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Department of Epidemiology and Public Health and Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production.
In contrast, Mac-1- cells were inefficient APCs during
EAE. Late in disease, after mice had partially recovered from clinical
signs of disease, there was a reduction in Ag-presenting capability
that correlated with decreased MHC-II and B7-1 expression.
Interestingly, although CNS APCs induced T cell cytokine production,
they did not induce proliferation of either T cell lines or CNS T
cells. This was attributable to production by CNS cells (mainly by
macrophages) of NO. T cell proliferation was restored with an NO
inhibitor, or if the APCs were obtained from inducible NO
synthase-deficient mice. Thus, CNS APCs, though essential for the
initiation of disease, also play a down-regulatory role. The mechanisms
by which CNS APCs limit the expansion of autoreactive T cells in the
target organ include their production of NO, which inhibits T cell
proliferation, and their decline in Ag presentation late in
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide-induced EAE in
C57BL/6 mice is a clinically chronic, though nonprogressing,
inflammatory demyelinating disease. Activated CD4 T cells invade the
CNS and secrete Th1 cytokines such as IFN- and TNF-
. Microglia
and infiltrating macrophages obtained from the CNS are also potent
sources of TNF- during EAE (1). Both inflammation and
cytokine production by T cells and Mac1+ cells
peak 20 days after immunization and then decline. By day 40, mice
exhibit a partial recovery from clinical signs of disease and have
almost no residual inflammation (1). T cells that invade
the CNS during EAE must interact with APCs in the target organ before
performing their effector functions, such as cytokine secretion.
Several criteria must be met to characterize CNS APCs as "successful." At a minimum, the cells must be present in the CNS during EAE and express MHC-II and costimulatory molecules such as B7. An essential role of B7/CD28 in the effector phase of MOG-induced EAE has been demonstrated (2), and B7 expression by CNS APCs can profoundly influence their ability to present Ag (3). Potential CNS APCs should be able to present not only exogenous, but also endogenous Ag. Miller et al. (4) demonstrated that plastic adherent cells derived from the CNS during either Theiler’s murine encephalomyelitis virus infection, or proteolipid protein-induced EAE are capable of presenting endogenous myelin Ags to T cell lines. However, these authors did not phenotype the APCs.
There are several potential APCs in the CNS. These include resident
cells such as microglia and astrocytes, and infiltrating cells such as
macrophages, B cells, and dendritic cells. Astrocytes have been shown
in vitro to be relatively inefficient APCs (reviewed in Ref.
5), and furthermore, they do not express MHC-II in vivo
during EAE (6, 7, 8). Both B cells (9) and
dendritic cells (10) are present in the CNS during
MOG-induced EAE, though their roles in CNS Ag presentation remain
unknown. Resident CNS microglia express MHC-II during EAE (6, 7), and some reports suggest they present Ag after stimulation
in vitro with cytokines such as IFN- (reviewed in Ref.
5). However, their ability to present Ag directly ex vivo
remains controversial (3). Macrophages invade the CNS
during EAE and play an essential role in pathogenesis, as
macrophage-depleted mice display reduced clinical signs of EAE
(11, 12, 13). Thus, both macrophages and microglia are
candidate CNS APCs and were examined here.
There appears to be a block in the ability of T cells to expand in the CNS during EAE. When Ohmori et al. (14) used bromodeoxyuridine (BrdU) incorporation as a measure of proliferation, they found that very few T cells in the CNS were BrdU+. The authors concluded that even those BrdU+ cells identified in the CNS had probably proliferated in the periphery. Several studies indicate that T cells isolated from the CNS during EAE proliferate poorly in ex vivo culture (15, 16, 17, 18). The lack of CNS T cell proliferation could be influenced by the characteristics of the APCs in the microenvironment of the CNS. Because an inhibition of T cell expansion may be an important mechanism limiting disease progression, it is crucial to determine why T cells do not proliferate in the CNS during EAE.
