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Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, IL-10, and IL-4
mRNA during the preclinical phase, and their levels continued to
increase throughout the duration of the chronic-progressive disease
course. These data correlated with the continued presence of both
CD4+ T cells and F4/80+ macrophages within the
CNS infiltrates. In contrast, SJL/J mice with
PLP139–151-induced R-EAE displayed a biphasic pattern of
CNS expression for the proinflammatory cytokines, IFN- and TNF-,
with expression peaking at the height of the acute phase and
relapse(s). This pattern correlated with dynamic changes in the
CD4+ T cell and F4/80+ macrophage populations
during relapsing-remitting disease progression. Interestingly, IL-4
message was undetectable until disease remission(s), demonstrating its
potential role in the intrinsic regulation of ongoing disease, whereas
IL-10 was continuously expressed, arguing against a regulatory role in
either disease. These data suggest that the kinetics of cytokine
expression together with the nature of the persistent inflammatory
infiltrates are major contributors to the differences in clinical
course between TMEV-IDD and R-EAE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the differences in the clinical course of these diseases, recent evidence suggests that the underlying immunopathologic mechanisms in these two models are similar. We have reported previously that in active PLP139–151-induced R-EAE, the acute disease episode is mediated by T cells specific for the disease-initiating PLP139–151 epitope, whereas the first clinical relapse is associated with reactivity to the non-cross-reacting PLP178–191 epitope, a process termed epitope spreading (8, 9). Recent data indicate that epitope spreading may also contribute to the chronic nature of TMEV-IDD. Available evidence indicates that myelin damage is initiated by TMEV-specific CD4+ T cells targeting CNS-persistent virus Ag, while the chronic stage of the disease involves CD4+ myelin epitope-specific T cells primed via epitope spreading (10).
It is well documented that CD4+ Th1 cells and their proinflammatory cytokines are suspected to be important in the pathogenesis of multiple sclerosis (11), and necessary for the induction of both TMEV-IDD (12, 13, 14, 15, 16, 17) and R-EAE (7, 18, 19). In contrast to the inductive phase, there is conflicting information regarding the ongoing disease process and its regulation in both models. In R-EAE, cytokines such as IL-4, IL-10, and TGF-ß have been implicated in disease resolution; however, these data are conflicting (20, 21, 22). In the present study, we therefore sought to characterize the nature of the persistent cellular infiltrate within the CNS in both TMEV-IDD and R-EAE and attempt to correlate it with the temporal changes in the pattern of cytokine expression and disease progression.
Using competitive quantitative PCR (CQ-PCR), we show that during
TMEV-IDD, the mRNA for IFN-, TNF-, IL-10, and IL-4 were first
detectable before or coinciding with the onset of clinical signs. These
data correlated with immunohistochemical evidence from spinal cord
sections that indicated both persistent macrophage and T cell
infiltrates as disease and demyelination progressed. The cytokine
profile and the nature of cellular infiltrate were markedly different
during the clinical course of R-EAE, wherein the maximal expression of
the proinflammatory cytokines IFN- and TNF- correlated with the
clinical peaks of the acute phase and relapse(s). Unexpectedly, we did
not find an association of IL-10 with remission, as was previously
described (21), but rather found an increase in IL-4 message that was
only detectable in mice recovering from the acute phase/relapse or
those in complete remission. Most interestingly, these observed
differences in cytokine expression correlated with dramatic changes in
both the CD4+ T cell and F4/80+ macrophage
populations within the CNS. Collectively, these data indicate that the
combination of the cellular infiltrate and temporal cytokine expression
within the CNS is largely responsible for the observed differences in
clinical course of TMEV-IDD and R-EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice, 6 to 7 wk old, were purchased from Harlan Laboratories (Indianapolis, IN). All mice were housed in the Northwestern animal care facility (Chicago, IL) and maintained on standard laboratory chow and water ad libitum. Severely paralyzed mice were afforded easier access to food and water.
Peptides
PLP139–151 (HSLGKWLGHPDKF) was purchased from Peptide Synthesis Core Laboratories (University of North Carolina, Chapel Hill). Amino acid composition was verified by laser desorption mass spectroscopy, and purity (>98%) was assessed by HPLC.
Induction and clinical evaluation of R-EAE
Mice were immunized with 40 µg PLP139–151 in CFA. Clinical severity was assessed daily and assigned a numerical grade of 0 to 5, as previously described (7). The data are plotted as the mean clinical score for each group of animals at the time of sacrifice. Each group of animals displayed clinical signs representative of the entire population.
Induction and clinical evaluation of TMEV-IDD
Mice were anesthetized with methoxyflurane (Mallinckrodt Veterinary, Mundelein, IL) and inoculated in the right cerebral hemisphere with 2.9 x 106 plaque-forming units of TMEV, strain BeAn 8386, in 30 µl DMEM. Mice were examined two to three times per week for the development of chronic gait abnormalities and spastic paralysis indicative of demyelination, and assigned a clinical score of 0 to 6, as follows: 0, asymptomatic; 1, mild waddling gait; 2, severe waddling gait, intact righting reflex; 3, severe waddling gait, spastic hind limb paralysis, impaired righting reflex; 4, severe waddling gait, spastic hind limb paralysis, impaired righting reflex, mild dehydration and/or malnutrition; 5, total hind limb paralysis, severe dehydration and/or malnutrition; and 6, death. The data are plotted as the mean clinical score for each group of animals at the time of sacrifice. Each group of animals displayed clinical signs representative of the entire population.
