
We next examined the molecular requirements for the induction of
IL-12 production by microglia during Ag presentation to OVA-specific,
I-Ad-restricted TCR transgenic Th1 cells. We have
previously shown that IFN--pretreated BALB/c microglia express
I-Ad, I-E, CD40, and CD54, but fail to express CD80 and
CD86 molecules (22). To assess the involvement of MHC class II and
adhesion/costimulatory molecules, IL-12 p75 production in microglia-Th1
cell cultures was examined in the presence of specific neutralizing
Abs.
Anti-I-Ad, but neither anti-I-E nor isotype control,
mAb completely inhibits the secretion of IL-12 p75 (Fig. 5) and IL-12 p40 (not shown) from
IFN--pretreated microglia cultured with OVA323–339 and
Th1 cells, indicating that the interaction between MHC class II/peptide
complexes on microglia and the specific TCR on T cells is critical for
IL-12 induction (Fig. 5). To further investigate this issue, we studied
whether cross-linking of MHC class II expressed on IFN--treated
microglia would induce IL-12 secretion. Microglia (2 x
104 cells/well) incubated for 48 h on plates coated
with anti-MHC class II (either anti-I-Ab,d or I-E)
mAbs, TSST-1, SEA, or SEB (5 µg/ml) in the presence of IFN- (100
U/ml) fail to secrete either IL-12 p75 or IL-12 p40 (results not shown
from four independent experiments). These data indicate that MHC class
II ligation does not directly signal IL-12 production in microglia;
rather, peptide/MHC-TCR interactions may promote T cell activation and
up-regulate IL-12-inducing molecules.
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) and IL-12 p40 (not shown) secretion. This latter finding is
consistent with previous data demonstrating that ligation of CD40 on
macrophages and dendritic cells plays a major role in IL-12 induction
(39, 40). To directly test the effect of CD40 ligation on IL-12
production, microglia (2 x 104 cells/well in 100 µl
of culture medium) were incubated on plates precoated with stimulatory
anti-CD40 mAbs (3/23 or HM40-3; 5 µg/ml) in the presence of
IFN- (100 U/ml) or were pretreated with IFN- for 24 h and
then exposed to soluble anti-CD40 mAb for 48 h. Both
plate-bound and soluble anti-CD40 mAbs stimulate the secretion of
IL-12 p40 (2.0 ± 1.2 and 3.0 ± 0.8 ng/ml, respectively;
mean ± SEM of five independent experiments performed in
duplicate), but not of IL-12 p75. Unstimulated microglia or microglia
exposed to isotype controls fail to secrete either IL-12 p75 or p40.
Thus, although signaling through CD40 may induce IL-12 p40, interaction
with Th1 cells is required for secretion of bioactive IL-12 p75
by microglia.
Neutralizing mAbs against CD54, CD80, CD86, and CD28, the
counter-receptor for CD80/CD86 molecules on T cells, have no effect on
the capacity of microglia to secrete IL-12 p75 during Ag-specific
interaction with Th1 cells (Fig. 5).
IFN- is required for IL-12 p75 production by microglia in response
to LPS or Staphylococcus aureus (16, 17). We therefore asked
whether IFN- secreted by Ag-activated Th1 cells (22) (Fig. 1) may
also be necessary for T cell-dependent IL-12 induction in microglia. As
shown in Fig. 5, addition of a neutralizing mAb against mouse IFN-
to microglia-Th1 cell cultures inhibits IL-12 p75 secretion by about
50%, indicating that Th1-derived IFN- cooperates with
contact-dependent signals to induce IL-12 in microglia. Similar results
were obtained when IFN- was neutralized with a hamster
anti-IFN- mAb (Fig. 5) or a rat anti-IFN- mAb (from
PharMingen; data not shown). The inability of anti-IFN- mAbs to
completely suppress IL-12 p75 production may be due to the pretreatment
of microglia with IFN-, which could already prime these cells for
IL-12 production. We have recently reported that microglia primed with
IFN- and then treated with LPS secrete less IL-12 p75 than microglia
treated simultaneously with IFN- and LPS (17). Thus, in both T
cell-dependent and T cell-independent responses, optimal IL-12
production by microglia appears to require the simultaneous presence of
IFN- and additional activating stimuli.