NO, potentially generated by CNS APCs, could inhibit T cell
proliferation in the CNS. Inducible NO synthase (iNOS) is expressed in
the CNS during EAE (19, 20). NO produced by peritoneal
macrophages, or microglial cell lines derived from neonatal mice, is a
potent inhibitor of proliferation of T cell clones, though cytokine
production remains unaffected (21). However, NO has a wide
range of biologic activities and has been suggested in various studies
to play either a pathogenic or even a protective role in EAE (22, 23 ; reviewed in Ref. 24). iNOS can be induced in
vitro by IFN- and TNF- (25, 26, 27), and these cytokines
are highly expressed in the CNS during EAE (1). Thus, NO
is a candidate repressor of CNS T cell proliferation.
In this study, we demonstrate that both macrophages and microglia derived from the CNS during EAE and used directly ex vivo express MHC-II, B7-1, and B7-2 and efficiently present both endogenous and exogenous Ag to CNS T cells and T cell lines. The ability of CNS cells to present Ag varied over the clinical course of EAE. A stabilization of clinical signs was correlated with a decline in Ag presentation and MHC and costimulatory molecule expression. In addition, CNS APCs produced NO, which inhibited proliferation but not cytokine production of CNS T cells and T cell lines. This could be overcome by culture with NO inhibitors, or when CNS APCs were derived from iNOS-/- mice. Thus, CNS infiltrating and resident APCs contribute both to the emergence and resolution of EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (B6) and B6-NOS2tm1 lau (iNOS-/-) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were 7–9 wk of age at the time of immunization. Mice were maintained and housed in the Yale animal care facilities, and all experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committee.
MOG peptide
MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) of murine origin was synthesized by the W. M. Keck Biotechnology Resource Center at Yale University. The peptide was purified by reverse-phase (C18) column HPLC, and a trifluoroacetic acid/acetonitrile gradient.
Active induction of EAE
EAE was induced by s.c. flank injections of 300 µg of MOG35–55 peptide in CFA (Difco, Detroit, MI) with 500 µg of Mycobacterium tuberculosis on days 0 and 7, supplemented by i.p. injections of 500 ng of pertussis toxin (List Biological, Campbell, CA) on days 0 and 2, as described previously (28). The mice were observed daily for clinical signs and scored on a scale of 0 to 5 (28). The disease index was calculated on day 30 by adding the daily average disease scores, dividing by the average day of disease onset, and multiplying by 100.
Cell lines
An anti-MOG p35–55-specific T cell line (T-MOG) generated from draining LN of MOG35–55-immunized mice (29) was kindly provided by Dr. Thomas Spahn (Westfälische Wilhelms University, Münster, Germany). OVA-specific T cell line 21C11 (30) was used as a control and designated T-OVA. T cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (all obtained from Life Technologies, Gaithersburg, MD). T cells were stimulated every 2 wk in 24-well plates with irradiated (2000 rad) spleen cells, 20 µg/ml MOG35–55, or 500 µg/ml OVA (Sigma, St. Louis, MO), and 5 U/ml recombinant human IL-2 (Boehringer Mannheim, Indianapolis, IN). Fresh medium supplemented with IL-2 was given on alternate weeks. All cells were incubated at 37°C in humidified air containing 10% CO2.
Isolation of CNS APCs and FACS analysis
To isolate cells from the CNS, mice were deeply anesthetized and perfused intracardially with RPMI 1640 medium (Life Technologies). Brain and spinal cord cell suspensions were incubated with 1 mg/ml collagenase II (Sigma), at 37°C for 20 min, and mononuclear cells were isolated by discontinuous Percoll (Pharmacia, Piscataway, NJ) gradient. For FACS staining, cells were washed in FACS buffer (1% FCS, 0.1% sodium azide in PBS) and, after blocking with purified rat, hamster, and goat IgG, were stained with directly conjugated Abs. For purification of CNS APC populations, CNS cells were pooled from four to five equivalently affected animals and stained with anti-Mac-1 and/or anti-CD45-CyChrome. Mac-1+ and Mac-1- or Mac-1+CD45high (macrophage) and Mac-1+CD45int (microglia) populations were collected by using FACStarPlus (Becton Dickinson, San José, CA). Abs used were anti-Mac-1-PE, anti-CD45 CyChrome, anti-IAb-FITC, anti-B7-1-PE, anti B7-2-PE, and anti-Mac-1-FITC (all obtained from BD PharMingen, San Diego, CA).