Isolation of RNA
At varying time points following induction of R-EAE or TMEV-IDD, mice (two per time point) were anesthetized and perfused through the left ventricle with 50 ml of PBS. Spinal cords were extruded by flushing the vertebral canal with PBS and then rinsed in PBS. Tissues were forced through a 100-mesh stainless steel screen to give a single cell suspension and pelleted by centrifugation (500 x g) for 5 min at 4°C. The pellets were resuspended vigorously in 16 ml 4 M guanidinium isothiocyanate/50 mM Tris-Cl (pH 7.5)/25 mM EDTA (Life Technologies, Gaithersburg, MD), and 1% 2-ME and 0.5% N-lauroylsarcosine (Sigma-Aldrich, St. Louis, MO). Shearing of DNA was facilitated by forcing the resulting suspension repeatedly through a 23-gauge needle. Total RNA was isolated by high-speed gradient centrifugation (27,000 rpm) of 8 ml lysate through a 3 ml 5.7 M CsCl pad using a SW41 swinging bucket rotor for 20 h at 4°C. The resulting RNA pellet was resuspended to a final concentration of 1 µg/µl with diethylpyrocarbonate-treated water and stored in aliquots at -70°C.
First strand cDNA synthesis
First strand cDNA was generated from 2 µg total RNA using Advantage-RT Kit (Clontech, Palo Alto, CA) using 20 pmol oligo(dT) primer, per the manufacturer’s provided protocol, in a total volume of 20 µl. Following first strand synthesis, each cDNA sample was brought to a final volume of 100 µl with distilled water.
CQ-PCR and densitometry
The PCR for all cytokines was performed with a previously described cytokine polycompetitor plasmid (pLOC) (23). The amount of pLOC, cDNA, and final MgCl2 concentration was determined empirically for each cytokine tested. Additions of pLOC ranged from 10 µl of a 0.5 ng/ml stock (IL-4) to 10 µl of a 250 ng/ml stock (hypoxanthine phosphoribosyltransferase; HPRT) per each 50-µl PCR reaction. Final PCR conditions included 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 2.5 to 5 mM MgCl2, 2 mM dNTPs, 100 pmols of each 5' and 3' gene-specific primer, 1 U Taq polymerase (Qiagen, Chatsworth, CA), and 5 to 10 µl diluted cDNA. The primers were synthesized by Life Technologies, and amplify both the competitor plasmid and wild-type cDNA. Cycling conditions were 94°C, 40 s; 60°C, 20 s; and 72°C, 40 s, for a total of 30 cycles, linked to a final 72°C extension program for 3 min and then to a final 4°C soak program. PCR products were run on an ethidium bromide-containing 2% agarose gel and illuminated using a UV light source, then photographed using Polaroid type 667 film. Gel images were then scanned into Adobe Photoshop using an Epson ES 1200-C scanner and imported as TIFF files into Kodak 1D Digital Science for densitometry. The sum intensity and band area were determined for each competitor/wild-type amplicon pair, and a ratio of wild-type/competitor intensity was determined for all cytokines and adjusted to each sample’s housekeeping gene, HPRT. Sum intensity values for each wild-type amplicon were also adjusted for the difference in size to that of the competitor band to account for increased ethidium bromide incorporation in the higher m.w. competitor band. Attomoles of target cDNA were determined by multiplying the adjusted wild-type/competitor ratio with the amount of pLOC added to each sample and then adjusted for the amount of cDNA used in each PCR amplification.
Immunohistochemistry and quantitation of CD4+ and F4/80+ cells
For immunohistochemistry, two mice per time point were perfused with PBS, as described in a previous Materials and Methods section. Spinal cords were dissected out and flash frozen in O.C.T. compound (Fisher, Itasca, IL). Random 6-µm sections from the lumbar region of the spinal cord were cut and fixed in acetone. For CD4 staining, slides were incubated with biotinylated anti-L3T4 (PharMingen, San Diego, CA), and for macrophage staining, with biotinylated anti-F4/80 (Caltag, San Francisco, CA), for 1 h at room temperature. All slides were then rinsed and processed through a secondary incubation with avidin-biotin-peroxidase complex (Vector Labs, Burlingame, CA). Color was developed using diaminobenzidine (DAB; Kirkegaard & Perry Laboratories, Gaithersburg, MD), counterstained using contrast green, dehydrated, and mounted in Accu-Mount-60 (Stephens Scientific, Riverdale, NJ). Following CD4 and F4/80 immunohistochemistry, for each time point, eight serial lumbar sections were analyzed using a grid reticle viewfinder at x100 magnification. At this magnification, each grid measures a 9-mm2 area, and represents approximately 5 to 7% of the total white matter. A total of twelve 9-mm2 areas was measured per cord representing an equal distribution between ventral, dorsal, and both lateral regions. Gray matter was not analyzed because neither R-EAE nor TMEV-IDD typically present with infiltration into this region of the CNS. Data are presented in tabular form as the mean number of positive cells ± the SEM for the entire 27-mm2 ventral, dorsal, and lateral regions analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL mice were monitored for the development of clinical signs of
demyelination following infection with TMEV and following priming with
PLP139–151/CFA (Fig. 1
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Clinical signs in R-EAE were first evident on day 13 postimmunization,
and disease symptoms rapidly increased, peaking on day 14.
