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of June 18, 2017.
Estrogen Treatment Down-Regulates TNF-α
Production and Reduces the Severity of
Experimental Autoimmune
Encephalomyelitis in Cytokine Knockout
Mice
Atsushi Ito, Bruce F. Bebo, Jr., Agata Matejuk, Alex
Zamora, Marc Silverman, Amber Fyfe-Johnson and Halina
Offner
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:542-552; ;
doi: 10.4049/jimmunol.167.1.542
http://www.jimmunol.org/content/167/1/542
Estrogen Treatment Down-Regulates TNF-␣ Production and
Reduces the Severity of Experimental Autoimmune
Encephalomyelitis in Cytokine Knockout Mice1
Atsushi Ito,2*‡ Bruce F. Bebo, Jr.,2*†‡ Agata Matejuk,*‡ Alex Zamora,‡ Marc Silverman,‡
Amber Fyfe-Johnson,‡ and Halina Offner3*‡
M
ultiple sclerosis (MS)4 is an organ-specific autoimmune disease of the CNS that strikes women 2–3
times more often than men (1–3). Sex-linked factors,
including genetic and hormonal influences, appear to be involved
in regulating the increased susceptibility of women to MS (4). A
number of observations support the idea that fluctuations in sex
hormone levels are related to changes in disease status. For instance, the initial incidence of MS usually occurs during the reproductive years (5). In addition, clinical disease often abates during pregnancy, a time distinguished by an increase in sex
hormones, and exacerbates postpartum when sex hormones are at
low levels (6, 7). There are also reports suggesting a link between
oral contraceptive use and a reduction in disability in women with
MS (8, 9), although the use of oral contraceptives does not appear
*Department of Neurology and †Neurological Sciences Institute, Oregon Health Sciences University, and ‡Neuroimmunology Research, Department of Veterans Affairs,
Portland, OR 97201
Received for publication December 18, 2000. Accepted for publication April
24, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health (AI42376
and NS23444, to H.O.), the National Multiple Sclerosis Society (RG3165 to B.F.B.;
RG3108 to H.O.), and the Department of Veterans Affairs. A.M. is a postdoctoral
fellow of the National Multiple Sclerosis Society (FA1397).
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: [email protected]
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.
Copyright © 2001 by The American Association of Immunologists
to reduce the risk of developing MS (10). Consequently, sex hormones such as estrogen are considered important regulators of disease activity and are the focus of ongoing clinical trials for the
treatment of MS (R. Voskuhl, unpublished observation).
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 –21). The increased severity of EAE in female mice is associated with elevated
levels of Th1 cytokines compared with those in males (18 –21).
0022-1767/01/$02.00
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A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought
to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis.
However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study
cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the
protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease
suppression was also seen in IFN-␥-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. The Journal
of Immunology, 2001, 167: 542–552.
The Journal of Immunology
543
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.
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 ␮g 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).
Materials and Methods
Proliferation assay
Mice
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 ⫻ 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 ␮Ci [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.
Antigens
Mouse myelin oligodendrocyte glycoprotein 35–55 (MOG35–55; MEVG
WYRSPFSRVVHLYRNGK) 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 ␮g MOG35–55 in saline and an
equal volume of CFA containing 400 ␮g 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.
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)
Intracellular staining for cytokines
Single-cell suspensions from spleen were prepared from immunized mice
and cultured at 10 ⫻ 106 cells/ml in stimulation medium containing 50
␮g/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-V␤8.1/8.2
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 CyChrome-labeled Abs before fixation and permeabilization. The cells were
subsequently stained with V␤8.1/8.2 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.
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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.
Histopathology
544
ESTROGEN REDUCES SEVERITY OF EAE IN CYTOKINE KNOCKOUT MICE
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 ⱕ 0.05 was considered significant.
Results
Estrogen treatment reduces the severity of EAE in C57BL/6 and
cytokine-deficient mice
Estrogen treatment reduces chemokine and chemokine receptor
mRNA expression in the CNS
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␣,
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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 cytokinedeficient 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).