Intracellular cytokine staining
CNS or spleen APCs (1 x 105) were
cultured with T-MOG or T-OVA cells (2 x
105) in 96-well plates with or without the
addition of 20 µg/ml exogenous MOG peptide or 500 µg/ml OVA
respectively. Cells were cultured for 5 h in the presence of the
protein transport inhibitor GolgiStop (BD PharMingen). Cells were
stained for surface markers and then were fixed, permeabilized, and
stained for intracellular IFN- by using a Cytofix/Cytoperm Kit (BD
PharMingen) as recommended by the manufacturer. Abs used were
anti-CD4-FITC and anti-IFN--PE (BD PharMingen).
Enzyme-linked immunospot (ELISPOT) analysis
ELISPOT for IFN- was performed as described (1).
ELISPOT plates (Millipore, Ann Arbor, MI) were coated with the capture
Ab for IFN-. CNS cells (1 x 105), which
include both T cells and APCs, were cultured for 24 h with 10
µg/ml MOG35–55. Irradiated spleen cells
(3 x 105) from unimmunized B6 mice were
added to some wells as an additional source of APCs. After washing the
plates with PBS to remove the cells, a biotinylated detection Ab for
IFN- was added. Bound secondary Abs were visualized by using
HRP-streptavidin (Dako, Carpinteria, CA) and 3-amino-9-ethylcarbazole.
Abs R4-6A2 and XMG1.2-biotin, (BD PharMingen) were used for capture and
detection of IFN-. Spot-forming cells were enumerated with the aid
of a dissecting microscope.
Proliferation assays
CNS or spleen cells (2 x 105) were cultured in 96-well plates in triplicate, with or without 50 µg/ml MOG35–55 peptide. In some cases, 0.5 mM of the NOS inhibitor N-monomethyl-L-arginine (L-NMA) or its inactive D-isomer (D-NMA; Alexis, San Diego, CA) was added to cultures. [3H]Thymidine (1 µCi/well) was added after 48 h, and cultures were harvested 18 h later.
Nitrite assay
Cells were cultured as described above, and supernatants were collected after 48 h. The accumulation of NO2-, a stable end product of NO formation, was used as a relative measurement of NO. Supernatant (100 µl) was incubated with 100 µl of Griess reagent (Sigma) for 15 min at room temperature. Optical absorbance was measured at 570 nm with a microtiter plate reader with sodium nitrite as the standard.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ag-presenting capability of CNS cells was evaluated by their
ability to stimulate IFN- production from T-MOG. Cells were isolated
from the CNS or spleen 20 days after immunization, at the peak of
clinical signs of disease. T-MOG cells were cultured with CNS or spleen
cells as APCs for 5 h in the presence or absence of exogenous
MOG35–55 peptide and stained for surface CD4 and
intracellular IFN-. The T-MOG cells, with their higher levels of CD4
expression, could be distinguished from spleen or CNS T cells by FACS
(932 vs 139 mean fluorescence intensity respectively). T-MOG produced
IFN- (19% IFN-+) in the absence of
exogenous MOG peptide when day 20 CNS cells were added (Fig. 1
). T-MOG produced higher levels of
IFN- (88% IFN-+) when exogenous MOG
peptide also was added with the CNS cells, demonstrating that during
EAE, cells in the CNS are highly efficient at presenting Ag. In
contrast, CNS cells isolated from normal healthy mice (which are almost
entirely Mac-1+), were nearly incapable of
presenting Ag, inducing only 1.5% or 5% of the T-MOG cells to produce
IFN- in the absence or presence of exogenous MOG peptide
respectively (Fig. 1). Only CNS APCs and not splenic APCs induced
IFN- from T-MOG in the absence of exogenous Ag (Fig. 1), suggesting
that CNS APCs from immunized mice also could present endogenous Ag
derived from the CNS.