Characteristic of R-EAE, this acute phase was followed by a remission
that lasted through day 19 and the subsequent reappearance of a
clinical relapse that peaked approximately day 26 postimmunization
(Fig. 1A). In contrast to R-EAE, the clinical progression of
TMEV-IDD proceeded along a much slower path. The initial clinical
impairment of a mild waddling gait was not apparent until approximately
33 days PI in TMEV-infected animals. Clinical signs then persisted in a
chronic nature of an ascending and spastic hind limb paralysis until
the termination of the experiment at 100 days PI (Fig. 1B).
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To better understand the observed differences in clinical course between these two disease models, we quantitated the phenotypic nature and localization of the CNS mononuclear infiltrates in situ at varying times during the course of disease using immunohistochemistry. Previous studies have demonstrated that CD4+ T cells are necessary for the induction of both TMEV-IDD (14, 15, 24) and R-EAE (7, 19) in SJL mice. Our analysis was therefore aimed at determining not only the nature and location of the initial infiltrate at disease onset, but also its dynamics as disease progressed.
During the course of R-EAE, the CD4+ T cell and
F4/80+ macrophage infiltrate displayed very dramatic
changes. The CD4+ population was scarcely detectable
(0.73–2.19 positive cells/spinal cord region) during the preclinical
phase at 5 to 6 days postimmunization (Fig. 2A), but increased by
approximately 20-fold in all analyzed areas of white matter at the peak
of the acute phase of disease 3 days thereafter (Fig. 2B and
Table I). This increase in the number of
CD4+ cells correlated with the rapid onset of clinical
disease (Fig. 1
A). As the mice progressed into a spontaneous
full remission approximately 18 days postimmunization, the
CD4+ T cell levels decreased dramatically to levels
(1.21–5.86 positive cells/region) only slightly greater than those
seen during the preclinical phase (Fig. 2C), but then
dramatically increased (18.67–41.83 positive cells/section) as the
mice began to clinically relapse (Fig. 2D). It is
interesting to note that as the clinical scores began to decline as the
mice entered remission (remitting), the number of CD4+
cells had already decreased significantly, despite maintaining a
clinical score of at least 1 (Table I). In addition, the amount and
pattern of the infiltrate were similar in both the acute phase and the
relapse, and were detectable to the same degree in all spinal cord
regions (ventral, lateral, and dorsal) analyzed, indicating a
widespread targeting of myelin destruction. The F4/80+
macrophage population progressed in a similar biphasic manner as
compared with the CD4+ T cells, peaking at the height of
the acute and relapsing clinical episodes. However, the relative
numbers of F4/80+ cells were two- to threefold increased
over the numbers of CD4+ T cells during both the acute
phase and the relapse (Fig. 3,
A–D, and Table I). The localization of the
F4/80+ macrophages in the CNS lesions generally overlapped
with the CD4+ T cells. However, one unexpected observation
was that during the first relapse there were significantly more
F4/80+ cells detected than during the acute phase. The
significance of this observation may relate to the onset of epitope
spreading of CD4+ T cell responses to endogenous myelin
epitopes and the subsequent induction of additional relapses.
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E and 3E and Table II). The numbers of CD4+ T
cells and F4/80+ macrophages increased by twofold by day 79
PI, and correlate with a corresponding increase in disease severity
(Fig. 1B). As disease progressed to day 104 PI, the numbers
of CD4+ T cells and F4/80+ macrophages remained
elevated, but had decreased slightly, most likely as a result of
dehydration due to severe paralysis. In contrast to R-EAE, the
localization of the infiltrate was not as widespread, but rather
restricted primarily to the ventral spinal cord region as the
infiltrates persisted at higher levels in this region. An increase in
CD4+ and F4/80+ cells was, however, detected in
all sections at various times during early disease progression. The
significance of this finding is not apparent, but may be related to the
progression of a natural viral-induced demyelinating disease, wherein
virus persists in CNS-resident macrophages/microglia versus an
experimentally induced demyelinating model as in R-EAE.
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Temporal analysis of Th1-dependent proinflammatory cytokine mRNA in TMEV-IDD and R-EAE by CQ-PCR
The data to this point indicate that the clinical presentation and
the temporal changes in the inflammatory infiltrates in TMEV-IDD and
R-EAE differ dramatically, despite the presence of similar immune
populations in roughly the same ratios. We next sought to determine
whether the pattern of proinflammatory cytokine expression in the
target organ of the CNS would provide additional insight into the
differences between these two demyelinating models. Total RNA was
isolated from the spinal cords of SJL mice at varying times after
induction of R-EAE or TMEV-IDD, and subjected to CQ-PCR to determine
message levels for the proinflammatory cytokines IFN- and TNF-.