MOG35–55-immunized C57BL/6 and cytokine-deficient mice
had numerous inflammatory and demyelinating lesions in the spinal cord at the peak of EAE, and no significant differences in the
number and size of the lesions were observed (Fig. 2). 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-␥.
FIGURE 1. Estrogen treatment reduces the severity of EAE in C57BL/6
and cytokine knockout (KO) mice. Mice were implanted with 2.5-mg timerelease pellets containing E2 1 wk before immunization with MOG35–55.
The animals were monitored daily for clinical paralysis and scored as outlined
in Materials and Methods. Each figure corresponds to one representative experiment of two or three total experiments for each knockout mouse strain. The
data from all the experiments are summarized in Table I.
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.
The expression of chemokine and chemokine receptor mRNA in
cytokine-deficient mice with EAE was often markedly different
from that in WT mice (Fig. 3). IL-4-deficient mice had reduced
expression of RANTES and MIP-1␣, but increased expression of
The Journal of Immunology
545
Table I. E2 suppresses EAE in C57BL/6 and cytokine-deficient micea
E2
Incidence
Onset
Mortality
Peak Score
CDI
B6
⫺
⫹
29/31
19/31
p ⫽ 0.005
10.9 ⫾ 1.9
20.8 ⫾ 3.7
p ⬍ 0.0001
3/31
0/31
p ⫽ 0.238
5.1 ⫾ 0.9
4.2 ⫾ 1.2
p ⫽ 0.222
64.7 ⫾ 27.0
15.1 ⫾ 17.0
p ⬍ 0.0001
IL-4KO
⫺
⫹
11/11
8/11
p ⫽ 0.214
11.5 ⫾ 2.3
20.6 ⫾ 4.9
p ⬍ 0.0001
3/11
0/11
p ⫽ 0.214
5.5 ⫾ 0.4
4.4 ⫾ 1.0
p ⫽ 0.561
76.7 ⫾ 20.1
18.4 ⫾ 22.2
p ⬍ 0.0001
IL-10KO
⫺
⫹
15/16
6/17
p ⫽ 0.001
12.3 ⫾ 1.3
22.0 ⫾ 4.3
p ⬍ 0.0001
2/16
0/17
p ⫽ 0.227
4.9 ⫾ 1.1
4.2 ⫾ 0.8
p ⫽ 0.977
53.0 ⫾ 27.2
6.5 ⫾ 10.9
p ⬍ 0.0001
IFN-␥KO
⫺
⫹
11/11
8/10
p ⫽ 0.214
13.6 ⫾ 2.6
21.8 ⫾ 2.7
p ⬍ 0.0001
1/11
1/10
p ⫽ 1.000
4.9 ⫾ 1.1
4.5 ⫾ 1.2
p ⫽ 0.999
58.6 ⫾ 22.4
19.3 ⫾ 15.9
p ⬍ 0.018
a
Significant differences in incidence and mortality between untreated and E2-treated mice were assessed by ␹2 analysis. Difference in onset was determined using the
two-tailed Student t test. Differences in peak score and cumulative disease index (CDI) were assessed by the Mann-Whitney test. KO, Knockout.
FIGURE 2. Estrogen-treated mice have fewer inflammatory CNS lesions. Spinal cords were recovered from mice at the peak of EAE (days 12–16
postimmunization), fixed in 10% phosphate-buffered
formalin, and embedded in paraffin as described in
Materials and Methods. Thin sections were stained
with either Luxol Fast Blue/periodic acid-Schiff/hematoxylin or silver nitrate and analyzed by light microscopy. Representative sections from the lumbar
spinal cord are presented with the dorsal side facing
the left. A, C57BL/6, LFB/HE; B, C57BL/6, silver;
C, C57BL/6 plus E2, LFB/HE; D, C57BL/6 plus E2,
silver; E, quantitative analysis of inflammatory lesions in untreated and estrogen-treated mice. KO,
knockout. ⴱ, Inflammatory foci were enumerated
from between 7 and 10 sections per spinal cord, at
least 2 spinal cords were examined per group.