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We next examined whether CNS cells could process and present
exogenously added protein Ags. CNS APCs were very efficient at
presenting exogenously added OVA to an OVA-specific T cell line (T-OVA,
67% IFN-+) and did not induce IFN- in the
absence of added OVA (2% IFN-+; Fig. 2A). Leupeptin, an Ag
processing inhibitor, prevented the stimulation of T-OVA (5%
IFN-+; Fig. 2A). To determine
whether the putative endogenous presentation to T-MOG was attributable
to MHC-MOG peptide complexes present on APCs in the CNS, or whether the
Ag was loaded during the isolation procedure, leupeptin was added to
cultures of T-MOG and CNS APCs. Leupeptin had no effect on the IFN-
production by T-MOG in the absence of added peptide, indicating that
the endogenous MOG35–55 was loaded on the APCs
in the CNS before the cells were isolated (Fig. 2B).
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To determine which CNS cells were presenting Ag, individual cell
populations were purified by FACS sorting. CNS cells were first sorted
into Mac-1+ and Mac-1-
populations. The Mac-1+ cells expressed MHC-II,
B7-1, and B7-2 (Fig. 3A). The
majority of the Mac-1+ population consisted of
macrophages and microglia, although a small number of cells (an average
of 5%) also expressed the dendritic cell marker CD11c. The
Mac-1- population contained mostly B cells and T
cells, and again a small number of CD11c+ cells
(an average of 4%). Mac-1- cells expressed B7-1
and B7-2, but not MHC-II (Fig. 3A). In this population, B7-1
and B7-2 were expressed predominately by CD4+ T
cells, as has been shown previously during EAE (31, 32).
Mac-1+ CNS cells presented both endogenous and
exogenous MOG to T-MOG (an average of 17% and 66%
IFN-+, respectively; Fig. 3B). In
contrast, Mac-1- cells did not present
endogenous Ag and were much less efficient at presenting exogenous
MOG35–55 (an average of 5%
IFN-+).
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B). Consistent with their ability to present Ag, both
macrophages
(Mac-1+CD45high) and
microglia (Mac-1+CD45int)
expressed MHC-II, B7-1, and B7-2 on day 20 (Fig. 3A). Decreased Ag presentation to T cell lines and CNS T cells by day 40
The Ag-presenting activity of CNS APCs was examined late in
disease, at a time when inflammation, cytokine production, and clinical
signs are reduced. Day 40 CNS APCs were less capable of presenting both
endogenous (5% IFN-+) and exogenous (26%
IFN-+) MOG to T-MOG, compared with their
stimulation of 15% and 80%, respectively, on day 20 (averaged data
from seven experiments; Fig. 4A). The ability of CNS APCs
to process and present whole proteins also declined. Day 40 APCs were
much less efficient at presenting exogenous OVA to T-OVA (8%
IFN-+) compared with day 20 (53%
IFN-+).
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-producing cells late in disease
(1) was a function of a decline in CNS Ag-presenting
ability. CNS cells, which include CNS T cells and APCs, were cultured
with MOG35–55 peptide with or without the
addition of irradiated spleen cells as an additional source of APCs.
The capability of CNS APCs was evaluated by comparing the number of
MOG-specific CNS T cells that produced IFN- in the presence or
absence of additional splenic APCs. IFN--producing CNS T cells were
enumerated by ELISPOT in this experiment, because it was not possible
to discriminate CNS T cells from the added irradiated splenic T cells
by FACS. On day 20, the number of responding MOG-specific CNS T cells
was high and was not significantly enhanced by the addition of
irradiated spleen cells, indicating that the capability of CNS APCs was
at its peak (Fig. 4B). However, on days 30 and 40, the
addition of irradiated spleen cells more than doubled the number of
responding CNS T cells (Fig. 4B). This further supports the
concept that late in disease, there is a decrease in the capability of
APCs in the CNS. Decreased expression of MHC-II and B7-1 on CNS APCs by day 40
To elucidate the mechanism of the decline in Ag-presenting
capability, the kinetics of MHC and costimulatory molecule expression
by Mac-1+ cells was examined.
