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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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, and TNF-
) and Th2 (IL-4) cells in
the spleen. However, only Th1 cells were apparent in the CNS. CD4 T
cells that produced IFN- or TNF- were present in the CNS by day 7
after immunization with MOG35–55, peaked at day 20, and
then waned. TNF- was also produced in the CNS by Mac-1+
cells. On days 7 and 10 after immunization, the TNF--producing
Mac1+ cells were predominantly microglia. By day 14, a
switch occurred in that the Mac1+ TNF--producing cells
had the phenotype of infiltrating macrophages. RANTES, IFN-inducible
protein 10 (IP-10), and monocyte chemotactic protein 1 chemokine mRNA
were detected in the CNS by day 8 after immunization. The early
presence of monocyte chemotactic protein 1 (MCP-1) in the CNS provides
a mechanism for the recruitment of macrophages. These data implicate
TNF- production by a continuum of T cells, microglia, and
macrophages at various times during the course of disease. The
importance of Th1 cytokines is highlighted, with little evidence for a
role of Th2 cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this communication, we use the myelin oligodendrocyte glycoprotein
(MOG) model of EAE to investigate several questions with an eye toward
understanding the mechanism by which the integrity of the BBB is broken
and the nature of the self-limiting inflammation. C57BL/6 mice are
immunized with MOG35–55 peptide in CFA and
treated with pertussis toxin and boosted once with the Ag. This
immunization regimen results in a reproducible disease course, which in
our previous studies demonstrated a 100% incidence of clinical signs
(1, 2). Clinical signs of disease are apparent by
approximately day 12, peak at 
20 days, and then stabilize, with
continued paralysis (1). MOG-induced EAE can be defined as
a chronic sustained disease characterized by inflammation and
demyelination. Inflammation in this model consists of T cells, B cells,
and macrophages (2). In this study, we show that the
presence of infiltrating cells precedes clinical signs by a few days,
peaks at 20 days, and then becomes less apparent.
The MOG model as first described in the rat by Linington and colleagues
(3, 4, 5) included the presence of Ab specific for the
protein. Data obtained with passive transfer experiments implicated
both arms of the immune system and were interpreted to indicate that T
cells were the initial culprits in breaching the BBB, but that
demyelinating Abs were critical for the clinical signs of paralysis.
This would suggest the importance of both Th1 (proinflammatory) and Th2
(Ab helper) CD4 T cells. However, this does not appear to be the case
in all species since gene-targeted mice that lack B cells or Ab are as
susceptible to MOG-induced EAE as their wild-type (WT) littermates
(2, 6). In addition, Ab titers to MOG correlate poorly
with clinical scores in WT mice (reviewed in Ref. 7), and
passive transfer of the disease does occur with T cells alone (see
Refs. 8, 9 ; A. E. Juedes and N. H. Ruddle,
manuscript in preparation). The importance of the Th1 cytokine
lymphotoxin (LT)- (1), but not IFN-
(10), and the lack of importance of the Th2 cytokine IL-4
(9, 11) are suggested by data obtained from gene-targeted
mice. However, in a mouse not subject to genetic manipulation, the
possibility remained that Th1 and Th2 cytokines do play a role in MOG
EAE. Th1 and Th2 cytokine mRNA have been detected in the CNS of mice
with EAE induced with recombinant human MOG (12). In this
study, we investigate whether murine MOG peptide induces both Th1 and
Th2 cytokine protein-producing cells and whether both of those cell
types can penetrate the BBB.