Figure 4A shows a
representative ethidium bromide-stained agarose gel profile of the
CQ-PCR for IFN-, and TNF- for both R-EAE and TMEV-IDD. In
CQ-PCR, the larger amplicon is that of the competitor, while the
lower band is that specifically amplified from target cDNA. The
concentration of competitor that was used was empirically determined
and was different for each cytokine examined. Densitometry analysis of
the resulting amplified products from IFN- and TNF- CQ-PCR
yielded the quantitative data shown in Figure 5 for spinal cord mRNA levels for mice
undergoing both R-EAE and TMEV-IDD.
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and TNF- mRNA
displayed a biphasic pattern of expression, with increased levels
coinciding with increasing incidence of clinical severity at the
day of onset of initial clinical signs, and at the first relapse (Fig. 5A). Expression of IFN- and TNF- was not detectable
during the preclinical period (5 days postimmunization); however, the
analysis of additional time points 1 to 2 days before the onset of
clinical signs revealed detectable levels of both proinflammatory
cytokines, despite the fact that the animals were still asymptomatic
(data not shown). In contrast, IFN- expression from
TMEV-IDD-affected mice was first detectable as early as day 14 PI,
several weeks before the onset of overt clinical signs (Fig. 5B). TNF- expression lagged slightly behind, being first
detectable at day 33 PI, approximately coincident with the day of
clinical disease onset. Once induced, the levels of both cytokines
continued to increase throughout the chronic phase of disease (last
time point examined, day 103 PI). Similar to IFN- and TNF-, the
expression pattern of lymphotoxin- paralleled the increases in
disease severity during both R-EAE and TMEV-IDD (data not shown).
Overall, the profile for the proinflammatory IFN- and TNF- again
correlated with the nature of the infiltrate and clinical course, being
biphasic in R-EAE and chronic-progressive in TMEV-IDD. Temporal analysis of Th2-dependent antiinflammatory cytokine mRNA expression in TMEV-IDD and R-EAE by CQ-PCR
Recent evidence suggests that the Th2 cytokines, IL-4 and/or
IL-10, may be involved in down-regulation of ongoing R-EAE (21, 22, 25, 26). Employing CQ-PCR, we determined the expression pattern of IL-4 and
IL-10 mRNA in the CNS during both R-EAE and chronic TMEV-IDD.
Representative ethidium bromide-stained agarose gel profiles for both
cytokines are shown in Figure 4B. Similar to the expression
pattern of the proinflammatory cytokines during TMEV-IDD, CQ-PCR
analysis revealed that both IL-4 and IL-10 were detectable before the
onset of clinical signs, 14 to 26 days PI, and their expression
continued throughout the chronic-progressive disease course (Fig. 6B). Overall, in both models,
the amount of detectable Th2-derived cytokine mRNA was approximately
10-fold lower than that observed for the proinflammatory cytokines.
Given this data, it would appear that in the TMEV model, the early and
prolonged expression of Th2 cytokines within the CNS is unable to
down-regulate the Th1 response thought to be responsible for
demyelination and clinical disease.
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A). In contrast to a
previous report (21) using semiquantitative RT-PCR suggesting that
IL-10 may be involved in mediating disease remission in the SJL mouse,
our data indicate that IL-10 expression does not correlate with the
onset of remission. As in TMEV-IDD, IL-10 was detectable at the time of
disease onset and remained elevated through the first remission and
subsequent relapse. In contrast, IL-4 expression was only detectable in
animals remitting from the acute clinical episode or in full remission
from acute disease, suggesting that expression of this cytokine may be
an important intrinsic mechanism involved in mediating disease
remission.
Previous reports have not quantitated the expression of the Th2
cytokines in the CNS of mice with R-EAE beyond the first remission.
Given our interesting observations on the potential regulatory role of
IL-4, we wanted to determine whether pro- and antiinflammatory cytokine
mRNA expression in the CNS or mice with R-EAE would continue to flux as
the animals progressed further in the disease course. Therefore, we
performed another R-EAE time course and followed animals through the
first relapse into a second remission and a subsequent second relapse.
Once again, the pattern of IL-10 expression did not increase or
decrease relative to the disease state, whereas IL-4 was strictly
correlated with disease remission (Fig. 7), and expression of IFN- and TNF-
correlated with increases in clinical severity during the acute phase
and first and second relapses (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical staining and quantitation of absolute cell numbers from the spinal cords of affected mice revealed that, for both models, the majority of the CNS cellular infiltrate was comprised of CD4+ T cells and F4/80+ macrophages. Interestingly, the increases in both of these populations correlated directly with increasing disease severity in R-EAE and TMEV-IDD. These data related to the CD4+ T cells are not surprising given previous reports of their essential role in the induction of both R-EAE (7, 19) and TMEV-IDD (14, 15). Our observations, however, further elaborate on the role of CD4+ T cells. The increased numbers of CD4+ T cells at the peak of acute phase and at the height of the first clinical relapse during R-EAE, as well as their continued presence throughout the chronic phase of TMEV-IDD, implicate them not only in disease induction, but also more importantly, in disease progression. This observation is supported by recent data from our lab demonstrating that PLP139–151, and subsequently PLP178–191-specific T cells, are responsible for mediating the initial acute phase and first clinical relapse of R-EAE, respectively, a phenomenon termed epitope spreading (8, 27). In addition, we and others have also previously demonstrated the importance of virus-specific CD4+ T cells in initiating myelin damage by targeting CNS-persistent virus in TMEV-infected SJL/J mice (12, 13, 15, 16, 17, 28, 29). In contrast, the chronic stage of TMEV-IDD also involves pathologic damage mediated by myelin epitope-specific CD4+ T cells that first arise 2 to 3 wk after the onset of clinical signs of disease via epitope spreading (10).
