| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Production and Reduces the Severity of Experimental Autoimmune Encephalomyelitis in Cytokine Knockout Mice1
,
*
Department of Neurology and
Neurological Sciences Institute, Oregon Health Sciences University, and
Neuroimmunology Research, Department of Veterans Affairs, Portland, OR 97201
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-deficient
mice. The decrease in disease severity was accompanied by a concomitant
reduction in the number of proinflammatory cytokine- and
chemokine-producing cells in the CNS. Although there was no apparent
increase in compensatory Th2 cytokine production in cytokine-deficient
mice, there was a profound decrease in the frequency of
TNF-
-producing cells in the CNS and the periphery. Therefore, we
propose that one mechanism by which estrogen protects females from the
development of cell-mediated autoimmunity is through a
hormone-dependent regulation of TNF-
production. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Th1 lymphocytes are thought to play a critical role in the initiation and expansion of CNS damage in MS and other cell-mediated autoimmune diseases (11). Most, if not all, cell-mediated autoimmune disorders remit during pregnancy (7, 12). In contrast, clinical signs of autoimmune diseases that involve Abs, such as lupus, may become worse during pregnancy (13). A number of immunological parameters are altered during pregnancy, among them a shift from a primarily proinflammatory Th1 response to an anti-inflammatory Th2 response (14). The shift toward Th2 immunity is thought to be beneficial for maintenance of the fetal allograft. It also might explain the remission of cell-mediated autoimmunity and the potential for exacerbation of humoral autoimmunity during pregnancy. Changes in immune function during pregnancy are probably induced by the profound increase in the production of sex hormones. High doses of estrogen have been shown to alter cytokine production by human myelin-specific T cells (15). Estrogen-induced changes in T cell cytokine production have also been demonstrated in experimental models of cell-mediated autoimmunity.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is induced by immunizing laboratory rodents with myelin proteins or peptides emulsified in CFA and serves as a useful model for MS (16). Immunization results in the induction of myelin-specific Th1 cells that home to the CNS, where they secrete inflammatory cytokines and chemokines, resulting in clinical paralysis and damage to the myelin sheath (17). Gender differences in some models of EAE parallel the gender dimorphism known for MS (18, 19, 20, 21). The increased severity of EAE in female mice is associated with elevated levels of Th1 cytokines compared with those in males (18, 19, 20, 21). Pregnancy has been shown to suppress the development of EAE (22, 23), and estrogen administered at levels equal to or greater than those in pregnancy have been shown to diminish clinical disease (24, 25). In recent studies from our laboratory, ovariectomy and loss of endogenous estrogen enhanced the severity of EAE, whereas treatment of mice with estrous and diestrous levels of exogenous estrogen effectively suppressed EAE (26, 27). Inhibition of EAE was accompanied by a drastic reduction in CNS cellularity and chemokine synthesis (27) in the absence of a significant shift toward Th2 cytokine synthesis (26).
The hypothesis that regulatory Th2 cytokines are important in
estrogen-induced suppression of EAE was tested in this study by
comparing EAE disease severity in cytokine-deficient mice treated with
and without estrogen. Estrogen effectively suppressed EAE in IL-4 and
IL-10 knockout mice. In addition, estrogen treatment of
IFN--deficient mice resulted in a significant reduction in disease
severity. Estrogen-treated mice had lower levels of cytokine and
chemokine production and had diminished levels of chemokine
receptor-positive cells in the CNS. These results suggest that
estrogen-mediated regulation of EAE can occur in the absence of
regulatory cytokines. The frequency of compensatory Th2 cytokines did
not increase in cytokine-deficient mice, nor did the frequency of
IFN--secreting cells significantly decrease. However, the frequency
of TNF--producing cells was profoundly diminished in
estrogen-treated animals. Since TNF- has been shown to play an
important role in the pathogenesis of EAE, we propose that one
mechanism by which estrogen suppresses disease is by reducing the
frequency and activity of TNF--producing cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female C57BL/6, IL-4 knockout
(B6.129P2-Il4tm1Cgn), IL-10 knockout
(C57BL/6-Il10tm1Cgn), and IFN- knockout
(B6.129S7-Ifngtm1Ts) mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). The mice were housed in the Animal
Resource Facility at Portland Veterans Affairs Medical Center in
accordance with institutional guidelines.
Antigens
Mouse myelin oligodendrocyte glycoprotein 35–55 (MOG35–55; MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid phase techniques and was purified by HPLC at Beckman Institute, Stanford University (Palo Alto, CA).
Estrogen treatment
Sixty-day release pellets containing 2.5 mg 17
-estradiol
(E2) or vehicle were implanted s.c. in the
scapular region behind the neck using a 12-gauge trochar as described
by the manufacturer (Innovative Research of America, Sarasota, FL). The
mice were implanted 1 wk before immunization with
MOG35–55. The concentration of
E2 expected in the serum is between 1500 and 2000
pg/ml, which is approximately 5 times less than the levels found during
pregnancy. E2 levels measured previously
(26) were equivalent to those reported by the
manufacturer.