The role of nonlymphoid cells such as CNS resident cells and
infiltrating macrophages and their products must also be considered in
elucidating the mechanisms of EAE. Several studies have addressed the
role of macrophages in EAE (13, 14, 15), although none used
the MOG model. Tran et al. (15) demonstrated the
importance of macrophages in an adoptive transfer model of myelin basic
protein (MBP)-induced EAE in SJL/J mice. Selective depletion of
peripheral macrophages by administration of mannosylated liposomes
containing dichloromethylene diphosphonate resulted in greatly reduced
clinical severity and reduced invasion of leukocytes into the
parenchyma. However, the CNS content of CD4 T cells and microglia was
not affected. Martiney et al. (13) used CNI-1493 to
inhibit macrophage activation in the same model of EAE, which also
resulted in reduced clinical severity and inflammation. In addition,
cytokine mRNA levels in the CNS were decreased after macrophage
inactivation; however, TNF- was not examined in that study. TNF-
mRNA production by resident microglia and infiltrating non-T
mononuclear CNS cells was demonstrated by Renno et al.
(16) in the SJL/J MBP model.
The nature of the cells that produce TNF- has not been addressed in
MOG EAE, and in fact evidence conflicts regarding a role for TNF- in
that disease. Liu et al. (17) found that TNF--/- mice
were even more susceptible to MOG-induced EAE than were WT mice. The
data of Suen et al. (1) suggest that LT- is crucial for
EAE and that the presence of TNF- does not compensate for its
absence. However, Korner et al. (18) showed that
MOG-immunized TNF--/- mice did have a slightly delayed time of
onset. There was also an interesting difference in the pattern of
inflammation in the CNS, manifested as a reduction in discrete
perivascular cuffs and limited expansion of cells into the
parenchyma.
The mechanisms by which the LT/TNF cytokines influence inflammation
most likely include both induction of adhesion molecules and
chemokines. Adhesion molecules such as VCAM-1 and ICAM-1 are
up-regulated in several models of EAE, including MOG (6, 19, 20, 21, 22). Murine LT- and TNF- are potent inducers of these
adhesion molecules in vitro and in vivo (23, 24, 25). The
expression of chemokines in EAE has been documented by many groups in
several models with varying results, although not in the MOG model
described here (summarized in Refs. 26 and
27). In many of these studies, analyses have been
conducted in the course of inflammation and do not provide a
distinction between those chemokines that contribute to inflammation
and those that are products of infiltrating cells. LT- and TNF-
can induce RANTES, monocyte chemotactic protein (MCP)-1, and
IP-10 from various cells lines in vitro (24, 28, 29), and
LT- has also been demonstrated to produce those chemokines in vivo
in a transgenic model (23). The possibility that such Th1
cytokines influence chemokine expression and later recruitment is
examined here. In this communication, EAE is considered as a dynamic
process. The cellular origin, the nature, and the temporal pattern of
cytokine production in the CNS is investigated and shown in the case of
TNF- to be produced by three different populations, T cells,
microglia, and macrophages. These data provide insight into the
mechanisms of this disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were 8–10 wk of age at the time of immunization.
MOG peptide
MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK), of murine origin, was synthesized by the W. M. Keck Biotechnology Resource Center at Yale University. The peptide was purified by reversed-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 MOG35–55 peptide in CFA (Difco, Detroit, MI) with 500 µg Mycobacterium tuberculosis on days 0 and 7, supplemented by i.p. injections of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA), as described previously (1). Control mice were immunized with 300 µg hen egg lysozyme (HEL) in CFA and injected with pertussis toxin as described above. The mice were observed daily for clinical signs and scored as described previously (1).
Isolation of total RNA/Northern blot analysis
Splenocytes were cultured at a concentration of 2 x 106 cells/ml, with or without 20 µg/ml MOG35–55. Cells were harvested 18 h later. Total RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD) according to the instructions provided by the manufacturer. Total RNA (10 µg) was electrophoresed in a 1% agarose/formaldehyde gel with 1 x MOPS as the running buffer. RNA was transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH), UV cross-linked, and hybridized to random-primed 32P-labeled probes overnight at 42°C as recommended by the manufacturer. After hybridization, the blots were washed twice in 2x standard saline citrate phosphate/EDTA (SSPE), which contained 0.2% SDS, for 30 min at room temperature, and1x SSPE and 0.1x SSPE containing 0.2% SDS for 30 min each at 65°C. The probed blots were exposed at -70°C to Hyperfilm MP (Amersham, Arlington Heights, IL).
cDNA probes
A 1.4-kb murine TNF- cDNA probe (a gift from Dr. Bruce
Beutler, University of Texas Southwestern Medical School, Dallas, TX)
was excised from the PBS vector with BamHI and
PstI. A 0.71-kb KpnI/HincII fragment
of the murine LT cDNA was used as the murine LT- probe as described
previously (30).