As stated previously, we also observed that increases in the
F4/80+ macrophage population correlated with increasing
disease severity and with periods of expression of high levels of
TNF- mRNA in the CNS. These results are consistent with previous
histologic evidence showing that activated macrophages/microglia play a
major role in myelin destruction in both disease models (30), and that
TNF- expression is a sensitive marker for activated macrophages
(31). Interestingly, we also observed that the numbers of macrophages
increased to two- to threefold the number of CD4+ T cells
as clinical disease progressed during both R-EAE and TMEV-IDD. This
result is interesting and may point to a pivotal role of this
population in the perpetuation of epitope spreading and the resulting
chronic demyelination. In this regard, we have demonstrated recently
that F4/80+, I-As+ macrophages/microglia
isolated from the spinal cords of mice 90 days PI with TMEV express
high levels of B7 costimulatory molecules (56), and endogenously
activate both virus-specific and myelin epitope-specific Th1 cells in
vitro in the absence of exogenously added Ag (unpublished observation).
Facilitation of epitope spreading by activated macrophages may occur
through continuing active phagocytosis of myelin debris and subsequent
processing and presentation of self epitopes within CNS lesions, as
well as by the production of chemokines (e.g., MIP-1 and MCP-1),
leading to the recruitment of additional peripheral macrophages and T
cells (32, 33), and the production of proinflammatory cytokines (e.g.,
TNF-, IFN-, and IL-12) active in the recruitment (34) and/or
local activation (35) of myelin epitope-specific Th1 cells.
Currently, there is no cell surface molecule that can distinguish
between infiltrating peripheral macrophages and CNS resident microglia.
Therefore, it is possible that our quantitation of F4/80+
cell numbers includes activated microglia as an additional correlate of
disease progression. Additionally, this pattern of increased
macrophages in the first relapse of R-EAE versus the acute phase
correlates well with published observations related to chemokine
production in R-EAE in the SJL mouse. We have shown that CNS expression
of MIP-1 correlated with acute disease development (32, 33), whereas
expression of MCP-1 did not. In contrast, MCP-1 production in the CNS
correlated with relapsing EAE development (33). Moreover,
anti-MIP-1, but not anti-MCP-1, inhibited development of
acute but not relapsing EAE, whereas anti-MCP-1 significantly
reduced the severity of relapsing EAE. This expression pattern is
consistent for a predominant role for MIP-1 in inducing T cell
infiltration into the CNS during disease initiation and a more
predominant role for MCP-1 in directing macrophage infiltration during
relapses.
Interestingly, in both models, the increases in the CD4+
population also paralleled increases in mRNA for both IFN- and
TNF-. These data correlate well with that of others who have
demonstrated previously the integral role of Th1 cells and
proinflammatory cytokines during disease initiation for R-EAE and
TMEV-IDD (18, 36, 37, 38, 39). Our data extend the role of the proinflammatory
cytokines by demonstrating their importance in the perpetuation of
ongoing disease. Additionally, the message for TNF- exceeded that
for IFN- in both models and supports our observations related to the
absolute cell numbers within the infiltrate. As discussed above, this
proinflammatory cytokine is most likely being produced by the
infiltrating F4/80+ macrophages (31), but activated
resident microglia and/or astrocytes within the CNS may also contribute
to its production (40, 41). The resulting increase in the
F4/80+ population during the first relapse of R-EAE also
correlates with the observed increase in TNF- mRNA in comparison
with levels achieved during acute disease. Interestingly, recent
reports have indicated that IFN- (42, 43) and TNF- (38, 44, 45)
are not necessary for the induction of R-EAE, whereas the role of
lymphotoxin- is still in question (38). It is important to note that
all of these studies utilized knockouts on the C57BL/6 background, a
strain that is genetically resistant to EAE induction in the absence of
treatment with pertussis toxin. Regardless, our results indicate that
in the SJL/J mouse the temporal appearance of CD4+ T cell
and F4/80+ macrophage populations, as well as the
expression kinetics of the proinflammatory cytokines IFN- and
TNF- are excellent predictors of the progression of the clinical
disease course of both R-EAE and TMEV-IDD.