Induction of EAE
C57BL/6 and cytokine-deficient mice were inoculated s.c. in the flanks with 0.2 ml of an emulsion containing 200 µg MOG35–55 in saline and an equal volume of CFA containing 400 µg Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Disease induction required i.v. administration of pertussis toxin on the day of immunization (25 ng/mouse) and 2 days later (67 ng/mouse). The mice were assessed daily for clinical signs of EAE according to the following scale: 0 = normal; 1 = limp tail or mild hindlimb weakness; 2 = moderate hindlimb weakness or mild ataxia; 3 = moderately severe hindlimb weakness; 4 = severe hindlimb weakness or mild forelimb weakness or moderate ataxia; 5 = paraplegia with no more than moderate forelimb weakness; and 6 = paraplegia with severe forelimb weakness or severe ataxia or moribund condition.
Histopathology
The intact spinal column was removed from mice during the peak of clinical disease and fixed in 10% phosphate-buffered formalin. The spinal cords were dissected after fixation and embedded in paraffin before sectioning. The sections were stained with either Luxol Fast Blue/periodic acid-Schiff/hematoxylin or silver nitrate and analyzed by light microscopy. Semiquantitative analysis of inflammation and demyelination was determined by examining at least 10 sections from each mouse.
RNase protection assay (RPA)
Spinal cords were recovered from animals at the peak of EAE
(days 12–16 postimmunization) and were frozen at -70°C until use.
Total RNA was extracted from frozen spinal cords using the STAT-60
reagent (Tel-Test, Friendswood, TX), and chemokine expression was
determined using the RiboQuant RPA kit (PharMingen, San Diego, CA)
according to the manufacturer’s instructions. Chemokine mRNA was
detected by hybridization with riboprobes specific for RANTES,
macrophage inflammatory protein-1 (MIP-1), monocyte
chemoattractant protein-1 (MCP-1), MIP-2, IFN-inducible protein of 10
kDa (IP-10), lymphotactin, and T cell activation Ag (TCA-3). Chemokine
receptor-specific riboprobes were used to detect CCR1, CCR1b, CCR2,
CCR3, CCR4, and CCR5. Cytokine mRNA was detected with riboprobes for
IL-4, IL-10, TNF-, lymphotoxin- (LT-), TNF-, and IFN-.
The sample loading was normalized using the housekeeping gene L32
included in each template set. RPA analysis was performed on 20 µg
total RNA hybridized with probes labeled with
[32P]UTP. After digestion of ssRNA, the RNA
pellet was solubilized and resolved on a 5% sequencing gel. Controls
included the probe set hybridized to transfer RNA only; appropriate
control RNA, which serves as an integrity control for the RNA sample;
and yeast transfer RNA as a background control. The gels were analyzed
using a phosphorimager (Bio-Rad, Hercules, CA), and the experimental
signal normalized to L32 using Quantity One software (Bio-Rad).
Proliferation assay
Draining lymph node (LN) and spleen cells were recovered from immunized mice at peak of clinical EAE (days 14–16 postimmunization) as previously described (28). The in vitro proliferative response was determined using a standard microtiter assay (29). Briefly, the cells were cultured in 96-well, flat-bottom tissue culture plates at 4 x 105 cells/well in stimulation medium alone (control) or with test Ags (i.e., MOG35–55) and incubated for 72 h at 37°C in 7% CO2. Wells were pulsed for the final 18 h with 0.5 µCi [methyl-3H]thymidine (Amersham, Arlington Heights, IL). The cells were harvested onto glass-fiber filters, and thymidine uptake was measured using a liquid scintillation counter. Results were determined from the means of triplicate cultures. Stimulation indexes were determined by calculating the ratio of Ag specific counts per minute to control counts per minute.
Intracellular staining for cytokines
Single-cell suspensions from spleen were prepared from immunized
mice and cultured at 10 x 106 cells/ml in
stimulation medium containing 50 µg/ml
MOG35–55. The cells were stimulated for 24
h, the last 5 h in the presence of brefeldin A. The cells were
then stained with anti-V8.1/8.2 TCR FITC for 30 min at 4°C
before fixation and permeabilization with Cytofix/Cytoperm solution
(PharMingen). The cells were then stained with anti-cytokine Abs
labeled with PE (anti-mouse IFN-, TNF-, IL-4, IL-10, and
IL-12; from PharMingen) for 30 min at 4°C. The cells were washed
twice in perm/wash buffer (PharMingen) and once in FACS staining buffer
(PBS, 1% BSA, and 0.05% NaN3) before two-color
FACS analysis on a FACScan instrument (Becton Dickinson, Sunnyvale, CA)
using CellQuest software (Becton Dickinson). For each experiment the
cells were stained with isotype control Abs to establish background
staining and to set the quadrants before calculating the percentage of
positive cells.
CNS mononuclear cells were isolated from perfused brain and spinal cord
by Percoll gradient centrifugation as described previously
(19). The cells were stimulated with
MOG35–55 peptide for 24 h, the last 5
h in the presence of brefeldin A. The cells were then stained with
anti-CD4 Cy-Chrome-labeled Abs before fixation and
permeabilization. The cells were subsequently stained with V8.1/8.2
TCR-FITC and the indicated cytokine-specific Ab coupled to PE and
analyzed by three-color flow cytometry. For each experiment the cells
were stained with isotype control Abs to establish background staining
and to set the quadrants before calculating the percentage of positive
cells.