Bioassay for TNF- and LT-
TNF-/LT- bioactivity was measured by using the WEHI 164
fibrosarcoma cell line as a cytotoxic target as described previously
(31). Briefly, serial dilutions of supernatants from
splenocytes cultured with or without MOG35–55
were incubated with WEHI 164 cells in 96-well microtiter plates for
48 h at 37°C. Target cell viability was then determined by
assessing their metabolic activity with the use of the MTT assay.
Enzyme-linked immunospot analysis
The enzyme-linked immunospot (ELISPOT) assay used was that
described by Tian et al. (32). To isolate cells from the
CNS, mice were deeply anesthetized and perfused intracardially with
RPMI 1640 medium (Life Technologies. In each experiment, cells were
pooled from two to three equivalently clinically affected mice. Brain
and spinal cord cell suspensions were incubated with collagenase II (1
mg/ml; Sigma, St. Louis, MO) at 37°C for 20 min, and mononuclear
cells were isolated by discontinuous Percoll (Pharmacia, Piscataway,
NJ) gradient. ELISPOT plates (Millipore, Ann Arbor, MI) were coated
with the appropriate capture Ab for IFN- or IL-4. Spleen or CNS
cells were plated with or without 10 µg/ml
MOG35–55. Plates were incubated at 37°C for
24 h (IFN-) or 48 h (IL-4) and washed with PBS to remove
cells. Biotinylated detection Abs for IFN- or IL-4 were added and
plates were incubated at 4°C overnight. Bound secondary Abs were
visualized using HRP-streptavidin (Dako, Carpinteria, CA) and
3-amino-9-ethylcarbazole. Abs R4–6A2/XMG1.2-biotin,
11B11/BVD6–24G2-biotin (PharMingen, San Diego, CA) were used for
capture and detection of IFN- and IL-4, respectively. Spot-forming
cells (SFC) were enumerated with the aid of a dissecting
microscope.
Flow cytometric analysis
Cells were isolated from the CNS as described above and cultured
at a density of 1–2 x 106 cells/ml at
37°C. Protein transport inhibitors (GolgiStop for IFN- staining or
GolgiPlug for TNF- staining) were applied as recommended by the
manufacturer (PharMingen). Cells were harvested 5 h later and
washed in FACS buffer (1% FCS and 0.1% sodium azide in PBS). After
blocking with purified rat, hamster, and goat IgG (Pierce, Rockford,
IL), cells were stained for surface markers with directly conjugated
Abs in FACS buffer. Cells were then fixed, permeabilized, and stained
for the intracellular cytokines TNF- or IFN- using a
Cytofix/Cytoperm kit (PharMingen) as recommended by the manufacturer.
Abs used were CD4-FITC, CD11b (Mac-1)-FITC, CD45-Cy-Chrome, IFN--PE,
and TNF--PE. All Abs were obtained from PharMingen.