In contrast to IFN- and TNF-, the expression of the Th2
cytokines, IL-4 and IL-10, did not correlate with the peaks of clinical
severity. Several previous studies have suggested a role for IL-10 in
R-EAE remission (21, 46). In contrast, our results, which employed
three separate time course experiments, indicated no such correlation,
in that IL-10 mRNA was expressed preclinically and increased as disease
progressed in both R-EAE and chronic-progressive TMEV-IDD. The reasons
for this discrepancy are unclear, but may relate to the use of an
myelin basic protein-adoptive transfer system to induce R-EAE in one
study (21) and the use of mRNA from CNS inflammatory infiltrates in a
Lewis rat EAE model in the other (46). We felt it necessary to analyze
mRNA from the entire spinal cord since cytokines such as TNF- and
IL-10 can also be produced by resident CNS populations such as
microglia and astrocytes, and could therefore contribute to the
resulting R-EAE or TMEV-IDD pathology (40, 41). Our data are consistent
with earlier reports in EAE demonstrating that administration of IL-10
failed to protect mice from the onset or progression of adoptive R-EAE
(25) and in the NOD mouse, wherein expression of transgene IL-10 in
pancreatic ß cells actually accelerated development of diabetes (47).
Current experiments employing immunohistochemistry and in situ PCR are
underway to determine which cell population(s) within the CNS produces
IL-10 during the course of autoimmune and virus-induced
demyelination.
The correlation of peak IL-4 mRNA expression during the first and second remissions is perhaps our most intriguing finding and further supports recent evidence for a role of this cytokine in down-regulation of autoimmune diseases. IL-4 has been reported to regulate EAE in a number of experimental settings, including the natural recovery from disease in the Lewis rat (48), and as an important mediator following tolerance induction by either oral administration of Ag (49) or immunization with altered peptide ligands (50). More recently, it has been reported that specific targeting of IL-4-producing neuroantigen-specific T cell hybridomas to the CNS led to the amelioration of R-EAE (22). Using similar methodology, Mathisen et al. showed that neuroantigen-specific Th2 clones producing constitutive IL-4 and transgene IL-10 could decrease EAE severity (26). Further support for a potential down-regulatory role of IL-4 has come from NOD mouse, in which it has been shown recently that targeted expression of IL-4 within pancreatic ß cells protected these normally susceptible NOD mice from the onset of both insulitis and diabetes (51).
Our findings showing the biphasic expression of IL-4 mRNA corresponding to disease remissions are the first demonstration that IL-4 may play an important role in mediating the intrinsic regulation in the PLP139–151-induced R-EAE system. It is important to note, however, that in the SJL/J mouse, IL-4 mRNA expression within the spinal cord does not always coincide with a decrease in clinical severity. IL-4 expression in TMEV-IDD was detectable as early as 14 days PI, several weeks before the onset of clinical signs, and yet all TMEV-infected animals went onto develop a chronic-progressive demyelinating disease. A similar pattern of IL-4 mRNA expression has also been observed in SJL/J mice infected with the more virulent DA strain of TMEV (52). Given that TMEV persists in macrophages/microglia within the CNS of infected SJL mice (53, 54, 55), it is possible that a local environment is established that favors the continued activation of Th1 responses, e.g., up-regulated IL-12, etc., in spite of the presence of Th2 cytokines.
Collectively, our findings support an important role for
CD4+Th1 cells and F4/80+ macrophages, as well
as the associated proinflammatory cytokines IFN- and TNF- as
important mediators not only of disease induction, but also of disease
progression during the distinct clinical courses of both R-EAE and
chronic-progressive TMEV-IDD. Our data also suggest a significant role
for IL-4, but not IL-10, in promoting R-EAE remission. Finally, our
data indicate that examination of the cytokine profile in the target
organ during the progression of autoimmune diseases may provide
insights into not only the underlying immunopathologic mechanism(s),
but also suggest possible therapeutic strategies aimed at complementing
the immune system’s intrinsic efforts to down-regulate the autoimmune
pathology.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611. E-mail address:
3 Abbreviations used in this paper: TMEV-IDD, Theiler’s murine encephalomyelitis virus-induced demyelinating disease; CNS, central nervous system; CQ-PCR, competitive quantitative PCR; EAE, experimental autoimmune encephalomyelitis; HPRT, hypoxanthine phosphoribosyltransferase; MCP, monocyte-chemotactic protein; MIP, macrophage-inflammatory protein; NOD, nonobese diabetic; PI, postinfection; PLP, proteolipid protein; R-EAE, relapsing EAE.
Received for publication May 14, 1998. Accepted for publication June 12, 1998.