Statistical analysis
Significant differences in incidence and mortality between
untreated and E2-treated mice were assessed by
2 analysis. Difference in onset was determined
using two-tailed Student’s t test. Differences in peak
score and cumulative disease index were assessed by the Mann-Whitney
test. Statistical significance of the frequency of cytokine-secreting
cells was analyzed using Student’s t test for comparisons
of two means. Differences in the expression of chemokine and cytokine
mRNA were also determined using Student’s t test.
p 
0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been previously established that estrogen, even at doses well below pregnancy levels, can induce a potent suppression of EAE (26, 27). However, the mechanisms by which estrogen suppresses this disorder are not completely understood. Estrogen has been shown to inhibit the production of proinflammatory cytokines and concomitantly enhance the production of anti-inflammatory cytokines (15, 30). The balance between IL-10 and IL-12 is thought to be of particular importance.
The role of regulatory cytokines in estrogen-induced protection from
EAE was examined in this study using cytokine-deficient mice.
Immunization with MOG35–55 resulted in the
induction of severe EAE in wild-type (WT) C57BL/6 mice, and no
differences in disease severity were found in similarly immunized
cytokine-deficient mice (Fig. 1
and Table I). C57BL/6 mice implanted with
E2-containing pellets had a lower incidence of
EAE and developed disease much later than untreated mice. However, EAE
that eventually developed in some E2-treated mice
was essentially equivalent in severity to that in untreated animals,
possibly due to early depletion of the E2
pellets. Nevertheless, treatment with E2 exerted
a profound reduction in both the incidence and the cumulative disease
index of EAE and significantly delayed the onset of symptoms in those
mice that eventually developed disease. Estrogen treatment had similar
effects on mice deficient in IL-4, IL-10, and IFN- (Fig. 1 and Table I). No statistically significant differences in the ability of
E2 to protect cytokine-deficient mice were found
(as determined by Fisher’s exact test).
|
|
). Healthy C57BL/6 and
cytokine-deficient mice that were treated with E2
before immunization did not have any detectable lesions in the spinal
cord (Fig. 2). Thus, it is apparent from these data that
E2 can suppress the development of both the
clinical and histopathologic manifestations of EAE in the absence of
IL-4, IL-10, and IFN-.
|
The egress of inflammatory cells into the CNS is a critical first
step in the development of EAE. Chemokines are low m.w. chemotactic
molecules that are thought to play an important role in the migration
and retention of immunocompetent cells in the CNS (31).
The influence of E2 treatment on chemokine and
chemokine receptor mRNA in the spinal cords of WT and
cytokine-deficient mice was measured using the RPA. Total RNA was
purified from spinal cords collected from mice at the peak of EAE (days
12–16 postimmunization), and chemokine/chemokine receptor-specific
mRNA was detected using radiolabeled riboprobes. Similar to our
recently published data (27), mRNAs coding for many of the
chemokine and receptor family members were detectable in the spinal
cords of WT C57BL/6 mice with EAE (Fig. 3). RANTES and IP-10 were expressed at
the highest levels, followed by MIP-1, MIP-2, and MCP-1. The levels
of TCA-3 mRNA were below the limits of detection for this assay. CCR5
was the most abundant chemokine receptor, followed by CCR1 and CCR2
(Fig. 3), whereas CCR1b, CCR3, and CCR4 were below the level of
detection.
|
). IL-4-deficient mice had reduced expression of
RANTES and MIP-1, but increased expression of MCP-1, whereas IL-10-
and IFN--deficient mice had reduced expression of all chemokines
tested except MCP-1. TCA-3 mRNA was only detectable in
IFN--deficient mice. The expression of CCR1, CCR2, and CCR5 was
nearly absent in IL-10-deficient mice, but was only moderately altered
in IL-4- and IFN--deficient mice. Thus, although distinct variations
in the pattern of chemokine or chemokine receptor expression occurred
in the different cytokine knockout mice, the development of EAE was not
significantly changed. These preliminary data provide evidence of the
complex interactions between chemokines and cytokines.
The expression of all chemokine and chemokine receptor mRNA was
significantly diminished or absent in both WT and cytokine-deficient
mice treated with E2 (Fig. 3). This effect is
probably the result of an E2-dependent decrease
in the trafficking of inflammatory cells into the CNS and possibly to
its ability to inhibit the production of key inflammatory factors.
Estrogen treatment reduced cytokine production in the CNS
The expression of cytokine mRNA in the spinal cords of mice at the
peak of EAE (days 12–16 postimmunization) was measured by RPA
analysis. mRNA encoding the proinflammatory cytokines IFN-, TNF-,
and LT- were the most abundant (Fig. 4A). However, differences in
the expression level of cytokine mRNA were apparent in the
cytokine-deficient mice. mRNA for both TNF- and IFN- were
substantially lower in IL-10 knockout mice compared with WT, but no
significant differences in LT- levels were noted. mRNA for all three
cytokines was profoundly lower in IFN- knockout mice compared with
WT mice. Surprisingly, low levels of IFN- mRNA were detected in
IFN--deficient mice. These mice were created by homologous
recombination of the first exon (32), leaving the second
exon intact. Although it has not been reported previously, it is quite
possible that an mRNA product coding for the second exon is expressed
and detected in our assay. Nevertheless, it is clear that lymphocytes
from these mice fail to make a functional IFN- protein when measured
by intracellular cytokine staining (Fig. 6C). In all groups
of mice, the levels of IL-4, IL-10, and TNF- (LT-) were below the
limits of detection. The levels of LT-, TNF-, and IFN- mRNA in
the spinal cords of E2-treated C57BL/6 and
cytokine knockout mice were significantly reduced compared with those
in untreated groups (Fig. 4A), with the exception of LT-
levels in IFN--deficient mice.