Th1 are superior to Th2 cells in stimulating PGE2 production by microglia
The prostanoid PGE2, which has been implicated in the
regulation of Th1 and Th2 responses, is produced by microglia and
astrocytes in response to cytokines or LPS (24, 25, 26). We therefore asked
whether microglia and astrocytes would produce PGE2 upon
Ag-dependent T cell stimulation. As shown in Fig. 6, interaction of unstimulated microglia
with Th1 or Th2 cells in the presence of OVA323–339 leads
to little or no secretion of PGE2, respectively. When
IFN--pretreated microglia are cultured with Th1 cells, substantial
amounts of PGE2 are secreted in the culture supernatants.
PGE2 production correlates with the number of microglia,
and it is maximal at 3 x 104 cells/well. The amount
of PGE2 secreted during Ag-specific interaction between
IFN--pretreated microglia and Th2 cells is about one-third of that
induced in the presence of Th1 cells (Fig. 6), suggesting either that
Th1 cells provide stronger signals for PGE2 production or
that products of activated Th2 cells inhibit PGE2
secretion. T cell-dependent PGE2 secretion by microglia is
optimally induced in the presence of 0.1–1 µM
OVA323–339 or 1–10 µM native OVA (Fig. 7). No PGE2 is secreted by
unstimulated or IFN--pretreated microglia in the presence of either
T cells or Ag alone (not shown), indicating that, as already shown for
IL-12, PGE2 induction involves Ag-specific interactions
between T cells and activated microglia. No or very little
PGE2 is detected in cell supernatants when T cells are
activated in the presence of spleen cells. No PGE2 is
released by Th1 or Th2 cells in the absence of microglia or by Th1 and
Th2 cells stimulated with activating anti-TCR or anti-CD3 Abs
in the absence of APC (not shown), indicating that microglia are the
sole source of PGE2 produced in microglia-T cell cultures.
Kinetic analysis of PGE2 secretion in microglia-Th1 cell
cultures shows that PGE2 becomes detectable after 6 h
and that the largest increase in PGE2 levels occurs between
8 and 24 h (Fig. 4). Similar kinetics are observed when microglia
are cultured with Th2 cells (not shown).
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-pretreated astrocytes and
Th1 cells (Fig. 6).
MHC class II/peptide-TCR interaction, but neither CD40-CD154
interaction nor IFN-, is required for T cell-dependent
PGE2 production by microglia
We next investigated the molecules involved in Ag-dependent
PGE2 production by microglia. As for IL-12, blockade of MHC
class II molecules with anti-I-Ad, but not with
anti-I-E or isotype control, mAb almost completely abolished
PGE2 production induced by I-Ad-restricted TCR
transgenic Th1 and Th2 cells (Fig. 8).
Cross-linking of MHC class II molecules by plate-bound anti-MHC
class II or superantigen (TSST-1, SEA, or SEB) did not induce
PGE2 production by microglia (results not shown from four
independent experiments). Thus, similarly to IL-12, PGE2
production requires the establishment of MHC class II/peptide-TCR
interactions, but it is not stimulated by signaling through MHC class
II molecules.
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). Microglia incubated for
48 h in the presence of plate-bound or soluble stimulatory
anti-CD40 mAbs (as described above) secrete little PGE2
(0.6 ± 0.2 and 0.5 ± 0.2 ng PGE2/ml,
respectively; mean ± SEM of five independent experiments
performed in duplicate). Unstimulated microglia or microglia exposed to
isotype controls fail to secrete PGE2. Together, these
findings indicate that signals triggered by CD40 ligation do not play a
crucial role in inducing PGE2 during microglia-T cell
interactions. PGE2 secretion is not affected by addition of
anti-CD54, anti-CD80, anti-CD86, or anti-CD28 mAbs to
microglia-T cell cultures.
To assess whether Th1-derived IFN- is required for induction of
PGE2 secretion by microglia, neutralizing anti-mouse
IFN- mAb was added to cultures of IFN--pretreated microglia and
Th1 cells in the presence of OVA323–339. At variance with
what was observed for IL-12, anti-IFN- mAb has no effect on
PGE2 production (Fig. 8). Anti-IFN- treatment also does
not affect PGE2 secretion in microglia-Th2 cultures. These
results indicate that IFN- is not required for PGE2
production and is also not responsible for the greater PGE2
induction by Th1 than by Th2 cells.