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and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations?. Acta Neurol. Scand. 78:318.[Medline]
) and lymphotoxin (TNF-ß). Curr. Opin. Immunol. 4:327.[Medline]
in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphoctye differentiation. J. Leukocyte Biol. 62:681.[Abstract]
production by myelin basic protein-specific T cell clones correlates with encephalitogenicity. Int. Immunol. 2:539. expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis: regulation by Th1 cytokines. J. Immunol. 154:944.[Abstract]
in murine astrocyte cell cultures. Immunology 78:399.[Medline]
gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
and lymphotoxin are not required for induction of acute experimental autoimmune encephalomyelitis. J. Exp. Med. 185:2177.This article has been cited by other articles:
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  A. Kroner, N. Schwab, C. W. Ip, S. Ortler, K. Gobel, K.-A. Nave, M. Maurer, R. Martini, and H. Wiendl Accelerated Course of Experimental Autoimmune Encephalomyelitis in PD-1-Deficient Central Nervous System Myelin Mutants Am. J. Pathol., June 1, 2009; 174(6): 2290 - 2299. [Abstract] [Full Text] [PDF]
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 I. L. King, T. L. Dickendesher, and B. M. Segal Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease Blood, April 2, 2009; 113(14): 3190 - 3197. [Abstract] [Full Text] [PDF]
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 M. B. Wax, G. Tezel, J. Yang, G. Peng, R. V. Patil, N. Agarwal, R. M. Sappington, and D. J. Calkins Induced Autoimmunity to Heat Shock Proteins Elicits Glaucomatous Loss of Retinal Ganglion Cell Neurons via Activated T-Cell-Derived Fas-Ligand J. Neurosci., November 12, 2008; 28(46): 12085 - 12096. [Abstract] [Full Text] [PDF]
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 L. Piccio, J. L. Stark, and A. H. Cross Chronic calorie restriction attenuates experimental autoimmune encephalomyelitis J. Leukoc. Biol., October 1, 2008; 84(4): 940 - 948. [Abstract] [Full Text] [PDF]
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 W. Elyaman, E. M. Bradshaw, Y. Wang, M. Oukka, P. Kivisakk, S. Chiba, H. Yagita, and S. J. Khoury Jagged1 and Delta1 Differentially Regulate the Outcome of Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2007; 179(9): 5990 - 5998. [Abstract] [Full Text] [PDF]
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B. Zhu, Y. Bando, S. Xiao, K. Yang, A. C. Anderson, V. K. Kuchroo, and S. J. Khoury CD11b+Ly-6Chi Suppressive Monocytes in Experimental Autoimmune Encephalomyelitis J. Immunol., October 15, 2007; 179(8): 5228 - 5237. [Abstract] [Full Text] [PDF]
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 D. J. Mekala, R. S. Alli, and T. L. Geiger IL-10-dependent infectious tolerance after the treatment of experimental allergic encephalomyelitis with redirected CD4+CD25+ T lymphocytes PNAS, August 16, 2005; 102(33): 11817 - 11822. [Abstract] [Full Text] [PDF]
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 N. M. Druzhyna, S. I. Musiyenko, G. L. Wilson, and S. P. LeDoux Cytokines Induce Nitric Oxide-mediated mtDNA Damage and Apoptosis in Oligodendrocytes: PROTECTIVE ROLE OF TARGETING 8-OXOGUANINE GLYCOSYLASE TO MITOCHONDRIA J. Biol. Chem., June 3, 2005; 280(22): 21673 - 21679. [Abstract] [Full Text] [PDF]
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A. P. Kohm, J. S. Williams, A. L. Bickford, J. S. McMahon, L. Chatenoud, J.-F. Bach, J. A. Bluestone, and S. D. Miller Treatment with Nonmitogenic Anti-CD3 Monoclonal Antibody Induces CD4+ T Cell Unresponsiveness and Functional Reversal of Established Experimental Autoimmune Encephalomyelitis J. Immunol., April 15, 2005; 174(8): 4525 - 4534. [Abstract] [Full Text] [PDF]
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D. J. Mekala, R. S. Alli, and T. L. Geiger IL-10-Dependent Suppression of Experimental Allergic Encephalomyelitis by Th2-Differentiated, Anti-TCR Redirected T Lymphocytes J. Immunol., March 15, 2005; 174(6): 3789 - 3797. [Abstract] [Full Text] [PDF]
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J. L. Croxford, H. A. Anger, and S. D. Miller Viral Delivery of an Epitope from Haemophilus influenzae Induces Central Nervous System Autoimmune Disease by Molecular Mimicry J. Immunol., January 15, 2005; 174(2): 907 - 917. [Abstract] [Full Text] [PDF]
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A. P. Kohm, J. S. Williams, and S. D. Miller Cutting Edge: Ligation of the Glucocorticoid-Induced TNF Receptor Enhances Autoreactive CD4+ T Cell Activation and Experimental Autoimmune Encephalomyelitis J. Immunol., April 15, 2004; 172(8): 4686 - 4690. [Abstract] [Full Text] [PDF]
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 E. L. Oleszak, J. R. Chang, H. Friedman, C. D. Katsetos, and C. D. Platsoucas Theiler's Virus Infection: a Model for Multiple Sclerosis Clin. Microbiol. Rev., January 1, 2004; 17(1): 174 - 207. [Abstract] [Full Text] [PDF]
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 J. Imitola, E. Y. Snyder, and S. J. Khoury Genetic programs and responses of neural stem/progenitor cells during demyelination: potential insights into repair mechanisms in multiple sclerosis Physiol Genomics, August 15, 2003; 14(3): 171 - 197. [Abstract] [Full Text] [PDF]