RNase protection assay
Mice were deeply anesthetized and perfused intracardially with PBS. Total RNA was isolated from the brains and spinal cords using Trizol reagent (Life Technologies) according to the manufacturer’s recommendations. Chemokine mRNA levels were determined using the Riboquant Multiprobe RNase Protection Assay System (PharMingen). 32P-labeled riboprobes were synthesized from the plasmid template set mCK-5 using T7 polymerase, after which the DNA template was digested with DNase. Total RNA (15 µg) was hybridized with the riboprobes overnight at 56°C. The following day, ssRNA species were removed by digestion with RNase A. The protected RNA species were phenol/chloroform extracted, ethanol precipitated, and electrophoresed on a 5% polyacrylamide gel. Protected chemokine probes were visualized by autoradiography of the dried gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The phenotype of MOG35–55-specific cells in
the spleen was analyzed by evaluating their production of
representative Th1 (LT-, TNF-, and IFN-) and Th2 (IL-4)
cytokines (33, 34, 35). Mice were immunized with
MOG35–55 in CFA and pertussis toxin according to
the protocol for EAE induction, which as noted above results in
clinical signs by day 12. Spleens were removed 14–20 days after the
initial immunization, and cells were cultured with or without
MOG35–55. Cytokine protein production was
analyzed using a WEHI assay (for TNF- and LT-) or an ELISPOT
assay (for IFN- and IL-4). LT- and TNF- production were not
evaluated by ELISPOT due to the lack of a commercially available Ab
that distinguishes between them. Supernatants from cells cultured with
MOG35–55 exhibited strong cytotoxic activity
against WEHI 164 cells, indicating the Ag-specific production of
TNF- or LT- protein (Fig. 1
A). The production of both
cytokines was confirmed at the mRNA level by Northern blot analysis.
RNA messages for both TNF- and LT- were detected after
stimulation with MOG35–55 (Fig. 1B).
MOG35–55-specific cells that produced IFN-,
and to a lesser extent IL-4, were also detected by ELISPOT analysis
(Fig. 2A). These data indicate
that Th1 cells were the predominantly primed T cell population,
although Th2 cells were also stimulated.
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To determine whether both Th1 and Th2 cells infiltrate the CNS,
ELISPOT assays were performed for IFN- and IL-4. Spleen or CNS
isolated cells were cultured in medium alone or stimulated with
MOG35–55 or anti-CD3. As noted above, both
IFN- and IL-4 protein-producing cells could be detected in the
spleen 20 days after immunization in response to
MOG35–55 or anti-CD3 stimulation (Fig. 2
A). IFN--producing cells were also present in the CNS of
MOG-immunized mice. They were apparent even in the absence of
additional exogenous Ag stimulation and especially at high numbers
after stimulation with MOG35–55. Their frequency
was 10-fold higher than that seen in the spleen (note difference in
scale, Fig. 2C). In contrast, no IL-4-producing cells could
be detected in the CNS, even when cells were stimulated in vitro with
anti-CD3. At 40 days after immunization, when the clinical signs of
disease are stabilized (1), IFN--producing cells were
still apparent in the CNS, although at a decreased frequency compared
with day 20. It was possible that this is due to a switch from a Th1 to
a Th2 phenotype. However, this is considered unlikely, because even at
this late time, no IL-4-producing cells were detected in the CNS,
whereas they were still apparent in the spleen (Fig. 2, B
and D). Therefore, there is little support for the concept
that Th2 cells contribute to either the induction or resolution of the
clinical signs of MOG EAE, as only Th1 cells could be found in the CNS
throughout the course of disease.
The phenotype of IFN-- and TNF--producing cells in the CNS
The frequency and phenotype of cytokine-producing cells in the CNS
was evaluated further using intracellular cytokine staining. Using this
technique coupled with staining for various cell phenotype markers, it
is possible to attribute cytokine production in the CNS to a particular
cell type and to evaluate several different cytokines. FACS analysis
also allows quantification of the actual number of cytokine-producing
cells in the CNS as disease progresses. Cells were isolated from the
CNS and cultured for 5 h with or without
MOG35–55 and a protein transport inhibitor.
Cells were analyzed by FACS after being stained for surface CD4 and
intracellular IFN-, TNF-, and IL-4 to assess the Th phenotype.