Mac-1+ cells isolated from unimmunized mice
displayed little to no expression of these molecules (Day 0; Fig. 5). Expression of MHC-II, B7-1, and B7-2
was up-regulated on Mac-1+ cells by day 14 and
peaked on day 20 at the time of peak Ag-presenting capability. By day
40, when APC activity declines, the expression of both MHC-II and B7-1
also was significantly reduced. On day 20, an average of 37% of
Mac-1+ cells expressed MHC-II, compared with only
5% on day 40 (Fig. 5). B7-1 expression also was reduced from an
average of 56% of Mac-1+ cells to 25%
B7-1+ by day 40, whereas B7-2 expression was not
significantly down-regulated. Thus, the kinetics of Ag-presenting
capability correlated with that of MHC-II and B7-1 expression.
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Having demonstrated that CNS T cells produce cytokines when
stimulated with CNS APCs, we evaluated their proliferative capacity.
CNS cells from MOG35–55 immunized mice did not
proliferate in response to Ag, though spleen cells from the same mice
did (Fig. 6). Neither the addition of
IL-2 nor exogenous spleen cells restored the ability of CNS cells to
proliferate. To determine whether this was attributable to an
inhibitory factor, we examined NO production in the CNS cultures. High
levels of NO were detected in the supernatants of cultures of CNS cells
from MOG-immunized mice by an assay for the accumulation of
NO2- (referred to as NO; Fig. 6A). NO levels peaked 20 days after immunization,
corresponding to the peak of clinical signs of disease and of TNF-
and IFN- production in the CNS (1). NO production was
reduced by day 40 (Fig. 6A) and was not detected in cultures
of cells isolated from the CNS of unimmunized mice (day 0, Fig. 6A). The high level of NO in cultures of CNS cells was
associated with a total absence of proliferation by CNS T cells at
every time point tested (Fig. 6A). In contrast, NO was not
found in cultures of spleen cells from MOG-immunized mice, and
those cells did proliferate well to MOG35–55 at
all time points tested (Fig. 6B).
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To further investigate whether NO production by CNS cells was
responsible for the absence of T cell proliferation, CNS cells were
cultured in the presence of the NO synthase inhibitor
L-NMA, or its inactive D-isomer. Treatment of
CNS cell cultures with L-NMA, but not with
D-NMA, resulted in a significant reduction in the amount of
NO in culture supernatants (Fig. 7A). In parallel, treatment
with L-NMA also restored the proliferative
capacity of CNS cultures (Fig. 7A) but had no effect on
spleen cell proliferation (Fig. 7B). Robust proliferation
was seen when MOG35–55 peptide and
L-NMA were added to CNS cultures, with a 200-fold
increase over cells cultured without L-NMA (Fig. 7A). A increase in proliferation (13-fold) also was detected
even when no exogenous peptide was added, presumably attributable to
the presentation of endogenous Ag by CNS APCs (Fig. 7A).
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A). The ability of
macrophages and microglia to induce proliferation of the T cell line
T-MOG was also examined. When unsorted CNS cells (macrophages and
microglia) were used as APCs for T-MOG, Ag-specific proliferation was
not detected. However, when purified microglia were used as APCs, T-MOG
cells proliferated robustly in response to Ag (Fig. 8B),
indicating that their ability to induce proliferation is actually
inhibited by the presence of macrophage-produced NO. In contrast,
purified macrophages induced proliferation of the T-MOG cells only in
the presence of L-NMA (Fig. 8B).
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A). Proliferation of
spleen cells from WT and iNOS-/- mice was
essentially comparable (Fig. 7B).
To determine whether the restored proliferation of cells obtained from
iNOS-/- mice was attributable to an effect on
Ag presentation, WT CNS T cells were evaluated for their ability to
proliferate in response to iNOS-/- CNS APCs and
Ag. When T cells were enriched from the CNS of WT mice by sorting for
the Mac-1- population, they proliferated
vigorously in response to iNOS-/- CNS APCs and
Ag (Fig. 7C). Taken together, these data indicate that the
CNS T cells were not anergic and were capable of proliferating in a
supportive microenvironment. In addition, the removal of NO was
sufficient to allow their proliferation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The scenario suggested by the data presented here is that MOG-specific
T cells are activated in the periphery and then enter the CNS and
activate resident microglia through production of
lymphotoxin-, TNF-, and IFN-. This is consistent with
the kinetics and cellular source of cytokine production in the CNS
(1) and the kinetics of MHC-II and B7 expression. Once
microglia are activated, they produce TNF- and present endogenous Ag
to T cells. Cytokines produced by T cells and microglia contribute to
further recruitment and activation of macrophages, which also produce
TNF-, express costimulatory molecules and MHC-II, and present Ag.