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 A. D. Salama, T. Chitnis, J. Imitola, M. J. I. Ansari, H. Akiba, F. Tushima, M. Azuma, H. Yagita, M. H. Sayegh, and S. J. Khoury Critical Role of the Programmed Death-1 (PD-1) Pathway in Regulation of Experimental Autoimmune Encephalomyelitis J. Exp. Med., July 7, 2003; 198(1): 71 - 78. [Abstract] [Full Text] [PDF]
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 J. P. Palma, D. Kwon, N. A. Clipstone, and B. S. Kim Infection with Theiler's Murine Encephalomyelitis Virus Directly Induces Proinflammatory Cytokines in Primary Astrocytes via NF-{kappa}B Activation: Potential Role for the Initiation of Demyelinating Disease J. Virol., June 1, 2003; 77(11): 6322 - 6331. [Abstract] [Full Text] [PDF]
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 T Seifert, B C Kieseier, S Ropele, S Strasser-Fuchs, F Quehenberger, F Fazekas, and H-P Hartung TACE mRNA expression in peripheral mononuclear cells precedes new lesions on MRI in multiple sclerosis Multiple Sclerosis, December 1, 2002; 8(6): 447 - 451. [Abstract] [PDF]
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A. P. Kohm, P. A. Carpentier, H. A. Anger, and S. D. Miller Cutting Edge: CD4+CD25+ Regulatory T Cells Suppress Antigen-Specific Autoreactive Immune Responses and Central Nervous System Inflammation During Active Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2002; 169(9): 4712 - 4716. [Abstract] [Full Text] [PDF]
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A. Y. Karulin, M. D. Hesse, H. C. Yip, and P. V. Lehmann Indirect IL-4 Pathway in Type 1 Immunity J. Immunol., January 15, 2002; 168(2): 545 - 553. [Abstract] [Full Text] [PDF]
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A. K. Singh, M. T. Wilson, S. Hong, D. Olivares-Villagomez, C. Du, A. K. Stanic, S. Joyce, S. Sriram, Y. Koezuka, and L. Van Kaer Natural Killer T Cell Activation Protects Mice Against Experimental Autoimmune Encephalomyelitis J. Exp. Med., December 17, 2001; 194(12): 1801 - 1811. [Abstract] [Full Text] [PDF]
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M. L. Jelachich and H. L. Lipton Theiler's Murine Encephalomyelitis Virus Induces Apoptosis in Gamma Interferon-Activated M1 Differentiated Myelomonocytic Cells through a Mechanism Involving Tumor Necrosis Factor Alpha (TNF-{alpha}) and TNF-{alpha}-Related Apoptosis-Inducing Ligand J. Virol., July 1, 2001; 75(13): 5930 - 5938. [Abstract] [Full Text] [PDF]
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O. S. Targoni, J. Baus, H. H. Hofstetter, M. D. Hesse, A. Y. Karulin, B. O. Boehm, T. G. Forsthuber, and P. V. Lehmann Frequencies of Neuroantigen-Specific T Cells in the Central Nervous System Versus the Immune Periphery During the Course of Experimental Allergic Encephalomyelitis J. Immunol., April 1, 2001; 166(7): 4757 - 4764. [Abstract] [Full Text] [PDF]
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Y. Katz-Levy, K. L. Neville, J. Padilla, S. Rahbe, W. S. Begolka, A. M. Girvin, J. K. Olson, C. L. Vanderlugt, and S. D. Miller Temporal Development of Autoreactive Th1 Responses and Endogenous Presentation of Self Myelin Epitopes by Central Nervous System-Resident APCs in Theiler's Virus-Infected Mice J. Immunol., November 1, 2000; 165(9): 5304 - 5314. [Abstract] [Full Text] [PDF]
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V. H. Secor, W. E. Secor, C.-A. Gutekunst, and M. A. Brown Mast Cells Are Essential for Early Onset and Severe Disease in a Murine Model of Multiple Sclerosis J. Exp. Med., March 6, 2000; 191(5): 813 - 822. [Abstract] [Full Text] [PDF]
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A. M. Girvin, M. C. Dal Canto, L. Rhee, B. Salomon, A. Sharpe, J. A. Bluestone, and S. D. Miller A Critical Role for B7/CD28 Costimulation in Experimental Autoimmune Encephalomyelitis: A Comparative Study Using Costimulatory Molecule-Deficient Mice and Monoclonal Antibody Blockade J. Immunol., January 1, 2000; 164(1): 136 - 143. [Abstract] [Full Text] [PDF]
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A. E. Juedes, P. Hjelmstrom, C. M. Bergman, A. L. Neild, and N. H. Ruddle Kinetics and Cellular Origin of Cytokines in the Central Nervous System: Insight into Mechanisms of Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., January 1, 2000; 164(1): 419 - 426. [Abstract] [Full Text] [PDF]
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S. A. Stohlman, L. Pei, D. J. Cua, Z. Li, and D. R. Hinton Activation of Regulatory Cells Suppresses Experimental Allergic Encephalomyelitis Via Secretion of IL-10 J. Immunol., December 1, 1999; 163(11): 6338 - 6344. [Abstract] [Full Text] [PDF]
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A. M. Girvin, K. B. Gordon, C. J. Welsh, N. A. Clipstone, and S. D. Miller Differential abilities of central nervous system resident endothelial cells and astrocytes to serve as inducible antigen-presenting cells Blood, May 15, 2002; 99(10): 3692 - 3701. [Abstract] [Full Text] [PDF]
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