The absence of an Ab specific to LT- prevented the analysis of that
cytokine by this technique. Background staining was very low for all
cytokines in cells isolated from control mice immunized with HEL and
pertussis toxin (Fig. 3A). As
expected from the ELISPOT data, no IL-4-producing T cells were apparent
in mice immunized with MOG. In confirmation and extension of the
ELISPOT analyses, MOG35–55-specific
IFN--producing CD4 T cells were detected in the CNS at several time
points after immunization. A representative day 20 experiment is shown
in Fig. 3A. MOG-specific T cells were present at a high
frequency, representing 17% of total CD4 T cells. IFN--producing T
cells were also detected in the CNS after culture with medium alone,
although at a much lower level (3%). These cells were presumably
activated by presentation of endogenous
MOG35–55.
MOG35–55-specific T cells in the CNS also
produced TNF-, as shown in Fig. 3A.
TNF-+ MOG-specific T cells were present at a
frequency similar to that of IFN-+ T cells.
CD4 T cells were the predominant source of IFN- in the CNS, although
there was a minor population of non-CD4 cells (<1% even at the peak
of disease). However, in addition to the T cells that produced TNF-,
we also noticed a substantial population of CD4-negative cells that
produced this cytokine. This population of CD4-negative cells expressed
Mac-1 (Fig. 3B), a marker for resident CNS microglia and
infiltrating macrophages. Mac-1+
TNF-+ cells were detected at a similar
frequency (8% of total cells) when cultured with or without
MOG35–55. This is in contrast to the
Mac-1-negative TNF-+ population, likely to be
CD4 T cells (Fig. 3B). Double staining with Mac-1 and CD4
revealed few cells that coexpressed these molecules (between 0.5
and 1%).
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and IFN- production in the CNS
The kinetics of cytokine production by different cell types was
analyzed throughout the course of disease. By days 7 and 10 after
immunization, a few IFN--producing CD4 Th1 cells could be detected
in the CNS by FACS analysis. These MOG-specific cells represented
3% of total CD4 T cells and were detected even before clinical
signs of disease were apparent (Fig. 4).
At these early time points, even though cells were detected by FACS,
there was relatively little inflammation apparent in the CNS by
hematoxylin and eosin staining. There was a marked increase in the
frequency of MOG35–55-specific T cells on days
15 and 20. The peak of MOG35–55-specific T cell
infiltration (17% of total CD4 T cells) occurred on day 20 and
declined thereafter. By day 40, the number of MOG-specific T cells was
significantly reduced to 6%. In fact, at this late time, there was a
striking reduction in all of the cell types that infiltrate the CNS. In
addition to the FACS data presented here, this was apparent by
immunohistochemistry and hematoxylin and eosin staining (data not
shown). TNF- production by T cells was also examined at selected
time points and was identical to that of IFN-. The kinetics of
TNF-+ Mac-1+ cell
accumulation in the CNS roughly paralleled that of CD4 Th1 cells.
Mac-1+ TNF-+ cells were
detected at early times (days 7 and 10), peaked at days 15 and 20, and
fell off at day 40 (Fig. 4).
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sequentially in the CNS
TNF-+ Mac-1+ cells
were detected early in the CNS, before the onset of clinical signs of
disease. Mac-1 stains both microglial cells as well as blood-derived
infiltrating macrophages. We reasoned that microglial-derived cytokines
could be important in the early phase of disease in recruiting other
cell types into the CNS. To distinguish between parenchymal microglia
and blood-derived macrophages, Mac-1+ cells were
stained with CD45. Parenchymal microglia are
Mac-1+ CD45low whereas
macrophages are Mac-1+
CD45high. The CD45high
population may also contain some activated microglia, since microglial
activation can result in an increase in CD45 expression
(36).