|
|
8.2+ Th cells was measured
by staining with anti-V8.2-FITC- and PE-labeled
cytokine-specific Abs. As shown in Fig. 4B, there was a
dramatic reduction in the frequency and staining intensity
(p < 0.0001) of TNF-- and IFN--producing
CD4+ T cells in the CNS of
E2-treated mice. Based on total cell numbers
recovered, E2 treatment caused a reduction of
proinflammatory cytokine-producing CD4+
Vb8.2+ T cells in the CNS from 29,000 to only 390
cells/mouse. A substantial reduction of proinflammatory
cytokine-producing CD4+,
Vb8.2- T cells was also observed (Fig. 4B). Taken together, these data confirm the RPA data
presented above and directly support the hypothesis that
E2 treatment inhibits the activation and
infiltration of proinflammatory cells into the CNS. Estrogen treatment failed to alter T cell proliferation and the expression of cell surface adhesion and activation Ags
Proliferation of draining LN cells from either untreated or
E2-treated mice was measured to determine whether
E2 could alter the ability of T lymphocytes to
recognize and respond to the immunizing Ag. LN cells were isolated from
three representative mice for each group, and the cells were pooled
before stimulation with MOG35–55 for 72 h.
The results shown in Fig. 5A
clearly illustrate that there was no effect of E2
treatment on the LN proliferation response to
MOG35–55 in WT and cytokine-deficient mice.
Similarly, E2 treatment did not alter the
response to Ag of splenocytes (data not shown). These results indicate
that E2 treatment prevents the development of EAE
without altering the ability of
MOG35–55-specific T cells to proliferate in
response to Ag.
|
B). Furthermore, no differences in activation markers
(CD69, CD25, Fas ligand, CD40 ligand, and CD28) were seen.
Estrogen treatment reduced the frequency of TNF--secreting cells
Recent studies have suggested that estrogen treatment promotes a
shift toward Th2 immunity that may be responsible for the suppression
of EAE (30). This hypothesis was addressed by assessing
the frequency of both pro- and anti-inflammatory cytokine-producing
cells in untreated and E2-treated mice using the
intracellular cytokine staining technique. Spleen cells were prepared
from untreated and E2-treated mice at the peak of
EAE (days 12–16 postimmunization) and stimulated with
MOG35–55 for 24 h, the last 6 h in the
presence of brefeldin A. The cells were stained with FITC-labeled
anti-V8.1/8.2 TCR Abs before fixation and permeabilization, and
then were stained with the indicated PE-labeled anti-cytokine Abs.
We focused on V8.1/8.2 TCR-bearing T cells because they are thought
to comprise a major population of the
MOG35–55-specific T cell responses in
H-2b mice (33).
The frequency of IFN-- and TNF--producing V8.1/8.2
TCR+ cells was similar in untreated C57BL/6 as
well as IL-4- and IL-10-deficient mice with EAE. However, the frequency
of TNF--producing cells was significantly lower in IFN- knockout
mice, and as expected, there were no detectable cells producing IFN-
in these mice (Fig. 6). The frequency of
TNF--producing V8.1/8.2 TCR+ cells was
significantly diminished in C57BL/6 mice (p =
0.004), IL-4 knockout mice (p = 0.06), and
IL-10 knockout mice (p = 0.001), but no further
reduction in the frequency of TNF--producing cells was observed in
E2-treated IFN- knockout mice (Fig. 6C). The diminution in staining intensity of cells from
E2-treated mice also suggests that these cells
also produce lower levels of TNF- compared with the untreated mice.
As the number of V8.1/8.2+ splenocytes
recovered from the intact and cytokine knockout mice was quite similar
(data not shown), it can be concluded that the total number of
TNF--producing, MOG-reactive lymphocytes in the spleens of
E2-treated mice was significantly reduced. The
frequency of V8.2- cells producing TNF-
was also reduced in all the E2-treated mouse
groups, suggesting that estrogen may influence cytokine production by
encephalitogenic or recruited T cells expressing different V genes as
well as other inflammatory cells, including macrophages.
The frequency of cells producing IFN-, IL-4, IL-10, and IL-12 was
also measured. Although there was a trend for
E2-treated mice to have a lower frequency of
IFN--producing cells (Fig. 6, B and C), these
values failed to attain statistical significance
(p > 0.05). Furthermore, the frequency of
IL-4-, IL-10-, and IL-12-producing cells was always below the limits of
detection for this assay. The failure to detect IL-4- and
IL-10-reactive cells suggests that E2 treatment
did not significantly shift the cytokine response toward Th2
production.
EAE was suppressed in TNF--deficient mice
The data presented above implicate TNF--producing cells as
probable contributors to induction of EAE. To further evaluate the
pathogenic contribution of TNF- in this model, the severity of EAE
was compared in TNF--deficient and WT control mice. Severe EAE
developed in the majority of WT mice after immunization with
MOG35–55 peptide (Table II). However, the incidence and severity
of EAE in TNF--deficient mice were greatly diminished. Not only did
fewer mice develop disease, but the mean peak disease score and the
cumulative disease index were also profoundly reduced (Table II). These
data confirm in a novel manner that TNF--producing cells are major
contributors to EAE induction, as has been established by others using
different approaches (34, 35, 36), and their regulation by
E2 provides an important new insight into the
regulatory effects of estrogen.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
knockout
mice. In conjunction with a lack of any detectable compensatory
cytokines, this finding provides strongevidence that these
cytokines may not be required for E2-mediated
suppression of EAE. Second, E2 treatment induced
a profound reduction in the frequency of TNF--producing cells in the
CNS and spleens of mice immunized with MOG35–55.