CNS APCs, particularly infiltrating macrophages, produce NO, which
serves to limit T cell expansion. These infiltrating macrophages are
presumably activated to produce NO after interaction with
cytokine-producing Th1 T cells in the CNS. In contrast, the
antiproliferative effects of NO are not as apparent in the spleen. Even
though macrophages are relatively abundant in the spleen, they are not
likely to be activated, as we saw no evidence for spontaneous cytokine
production or the presentation of endogenous Ag in the spleen. Though
CNS APCs are crucial for the initiation of disease, they also play a
down-regulatory role. CNS APCs may limit the expansion of autoreactive
T cells by at least two mechanisms: NO production, and late in disease,
down-regulation of their MHC-II and B7-1, resulting in decreased Ag
presentation. Thus, the presence and activity of APCs in the CNS
regulates both inflammation and the clinical course of EAE.
The inflammatory response that occurs in the CNS during EAE was
instrumental in up-regulating Ag presentation, as CNS APCs isolated
from healthy mice were relatively inefficient at presenting Ag directly
ex vivo. The identity of the cell that presents Ag to the initial
invading T cells is unclear. Evidence from a rat EAE model indicates
that perivascular macrophages could be the initial APC during EAE. When
this small population of macrophages was enriched from the CNS of
healthy rats they could present endogenous and exogenous Ag to T cell
lines in a proliferation assay (34). Several authors have
investigated the Ag-presenting capacity of microglia. Microglia
isolated from healthy rats and used directly ex vivo are relatively
inefficient APCs (34), though activation in vitro with
cytokines such as IFN- confers Ag-presenting capability to microglia
(reviewed in Ref. 5). The studies presented here are among
the first to investigate the Ag-presenting capacity of microglia
isolated ex vivo from the inflamed CNS during EAE. In another situation
of CNS inflammation, during graft-vs-host disease in rats, microglia
are activated by infiltrating T cells and express MHC-II, but not the
costimulatory molecules B7-1 or B7-2. Microglia isolated from these
rats can activate T cells to produce cytokines, but the T cells do not
proliferate, rather, they undergo apoptosis (3). Here we
demonstrate that microglia isolated from mice with EAE and analyzed
directly ex vivo, express MHC-II and the costimulatory molecules B7-1
and B7-2, and are efficient at presenting Ag to both CNS T cells and T
cell lines.
Microglia induced Ag-specific cytokine production and proliferation of T cell lines, whereas macrophages induced only cytokines. This was attributable to production of high levels of NO by the macrophages. Even though purified microglia could induce Ag-specific T cell proliferation, when macrophages and microglia were used together as APCs, proliferation was inhibited. This suggests that the outcome of a T cell’s interaction with a particular APC in the CNS may be influenced by the inflammatory milieu and the balance between macrophages and microglia, which does change during the course of EAE (1). In the studies presented here, the inhibitory effects of NO could be overcome by NOS inhibitors or in iNOS-/- mice. The antiproliferative effects of NO in the CNS may provide a partial explanation for the more severe disease exhibited by iNOS-/- mice (22, 23).
The fact that NO is induced by inflammatory cytokines such as IFN-
and TNF- (25, 26, 27) and yet negatively regulates
inflammation, provides a partial explanation for the seemingly
divergent and conflicting roles of certain cytokines in autoimmune
inflammatory diseases. For instance, TNF- exacerbates diabetes in
NOD mice if given early in disease, but inhibits if administered late
(37). It is possible that in this case it contributes to
the entrance of inflammatory cells into the target organ early in
disease, but through induction of NO limits their expansion. Inhibition
of CNS T cell proliferation by NO also may explain the severity of EAE
in several knockout mice that might have been expected to be protected:
IFN-R-/-,
IFN--/-, and, in some reports,
TNF--/- mice are more sensitive to EAE
(38, 39, 40). Consistent with this, peritoneal exudate cells
from IFN-R-/- mice induce enhanced
Ag-specific T cell proliferation compared with WT mice because of their
reduced production of NO (26). In addition, T cells from
IFN--/- mice demonstrate enhanced in vivo
proliferation as measured by BrdU incorporation (41).