On days 7 and 10 after immunization, >60% of the
Mac-1+ cells were CD45low
microglia, and they were the primary source of
Mac-1+-derived TNF- (Fig. 5). The TNF- production by
Mac-1+ cells in the CNS was dependent upon
immunization with MOG35–55, since few cells
isolated from mice immunized with HEL and supplemented with pertussis
toxin were TNF-+ (Fig. 5A). Around
day 14 or 15 after MOG immunization, there was a switch in the
proportion of CD45high
Mac-1+ cells, which increased to 80% (Fig. 5A). These cells produced TNF- (Fig. 5B). The
influx of macrophages into the CNS closely paralleled the pattern of T
cell infiltration, with day 15 also marking a large increase in the
number of MOG35–55-specific T cells in the CNS.
Macrophage infiltration continued, and, by day 20, most of the
Mac-1-derived TNF- was produced by CD45high
macrophages. By day 40, the frequency of Mac-1+
CD45high cells was reduced, and this resulted in
a corresponding decrease in the percentage of TNF- that was derived
from this population (Fig. 5B). Note that late in the
disease, there was a low level of TNF- present in the CNS (Fig. 4),
and this reduced amount was produced by resident microglia (Fig. 5B).
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Experiments were conducted to investigate the mechanism by which
the large influx of macrophages into the CNS occurred. We investigated
the possibility that there was an up-regulation of a
macrophage-specific chemokine in the CNS at an early time after
immunization. RNase protection assays on total RNA isolated from the
brain and spinal cord performed day 8 after immunization with
MOG35–55 showed expression of MCP-1, RANTES, and
IP-10 (Fig. 6). These chemokines were
apparent even before extensive mononuclear cell infiltration and could
be of parenchymal origin. The increase in CNS chemokine expression was
dependent upon immunization with MOG, since neither normal animals
(Fig. 6) nor those immunized with CFA and treated with pertussis toxin
(data not shown) expressed any of the chemokines detected with this
probe set. With overexposure of the autoradiogram, MIP-1 was also
weakly detected in one experiment 10 days after immunization with
MOG35–55. At later time points, the pattern of
chemokine expression was considerably more complex in that mRNAs for
many additional chemokines, including MIP-1, MIP-1ß, and MIP-2,
could be detected (data not shown). The array was quite similar to that
seen when RNA was analyzed from total lymph node cells, suggesting that
later in disease, the infiltrating cells themselves were contributing
to chemokine expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
from different cell types at distinct times in the disease process.
Early on, CD4 T cells and microglia predominate, whereas later there is
continued expression by CD4 T cells and a switch to macrophages. At the
peak of disease, CD45high macrophages outnumber
microglia. This has also been demonstrated to occur in rat MBP-induced
EAE using bone marrow chimeras, where hematogenous donor macrophages
outnumbered resident host-derived microglia in the spinal cord 4- to
7-fold (37). The early expression of the chemokines MCP-1,
RANTES, and IP-10 in the CNS provides a mechanism for the later influx
of macrophages and T cells. All three of these chemokines attract T
cells (38, 39, 40). In addition, MCP-1, and to a lesser extent
RANTES and IP-10, also attract monocytes (41, 42, 43). The
analysis of cytokines in the CNS reveals production of IFN- and
TNF- by CD4 Th1 cells that parallels the disease course with a peak
around day 20 and then a gradual waning. These data do not reveal
substantial production of these cytokines by CD8 cells, NK cells, or B
cells. Cytokine production by astrocytes was not examined here, since
the protocol for isolating CNS mononuclear cells excludes them
(44). The kinetics of TNF- production is particularly
interesting in that this cytokine was produced not only by infiltrating
T cells, but also early on by microglia. There was a later switch to
TNF- production by macrophages and then the decline seen in T cells.