This novel observation coupled with the established importance of
TNF- for the induction of EAE in this model provides strong evidence
that the decreased frequency of TNF--producing cells induced by
treatment with E2 constitutes a major regulatory
pathway. Our study differs from previously published studies that reported treatment with estrogen at levels equivalent to or exceeding pregnancy levels suppress the development of EAE (24, 25, 30), and that the primary mechanisms by which this occurs is through immune deviation toward Th2 cytokines (15, 37, 38). It is quite possible that the dose and form of estrogen has a critical role in determining the mechanisms of disease inhibition. In support of this argument, estrogen regulation of human T cell cytokine production appears to be acutely dependent on the dose of hormone (15). It remains to be determined if this is an adequate explanation for the differences between this study and others.
Most functional knockout mice are bred onto the C57BL/6 strain, which
is highly susceptible to EAE induced by immunization with
MOG35–55 (39). It is clear that T
cells producing Th2 cytokines in this model are nonencephalitogenic and
also to confer protection against EAE. Mice deficient in IL-10
production have a higher incidence of EAE and develop a more aggressive
form of disease (40). In addition, overexpression of IL-10
suppresses the development of disease (40, 41). A number
of reports also suggest that IL-4 can suppress Th1 responses and
protect mice from EAE (42, 43), although studies using
IL-4 knockout mice have shed some doubt about the importance of this
cytokine for protection in this model (40, 44).
Surprisingly, neutralization of IFN- may cause mice to develop more
severe EAE (45, 46, 47), implicating this cytokine in EAE
regulation as well as pathogenesis. It is clear that there are
limitations to be considered when using cytokine knockout mice, since
many cytokines have pleiotropic functions and can be duplicated by
other cytokines. Definitive answers may have to wait until the next
generation of provisional knockout mice become available in which both
the temporal and spatial expression of the target genes can be
regulated.
Our previous studies demonstrated that low dose E2 therapy profoundly inhibited EAE induced in several mouse strains, with effective doses ranging from pregnancy to diestrous levels of E2 (26, 27). Moreover, we found that a primary effect of estrogen was to inhibit the trafficking of inflammatory cells into the CNS, thereby decreasing expression of proinflammatory cytokines, chemokines, and chemokine receptors in the affected target organ (27). In the current study treating mice with low doses of E2 (2 ng/ml, or approximately one-fifth the levels found during pregnancy in the mouse) clearly demonstrate the efficacy of this treatment regimen in all the cytokine knockout mice tested. Furthermore, the diminution of disease occurred in the absence of compensatory increases in other Th2 cytokines, suggesting that these cytokines may not be important for low dose E2 regulation of EAE. The lack of a Th2 cytokine shift also makes it less likely that E2 therapy could induce pathogenic anti-myelin Abs. This is an important point, since pathogenic anti-myelin Abs may play an important role in MOG-induced EAE (48).
Consistent with our previous report (27) and the results
of others (49, 50), RPA analysis of CNS tissue detected
enhanced expression of the chemokines RANTES, IP-10, MIP-1, MIP-2,
and MCP-1; the chemokine receptors CCR1, CCR2, and CCR5; and the
cytokines LT-, TNF-, and IFN- in mice with EAE. The mRNA
expression levels of selected chemokines, chemokine receptors, and
cytokines in the CNS of IL-10 and IFN- knockout mice were
substantially different from those in intact mice even though EAE
disease severity was comparable. These observations suggest that
redundancies in the encephalitogenic cascade (i.e., the unexpected
appearance of TCA-3 message in IFN- knockout mice) may allow for
full expression of EAE even in the absence of certain components.
Considerable reductions in the expression of all detectable cytokines,
chemokines, and chemokine receptors in the CNS occurred in intact and
cytokine knockout mice treated with E2. This
reduction in mRNA expression was clearly influenced by the lower number
of recruited inflammatory cells found in the CNS of
E2-treated mice. However,
E2 also considerably reduced the intracellular
staining intensity and percentage of cells producing IFN- and
TNF- in both V8.2+ and
V8.2- T cells that were recovered from CNS
tissue of WT mice with EAE. These results prompted a more extensive
search for systemic effects of E2 that could
account for its potent inhibitory effects on EAE. However, no
significant differences in the expression of adhesion receptors or
activation Ags were found. Furthermore, no differences in the ability
of peripheral lymphocytes to proliferate to the immunizing Ag were
detected.
There are many studies that support the critical involvement of TNF-
in EAE. Elevated expression of TNF- can be found in the CNS during
acute episodes of disease (51), and T cell clones that
produce TNF- transfer EAE more effectively (34).