Since we have previously shown that IL-10 down-regulates
PGE2 production by LPS-treated rat microglia (41), we
assessed whether IL-10 released during activation of Th2 cells (Fig. 1)
could be responsible for the low levels of PGE2 produced
during Ag-specific interaction between microglia and Th2 cells.
Addition of neutralizing anti-IL-10 mAb, but not of an isotype
control, causes a twofold increase in the amount of PGE2
produced by microglia in the presence of Th2 cells (Fig. 8). As
expected, anti-IL-10 mAb does not influence PGE2
production triggered by Th1 cells, which fail to secrete IL-10 upon
activation (Fig. 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR ligation by MHC class II/peptide complexes is absolutely required
for T cell-induced IL-12 and PGE2 secretion by microglia
because production of both mediators is inhibited by anti-I-A
neutralizing mAb and requires pretreatment of microglia with IFN-,
which up-regulates MHC class II molecules. However, signaling through
MHC class II molecules is not directly involved in IL-12 and
PGE2 production by microglia. Interaction of TCR with MHC
class II/peptide complexes is rather required to augment cell-cell
interactions and/or to trigger T cell activation, which, in turn,
provides IL-12- and PGE2-inducing stimuli.
Interactions between CD40 on IFN--treated microglia and CD154 on Th1
cells are essential for up-regulation of IL-12 p75 secretion, as
recently observed in dendritic cells and macrophages (39, 40, 42).
Blockade of the CD40-CD154 pathway with anti-CD154 mAb abolishes
IL-12 secretion and reduces IFN- secretion by Th1 cells activated
with microglia (22). However, CD40 ligation by stimulatory
anti-CD40 mAbs, although inducing IL-12 p40, is not a sufficient
stimulus for IL-12 p75 secretion by microglia. This indicates that
IL-12 p75 induction requires stronger or additional signals that are
delivered during microglia-Th1 interactions. The finding that
neutralization of IFN- partially blocks Th1-driven IL-12 p75
secretion indicates that IFN- is involved in optimal induction of
bioactive IL-12 in microglia, as shown for LPS-stimulated IL-12
production (16, 17). In this respect microglia are similar to
macrophages (43), whereas in dendritic cells T cell-driven IL-12
production does not depend on IFN- (42). Secretion of IL-10, which
inhibits IL-12 production (17), may explain why Th2 cells, which
express CD154 (42), fail to induce IL-12 production by microglia.
Adoptive transfer studies and analysis of T cell cytokine expression in total CNS and in inflammatory cell infiltrates have established the crucial role of Th1 cells in triggering EAE (44, 45, 46). The demonstration of IL-12 and Th1-type cytokines in active lesions of MS brains suggests that Th1 cells may initiate and/or propagate the inflammatory response leading to macrophage/microglia activation and demyelination (6, 47, 48). Furthermore, expression of CD40 on activated microglia and macrophages and of CD154 on T cells present in MS lesions (12) suggests that the CD40-CD154 pathway is involved in the stimulation of Th1 responses. Our findings that microglia can efficiently restimulate Th1 responses and secrete IL-12 via Ag-specific and CD40-CD154 interactions with Th1 cells support the possibility that microglia can reactivate Th1 cells in vivo and further skew the immune response within the CNS toward the Th1 pathway (13). Conversely, neither T cells (this study) nor proinflammatory stimuli (16, 17) induce IL-12 production by astrocytes, which is consistent with their poor capacity to activate Th1 cells.
The present data raise the possibility that Th1 cells reactivated
within the CNS may stimulate microglia to secrete PGE2
through signals different from those required to induce IL-12
secretion. Although CD40 ligation on microglia by activating
anti-CD40 mAbs induces modest PGE2 production,
anti-CD154 mAb does not inhibit PGE2 secretion during
Ag-specific interaction with Th1 or Th2 cells, indicating that the
CD40-CD154 pathway is not critically involved, unlike for IL-12.
IFN- is also not essential for Th1-driven PGE2
production, and this may explain why microglia produce PGE2
upon interaction with Th2 cells that fail to secrete IFN-.