The population defined here as CD45high
macrophages may also contain some activated microglia, since microglial
activation can result in an increase in CD45 expression
(36). However, we consider this to be unlikely since the
same study demonstrated that the extent of the increase in CD45
expression is not sufficient to result in an overlap of the two
populations. Several previous studies have addressed cytokine and chemokine production in EAE, although most have considered them in non-MOG models, and none have examined them in detail at the kinetic and cellular level employed here. Our results indicating a poor representation of Th2 cytokines in the spleen, and the absence in the CNS, once again suggest that MOG EAE in the mouse is not dependent on Ab production (2, 6). Several studies have suggested that IL-4 plays a down-regulatory role in EAE (45, 46, 47), although none were conducted with the MOG model. Our studies with that model do not support a role for IL-4 and are in agreement with that of Di Rosa et al. (48), who showed that IL-4 production by CNS-infiltrating cells was low or undetectable during remission of EAE induced with PLP139–157 in SJL mice. We have not found convincing evidence that certain other cytokines that have been implicated either positively or negatively in EAE play a role in mice not subject to genetic manipulation. These additional cytokines include IL-6 and IL-10. IL-6-/- mice are completely resistant to clinical signs of the disease (6, 49, 50, 51), whereas IL-10-/- mice are even more susceptible, developing a more severe EAE than their WT littermates. Using the FACS analysis employed here, we have not detected any significant expression of IL-6 or IL-10 protein in the CNS.
Other studies have evaluated cytokine mRNA production in the CNS during
EAE. Renno et al. (16) evaluated cytokine mRNA by RT-PCR
from cells isolated from the CNS 17 days after immunization of SJL mice
with MBP. They report that T cells in the CNS produce IFN- and IL-2,
but not TNF-. This is in contrast to our studies that demonstrate
TNF- protein production by CD4 T cells. In agreement with our
studies, Renno et al. (16) demonstrate TNF- mRNA in CNS
microglia and macrophages. However, they did not detect the switch in
populations noted in our studies, in all probability because they
concentrated on only one time point. Okuda et al. (12)
examined cytokine mRNA expression in the CNS of mice with EAE induced
by immunization with recombinant human MOG. Similar to our studies,
they detected the presence of mRNA for the Th1 cytokines TNF-,
LT-, and IFN-. However, contrary to our studies, they reported
the presence of the Th2 cytokines IL-4 and IL-10 at the severe early
phase of the disease and also noted a further increase in IL-10 in the
chronic late phase of disease. This discrepancy could be due to the
different Ags employed, or to the different techniques used to examine
cytokine expression. Okuda et al. (12) used PCR-based
methods, which are extremely sensitive, and examined cytokine mRNA and
not protein. Additionally, they were using recombinant human MOG
protein and not murine peptide to induce disease.
As noted above, several studies have evaluated chemokines in EAE, but
none thus far in the model of MOG EAE described here. The general
conclusion of these previous studies is that several chemokines are
activated in a pattern that varies temporally and spatially. In several
studies, MIP-1 is detected after disease onset and shown to
correlate with EAE severity, with less emphasis placed on MCP-1,
RANTES, and IP-10 (52, 53). In addition, disease induced
by adoptive transfer of PLP T cells can be prevented by treatment with
an Ab to MIP-1, suggesting a crucial role for this chemokine
(52). In other studies, the chemokines MCP-1, RANTES, and
IP-10 are activated. In the report by Berman et al. (54),
MCP-1 protein was detected at the onset of inflammation in
myelin-induced EAE in the Lewis rat . The MCP-1 expression was
interpreted to correlate with the late influx of macrophages. These
authors conclude that MCP-1 is a mediator of macrophage infiltration in
EAE, a conclusion supported by our data. Two other studies implicate
MCP-1 in multiple sclerosis (55, 56). An additional study
using a variant of the MOG model implicates C10 in macrophage
recruitment (27). This chemokine was not analyzed here.
The cellular origin of chemokines in all models and the nature of
several additional lymphoid organ specific chemokines has not been
determined.