Moreover, blockade of TNF- with neutralizing Abs or metabolic
inhibitors ameliorates signs of EAE, whereas overexpression of TNF-
exacerbates disease (36, 52, 53, 54). In contrast to a
previously published report (55), we found that the
severity of EAE in TNF- knockout mice was significantly less severe
than that in wild-type controls. To the best of our knowledge, this is
the first study of EAE in this particular strain of knockout mice. This
observation differs from other published reports in that these mice
were derived from a first generation backcross between C57BL/6 and
129.J mice (56). Differences in the genetic background may
help explain the differences in the severity of EAE between the various
models. In any event, these experiments support the hypothesis that a
reduction in the frequency of TNF--producing cells is a significant
component of the mechanism by which E2
inhibit EAE.
Previous studies from our laboratory and others have failed to detect
secreted TNF- by Ag-stimulated LN or spleen cells using standard
immunoassay techniques. For reasons that are not completely clear,
intracellular staining is a much more sensitive technique for the
detection of many cytokines, including TNF-, and undoubtedly
facilitated our novel observation. Systemic E2
regulation of TNF- production could account for much of the observed
reduction in EAE severity by reducing the ability of encephalitogenic
cells to enter the CNS, suppressing the recruitment and activation of
other inflammatory cells, and/or inhibiting TNF--induced damage to
myelin-producing oligodendrocytes.
Estrogen exerts its effects on cells by binding to two distinct
estrogen receptors, ER- and ER- (57). The activation
of gene transcription by estrogen requires ER dimerization and binding
to an estrogen response element (ERE), followed by activation of
transcription factors and cofactors (58). ERs are
expressed in a variety of immunocompetent cells, including T cells and
macrophages (21, 59, 60), and EREs have been identified in
a number of immunologically relevant genes. For example, an ERE has
been mapped to the 5'-flanking sequence of the IFN- gene, and
estrogen has been shown to induce transcription of genes containing
this ERE (61). Recently, an E2
inhibitory element was mapped to the TNF- promoter
(62). Interestingly, ER- was more potent than ER- at
repressing the TNF- promoter (62). Estrogens have been
shown to decrease the expression of TNF- in a number of cells
(63, 64), and the identification of an estrogen-sensitive
repressor in the TNF- promoter explains these observations. It is
possible that estrogen binds to ER- expressed in T cells and
macrophages and represses the production of TNF-. However, this
hypothesis remains to be tested.
In conclusion, our data strongly support the idea that down-regulation
of TNF- production is a major pathway by which estrogen ameliorates
cell-mediated autoimmunity. However, at this time, these data do not
exclude a role for other potential mechanisms. Estrogen, at the doses
used in this study, also reduced the production of IFN-, although
this effect was much less pronounced than the effect on TNF-
production. The ability of estrogen treatment to alter the expression
of other proinflammatory cytokines, such as IL-1, IL-6, and LT-, has
yet to be established in this system. Furthermore, the consequences of
estrogen treatment on the production of regulatory cytokines such as
TGF- would further enhance our understanding of this potent
treatment for the suppression of EAE. It is important to determine the
entire pathway by which estrogen suppresses disease so that the
implications of this therapy for women with MS can be completely
understood. This information will also be valuable when considering the
application of combination therapies that include estrogen.
|
Footnotes |
|---|
2 A.I. and B.F.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Halina Offner, Neuroimmunology Research R&D-31, Veterans Affairs Medical Center, 3710 S.W. U.S. Veterans Hospital Road, Portland, OR 97201. E-mail address: offnerva{at}ohsu.edu
4 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; E2, 17-estradiol; MBP, myelin basic protein; MIP-1, macrophage-inflammatory protein-1; IP-10, IFN-inducible protein of 10 kDa; MCP-1, monocyte chemoattractant protein-1; TCA-3, T cell activation Ag; LN, lymph node; WT, wild type; RPA, RNase protection assay; LT-, lymphotoxin-; ER, estrogen receptor; ERE, estrogen response element.
Received for publication December 18, 2000. Accepted for publication April 24, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-ethinylestradiol: an immunological study in the Lewis rat. Int. Arch. Allergy Immunol. 102:133.[Medline]
-Estradiol inhibits cytokine and chemokine
receptor mRNA expression in the central nervous system of female mice
with experimental autoimmune encephalomyelitis. J. Neurosci.
Res. In press.