Preliminary experiments using neutralizing anti-TNF- Abs suggest
that TNF- secreted during Th1 and Th2 activation may be involved in
the induction of PGE2 secretion by microglia (F. Aloisi,
and L. Minghetti, unpublished observations). The present finding that
neutralization of IL-10 in microglia-Th2 cell cultures increases
PGE2 production indicates that Th2 cells inhibit the
production of both pro- and anti-inflammatory mediators by
microglia. This may perhaps account for the controversial results on
the inhibition (49, 50) or lack of inhibition (51) of EAE following
IL-10 administration.
The lipid mediator PGE2 inhibits Th1 cytokine secretion (28, 29, 30), IL-12R expression (32), and IL-12 production by APC, including microglia (17), and may thus favor Th2 responses that could play a role in down-regulating CNS inflammation (45, 52, 53). Consistent with this view, PGE2 has been detected within the CNS during the spontaneous recovery from EAE and following oral administration of myelin basic protein (33). Increased PGE2 levels have also been detected in the brain of MS and HIV-1-infected patients (54, 55).
The low amounts of PGE2 secreted in astrocyte-T cells
cultures suggest that mechanisms other than Ag-specific interactions
with T cells may be more relevant in inducing PGE2
secretion by astrocytes. Previous studies have shown that IL-1 is the
most effective stimulus for PGE2 production by astrocytes
(56, 57) and that IFN- potentiates the effect of IL-1 (F. Aloisi and
L. Minghetti, unpublished observations). During Ag presentation within
the CNS, IL-1 and IFN- secreted by activated microglia and Th1
cells, respectively, could induce PGE2 secretion by
astrocytes and contribute to the down-regulation of Th1 responses.
Whether PGE2 produced by microglia or astrocytes is
involved in the control of CNS inflammation is currently being
addressed by examining the intracerebral expression of
cyclo-oxygenase-2, the inducible enzymatic isoform responsible for PG
synthesis in activated glial cells (26), at different stages of EAE.
The balance between secretion of IL-12 and PGE2 by Ag presenting microglia may regulate the development of Th1 responses within the CNS. The kinetics of secretion of IL-12 p75 and PGE2 reported in the present study during Th1-microglia interaction suggest that early production of IL-12 could be important for initial Th1 activation, whereas delayed production of IL-12 may represent a negative feedback mechanism limiting Th1 activation. Blockade of CD40-CD154-mediated cellular interaction was recently shown to prevent and reduce disease severity in experimental autoimmune diseases, including EAE (12, 58), and has been proposed as a possible therapy for the treatment of putative Th1-mediated diseases, such as MS. Our data imply that blocking the CD40-CD154 pathway should have the additional advantage of selectively inhibiting IL-12 production while not affecting the ability of CNS APC (and possibly of other APC) to produce PGE2, a mediator with inhibitory activity on Th1 responses.
Based on the present data, we would propose the following scenario for
the regulation of immune responses during CNS infection or
immunopathology: 1) peripherally activated T cells specific for viral
or CNS Ags cross the blood-brain barrier and enter the CNS parenchyma;
2) quiescent microglia respond to IFN- secreted by infiltrating Th1
cells by up-regulating expression of MHC class II and
adhesion/costimulatory molecules, including CD40, and acquire the
capacity to efficiently present Ag, leading to enhanced Th1-type
cytokine secretion; 3) activated Th1 cells, which express CD154,
recognize Ag on microglia and induce IL-12 secretion by microglia via
ligation of CD40 and IFN- secretion, thereby providing a potentially
important amplification loop for Th1 activation; 4) following
interaction with Th1 cells, microglia also secrete PGE2,
which down-regulates Th1 responses; 5) Ag-specific interaction between
microglia and Th2 cells stimulates secretion of PGE2, but
not of IL-12 by microglia while inducing Th2 activation, contributing
to the down-regulation of Th1 responses; and 6) astrocytes, which
reactivate preferentially Th2 cells, may further contribute to the
down-regulation of Th1 responses.
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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; EAE, experimental allergic encephalomyelitis; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B.
Received for publication April 27, 1998. Accepted for publication October 26, 1998.
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