The data provided here, taken along with those obtained with
gene-targeted mice, provide a framework for elucidating the process of
inflammation in EAE. We define three nearly simultaneous early events:
Th1 T cell invasion, chemokine activation, and TNF- production by
microglia. Which event is first? Are the processes independent? We
propose that the early infiltrating Th1 cells are key to the later
events. Frei et al. (57) have shown that IFN-, one of
the Th1 products analyzed here, activates microglia to produce TNF-,
and Renno et al. (16) have demonstrated that Th1 cytokine
supernatants induce TNF- mRNA from CD45low CNS
cells. We suggest that MOG-activated T cells enter the CNS and activate
microglia which produce TNF-, leading to the induction of VCAM and
ICAM. These adhesion molecules facilitate the traffic of mononuclear
cells across the endothelium. This may explain the phenomenon seen in
MOG-immunized TNF--/- mice. It may be that the initial production
of TNF- is crucial for the movement of cells into the parenchyma. In
its absence, cells remain in the perivascular region. Nearly concurrent
with T cell invasion and TNF- production by microglia is the
induction of chemokines. As noted above, LT- and TNF- induce
MCP-1 in vitro, and these cytokines are likely to induce MCP-1 to
attract the later influx of macrophages. Macrophages also produce
TNF-, inducing more adhesion molecules and infiltration.
One question not answered here is how the process of inflammation in EAE is limited. Despite the continued presence of Th1 Ag-specific cells in the spleen at 40 days, these cells are no longer found at high levels in the CNS. There are several nonmutually exclusive explanations for this limitation. Although IL-4 was not induced late in disease, it is possible that other anti-inflammatory cytokines such as TGF-ß may be induced. This could occur in combination with a lack of recruitment into the CNS late in disease due to a decrease in the expression of chemokines and adhesion molecules. Another possibility is the exhaustion of infiltrating cells due to apoptosis. Many studies have demonstrated apoptosis of infiltrating cells in the CNS of animals with EAE (58, 59, 60). Recently, a study was published that demonstrated a role for the Fas ligand in the recovery from EAE. Sabelko-Downes et al. (61) showed that Fas ligand-deficient recipients of WT MBP-specific lymphocytes developed prolonged clinical signs of disease. In addition, these mice had an increased number of CD4 T cells present in the CNS late in disease, suggesting a role for Fas ligand in curtailing the expansion of activated Fas+ lymphocytes. It has also been postulated that B cells or their products could play a protective immunoregulatory role in EAE (reviewed in Ref. 7). The limitation of inflammation seen in the CNS could also be the result of a decrease in Ag-presenting capability due to lower levels of MHC II or costimulatory molecules. The capabilities of APC in the CNS at various times in disease are currently under investigation.
The data provided here emphasize that EAE is a dynamic process with
ordered sequential contributions by individual cells and their
products. One of the more fascinating observations is that TNF- is
produced by three different populations of cells at different times in
the disease. The data provide several potential cellular, chemokine,
and cytokine targets for therapeutics that affect the disease at
different times in the process, allowing the lessons learned from this
model to be applied to human inflammatory diseases.
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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. E-mail address:
3 Abbreviations used in this paper: BBB, blood-brain barrier; EAE, experimental autoimmune encephalomyelitis; LT, lymphotoxin; MCP, monocyte chemotactic protein; MBP, myelin basic protein; PLP, proteolipid protein; MOG, myelin oligodendrocyte glycoprotein; WT, wild type; IP-10, IFN-inducible protein 10; SSPE, standard saline citrate phosphate/EDTA; ELISPOT, enzyme-linked immunospot; HEL, hen egg lysozyme; SFC, spot-forming cell.
Received for publication August 8, 1999. Accepted for publication October 13, 1999.
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References |
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) exhibited
cytotoxic activity when cultured with WEHI 164 cells. B,
Northern blot analysis of total RNA isolated from splenocytes cultured
in the presence or absence of MOG35–55. Blots were
hybridized with probes specific for murine TNF-
)
and IL-4 (
) after MOG35–55 or anti-CD3 stimulation
on day 20 (A) or day 40 (B) after
immunization. CNS-isolated cells produced SFC for only IFN-