genes. Science 259:1739. expression of encephalitogenic cells. Eur. J. Immunol. 25:1951.[Medline]
gene are susceptible to the induction of experimental autoimmune encephalomyelitis. J. Immunol. 156:5.[Abstract]
confers resistance to experimental autoimmune encephalomyelitis. Eur. J. Immunol. 26:1641.[Medline]
plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
or its CCR5 receptor. Eur. J. Immunol. 30:1410.[Medline]
expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental autoimmune encephalomyelitis: regulation by Th1 cytokines. J. Immunol. 154:944.[Abstract]
. Proc. Natl. Acad. Sci. USA 92:11294. and lymphotoxin are not required for induction of acute experimental autoimmune encephalomyelitis. J. Exp. Med. 185:2177.-deficient mice: a critical requirement for TNF in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184:1397. and . Endocrinology 138:863. promoter. J. Immunol. 146:4362.[Abstract]
transcription requires estrogen receptor activation function-2 and is enhanced by coactivators. Proc. Natl. Acad. Sci. USA 96:15161.This article has been cited by other articles:
|
|
 |
|
  F. E. Nashold, K. M. Spach, J. A. Spanier, and C. E. Hayes Estrogen Controls Vitamin D3-Mediated Resistance to Experimental Autoimmune Encephalomyelitis by Controlling Vitamin D3 Metabolism and Receptor Expression J. Immunol., September 15, 2009; 183(6): 3672 - 3681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Langer-Gould, S. M. Huang, R. Gupta, A. D. Leimpeter, E. Greenwood, K. B. Albers, S. K. Van Den Eeden, and L. M. Nelson Exclusive Breastfeeding and the Risk of Postpartum Relapses in Women With Multiple Sclerosis Arch Neurol, August 1, 2009; 66(8): 958 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang, B. Dehghani, Y. Li, L. J. Kaler, T. Proctor, A. A. Vandenbark, and H. Offner Membrane Estrogen Receptor Regulates Experimental Autoimmune Encephalomyelitis through Up-regulation of Programmed Death 1 J. Immunol., March 1, 2009; 182(5): 3294 - 3303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Fairweather, S. Frisancho-Kiss, and N. R. Rose Sex Differences in Autoimmune Disease from a Pathological Perspective Am. J. Pathol., September 1, 2008; 173(3): 600 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Straub The Complex Role of Estrogens in Inflammation Endocr. Rev., August 1, 2007; 28(5): 521 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J Ma, E. A Guzman, A. DeGuzman, H K. Muller, A. M Walker, and L. B Owen Local cytokine levels associated with delayed-type hypersensitivity responses: modulation by gender, ovariectomy, and estrogen replacement J. Endocrinol., May 1, 2007; 193(2): 291 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Polanczyk, C. Hopke, A. A. Vandenbark, and H. Offner Treg suppressive activity involves estrogen-dependent expression of programmed death-1 (PD-1) Int. Immunol., March 1, 2007; 19(3): 337 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Gregg, V. Shikar, P. Larsen, G. Mak, A. Chojnacki, V. W. Yong, and S. Weiss White Matter Plasticity and Enhanced Remyelination in the Maternal CNS J. Neurosci., February 21, 2007; 27(8): 1812 - 1823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. J. Morales, K. K. Loo, H.-b. Liu, C. Peterson, S. Tiwari-Woodruff, and R. R. Voskuhl Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis. J. Neurosci., June 21, 2006; 26(25): 6823 - 6833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Miyake, K. Sasaki, K. Ide, Y. Matsukura, K. Shijima, and D. Fujiwara Highly Oligomeric Procyanidins Ameliorate Experimental Autoimmune Encephalomyelitis via Suppression of Th1 Immunity J. Immunol., May 15, 2006; 176(10): 5797 - 5804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Vegeto, S. Belcredito, S. Ghisletti, C. Meda, S. Etteri, and A. Maggi The Endogenous Estrogen Status Regulates Microglia Reactivity in Animal Models of Neuroinflammation Endocrinology, May 1, 2006; 147(5): 2263 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Li, L. Strauss, A. Kaatrasalo, A. Mayerhofer, I. Huhtaniemi, R. Santti, S. Makela, and M. Poutanen Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis Endocrinology, March 1, 2006; 147(3): 1271 - 1277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. Lambert, E. M. Curran, B. M. Judy, G. N. Milligan, D. B. Lubahn, and D. M. Estes Estrogen Receptor {alpha} (ER{alpha}) Deficiency in Macrophages Results in Increased Stimulation of CD4+ T Cells while 17{beta}-Estradiol Acts through ER{alpha} to Increase IL-4 and GATA-3 Expression in CD4+ T Cells Independent of Antigen Presentation J. Immunol., November 1, 2005; 175(9): 5716 - 5723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Spach and C. E. Hayes Vitamin D3 Confers Protection from Autoimmune Encephalomyelitis Only in Female Mice J. Immunol., September 15, 2005; 175(6): 4119 - 4126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Pasche, S. Kalaydjiev, T. J. Franz, E. Kremmer, V. Gailus-Durner, H. Fuchs, M. Hrabe de Angelis, A. Lengeling, and D. H. Busch Sex-Dependent Susceptibility to Listeria monocytogenes Infection Is Mediated by Differential Interleukin-10 Production Infect. Immun., September 1, 2005; 73(9): 5952 - 5960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang, S. Cao, M. Nagamani, K. E. Anderson, J. J. Grady, and L.-J. W. Lu Decreased Circulating Levels of Tumor Necrosis Factor-{alpha} in Postmenopausal Women during Consumption of Soy-Containing Isoflavones J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 3956 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. van den Broek, J. G. Damoiseaux, M. H De Baets, and R. M. Hupperts The influence of sex hormones on cytokines in multiple sclerosis and experimental autoimmune encephalomyelitis: a review Multiple Sclerosis, June 1, 2005; 11(3): 349 - 359. [Abstract] [PDF]