Even though there is a decline in NO production in the CNS during the later stages of EAE, there is neither a surge of proliferating T cells nor sustained clinical signs and inflammation. This indicates that NO is not the only suppressive mechanism operative in this disease. Further studies with iNOS-/- mice will determine whether or not they exhibit relapses (not generally seen in WT mice in this model), or even convert to a chronic progressive type. Other events that could contribute to a lessening of clinical signs and inflammation include a reduction in the Ag-presenting capacity of both macrophages and microglia. The reduced expression of MHC-II and costimulatory molecules by CNS APCs may contribute to the absence of T cell proliferation in the CNS late in disease, even though only low levels of NO are produced at that time. In addition, the expression of other costimulatory molecules by CNS APCs is important in EAE. Mice deficient in inducible costimulatory molecule (ICOS), the receptor for B7-h (42) exhibit a more intense EAE than WT mice (43). This is most likely attributable in part to their diminished production of the Th2/Th3 cytokine IL-13, a potent down-regulatory molecule during EAE (44). It remains to be determined whether B7-h/ICOS is acting in the CNS, in the periphery, or both. Another accessory molecule, OX2/OX2R, appears to be important specifically in the CNS. OX2 is expressed on neurons, whereas OX2R is expressed on macrophages, and to a lesser extent, microglia (45). OX2-/- mice, or mice subject to OX2-OX2R blockade, exhibit an earlier onset and more severe clinical signs at the acute phase of disease (45, 46). These data suggest that OX2R could participate in down-regulating the activity of CNS macrophages and microglia.
One possibility for the down-regulation of MHC-II and costimulatory
molecules in the CNS late in disease is that this occurs through NO,
because IFN--induced MHC-II expression on macrophages is inhibited
by NO in vitro (47). However, our data do not support this
hypothesis because the up-regulation of NO and MHC-II in the CNS follow
identical kinetics. The mechanisms for the down-regulation of Ag
presentation may involve T cells, and a balance of Th1 and Th2/Th3
cytokines. T cell cytokines such as TNF- and IFN- are likely to
be important for the initiation of inflammation and Ag presentation in
the CNS. The decrease in these cytokines late in disease
(1) could play a role in the down-regulation of Ag
presentation and the resolution of inflammation. The expression of Th2
cytokines such as IL-10, TGF-
, or IL-13 may also be important. IL-10
and TGF- have been reported to inhibit MHC-II and costimulatory
molecule expression on microglia (48, 49, 50), and in
addition, IL-10-deficient mice are more susceptible to EAE with reduced
recovery than WT mice (51, 52). Dissecting the complexity
of the interaction of T cells and target organ APCs during autoimmune
diseases like multiple sclerosis will be important for effectively
targeting therapies to appropriate cell types.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy H. Ruddle, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, P.O. Box 208034, New Haven, CT 06520-8034.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; BrdU, bromodeoxyuridine; iNOS, inducible NO synthase; ELISPOT, enzyme-linked immunospot; L-NMA, N-monomethyl-L-arginine; D-NMA, N-monomethyl-D-arginine; WT, wild type; B6, C57BL/6; ICOS, inducible costimulatory molecule.
Received for publication December 22, 2000. Accepted for publication February 14, 2001.
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) or absence (
) of exogenously added MOG peptide before
staining for CD4 and IFN-
) or also with irradiated spleen cells as an additional source
of APCs (
). Data are graphed
as the percent of Mac-1+ cells positive for the relevant
marker. Data are an average of two to four experiments per time point,
with SDs shown.
). For proliferation, data are graphed as stimulation index
above culture in medium alone. Representative from one of several
experiments.
) or absence (