|
|
|
|
|
|
M M. Elloso, K. Phiel, R. A Henderson, H. A Harris, and S. J Adelman Suppression of experimental autoimmune encephalomyelitis using estrogen receptor-selective ligands J. Endocrinol., May 1, 2005; 185(2): 243 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Matejuk, C. Hopke, A. A. Vandenbark, P. D. Hurn, and H. Offner Middle-Age Male Mice Have Increased Severity of Experimental Autoimmune Encephalomyelitis and Are Unresponsive to Testosterone Therapy J. Immunol., February 15, 2005; 174(4): 2387 - 2395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Adamski and E. N. Benveniste 17{beta}-Estradiol Activation of the c-Jun N-Terminal Kinase Pathway Leads to Down-Regulation of Class II Major Histocompatibility Complex Expression Mol. Endocrinol., January 1, 2005; 19(1): 113 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-J. Shim, M. Warner, H.-J. Kim, S. Andersson, L. Liu, J. Ekman, O. Imamov, M. E. Jones, E. R. Simpson, and J.-A. Gustafsson Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren's syndrome PNAS, August 24, 2004; 101(34): 12628 - 12633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Garidou, S. Laffont, V. Douin-Echinard, C. Coureau, A. Krust, P. Chambon, and J.-C. Guery Estrogen Receptor {alpha} Signaling in Inflammatory Leukocytes Is Dispensable for 17{beta}-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2004; 173(4): 2435 - 2442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Adamski, Z. Ma, S. Nozell, and E. N. Benveniste 17{beta}-Estradiol Inhibits Class II Major Histocompatibility Complex (MHC) Expression: Influence on Histone Modifications and CBP Recruitment to the Class II MHC Promoter Mol. Endocrinol., August 1, 2004; 18(8): 1963 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Lambert, E. M. Curran, B. M. Judy, D. B. Lubahn, and D. M. Estes Estrogen receptor-{alpha} deficiency promotes increased TNF-{alpha} secretion and bacterial killing by murine macrophages in response to microbial stimuli in vitro J. Leukoc. Biol., June 1, 2004; 75(6): 1166 - 1172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.J. Fasouliotis, S.D. Spandorfer, S.S. Witkin, G. Schattman, H.C. Liu, J.E. Roberts, and Z. Rosenwaks Maternal serum levels of interferon-{gamma} and interleukin-2 soluble receptor-{alpha} predict the outcome of early IVF pregnancies Hum. Reprod., June 1, 2004; 19(6): 1357 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Koski, S. Hila, and G. E. Hoffman Regulation of Cytokine-Induced Neuron Death by Ovarian Hormones: Involvement of Antiapoptotic Protein Expression and c-JUN N-Terminal Kinase-Mediated Proapoptotic Signaling Endocrinology, January 1, 2004; 145(1): 95 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. D. Fillmore, E. P. Blankenhorn, J. F. Zachary, and C. Teuscher Adult Gonadal Hormones Selectively Regulate Sexually Dimorphic Quantitative Traits Observed in Experimental Allergic Encephalomyelitis Am. J. Pathol., January 1, 2004; 164(1): 167 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-b. Liu, K. K. Loo, K. Palaszynski, J. Ashouri, D. B. Lubahn, and R. R. Voskuhl Estrogen Receptor {alpha} Mediates Estrogen's Immune Protection in Autoimmune Disease J. Immunol., December 15, 2003; 171(12): 6936 - 6940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Latham, A. Zamora, H. Drought, S. Subramanian, A. Matejuk, H. Offner, and E. F. Rosloniec Estradiol Treatment Redirects the Isotype of the Autoantibody Response and Prevents the Development of Autoimmune Arthritis J. Immunol., December 1, 2003; 171(11): 5820 - 5827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Soldan, A. I. A. Retuerto, N. L. Sicotte, and R. R. Voskuhl Immune Modulation in Multiple Sclerosis Patients Treated with the Pregnancy Hormone Estriol J. Immunol., December 1, 2003; 171(11): 6267 - 6274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Polanczyk, A. Zamora, S. Subramanian, A. Matejuk, D. L. Hess, E. P. Blankenhorn, C. Teuscher, A. A. Vandenbark, and H. Offner The Protective Effect of 17{beta}-Estradiol on Experimental Autoimmune Encephalomyelitis Is Mediated through Estrogen Receptor-{alpha} Am. J. Pathol., October 1, 2003; 163(4): 1599 - 1605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cenci, G. Toraldo, M. N. Weitzmann, C. Roggia, Y. Gao, W. P. Qian, O. Sierra, and R. Pacifici Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-{gamma}-induced class II transactivator PNAS, September 2, 2003; 100(18): 10405 - 10410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Vandenbark, C. Rich, J. Mooney, A. Zamora, C. Wang, J. Huan, L. Fugger, H. Offner, R. Jones, and G. G. Burrows Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice J. Immunol., July 1, 2003; 171(1): 127 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ito, A. Matejuk, C. Hopke, H. Drought, J. Dwyer, A. Zamora, S. Subramanian, A. A. Vandenbark, and H. Offner Transfer of Severe Experimental Autoimmune Encephalomyelitis by IL-12- and IL-18-Potentiated T Cells Is Estrogen Sensitive J. Immunol., May 1, 2003; 170(9): 4802 - 4809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Subramanian, A. Matejuk, A. Zamora, A. A. Vandenbark, and H. Offner Oral Feeding with Ethinyl Estradiol Suppresses and Treats Experimental Autoimmune Encephalomyelitis in SJL Mice and Inhibits the Recruitment of Inflammatory Cells into the Central Nervous System J. Immunol., February 1, 2003; 170(3): 1548 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Matejuk, J. Dwyer, A. Zamora, A. A. Vandenbark, and H. Offner Evaluation of the Effects of 17{beta}-Estradiol (17{beta}-E2) on Gene Expression in Experimental Autoimmune Encephalomyelitis Using DNA Microarray Endocrinology, January 1, 2002; 143(1): 313 - 319. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