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
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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.
546
ESTROGEN REDUCES SEVERITY OF EAE IN CYTOKINE KNOCKOUT MICE
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FIGURE 3. Chemokine and chemokine receptor (CCR) mRNA expression in the spinal cords of untreated and estrogen-treated mice determined by
RPA. Spinal cords were recovered from mice at the peak of clinical disease (days 12–16 postimmunization), and total RNA was purified. The RPA was
performed using chemokine and chemokine receptor-specific riboprobes labeled with 32P, and the protected fragments were separated on a sequencing gel.
Quantitation was performed using a phosphorimager as described in Materials and Methods. Data are presented as the mean ⫾ SD from two or three
experiments per group. ⴱ, Significant differences between untreated and E2-treated groups were determined using Student’s t test (p ⱕ 0.05). KO, Knockout.
The Journal of Immunology
Intracellular staining of CNS mononuclear cells with anticytokine Abs was also performed. Mononuclear cells were recovered from the brain and spinal cords of perfused mice at the peak
of clinical disease. The total number of mononuclear cells recovered from the perfused CNS of untreated mice was 4 –5 times
higher than the number isolated from E2-treated mice. These cells
were stained with anti-CD4 Cy-Chrome-labeled Abs, and the frequency of cytokine-producing V␤8.2⫹ Th cells was measured by
staining with anti-V␤8.2-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.
FIGURE 4. Cytokine production is reduced in the CNS of estrogen-treated mice. A, Total RNA from the spinal cords of mice at the peak of EAE (days
12–16 postimmunization) was purified and used to measure cytokine-specific mRNA levels by RPA. Data are presented as the mean ⫾ SD from two
different experiments per group. ⴱ, Significant differences (p ⱕ 0.05) between the untreated and estrogen-treated mice were calculated using a two-tailed
Student t test. B, CNS mononuclear cells were isolated from the brains and spinal cords of perfused mice at the peak of EAE and stimulated for 24 h with
MOG35–55. Brefeldin A was added for the last 5 h, and cytokine production of fixed and permeabilized cells was measured by intracellular staining. The
cells were gated on CD4-Cy-Chrome-positive T lymphocytes. The numbers in the plots refer to the proportion of V␤ 8.2⫹ T cells that express the indicated
cytokine. ⴱ, Significant differences between untreated and E2 treated mice as determined by Student’s t test (p ⱕ 0.01). KO, knockout.
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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.
547
548
ESTROGEN REDUCES SEVERITY OF EAE IN CYTOKINE KNOCKOUT MICE
Estrogen treatment reduced the frequency of TNF-␣-secreting
cells
Proliferation of draining LN cells from either untreated or E2treated 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 MOG35–55specific T cells to proliferate in response to Ag.
The regulation of cell adhesion molecules is another possible
mechanism by which estrogen treatment controls the migration of
inflammatory cells into the CNS. The expression of cell surface
adhesion and activation/memory Ags was determined by staining
with fluorochrome-labeled Abs and flow cytometry. No significant
differences in the expression of very late Ag-4, CD44, or CD62L
were detected between LN cells from E2 vs control mice with EAE
(Fig. 5B). Furthermore, no differences in activation markers
(CD69, CD25, Fas ligand, CD40 ligand, and CD28) were seen.
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-V␤8.1/8.2 TCR Abs before fixation and permeabilization, and then were stained with the indicated PE-labeled
anti-cytokine Abs. We focused on V␤8.1/8.2 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 V␤8.1/8.2
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 V␤8.1/8.2
TCR⫹ cells was significantly diminished in C57BL/6 mice ( p ⫽
FIGURE 5. Estrogen treatment fails to alter T cell proliferation and the expression of
cell adhesion and activation markers. The
mice were treated with 17␤-estradiol 1 wk
before immunization with MOG35–55. A, LN
cells were recovered from three representative mice for each group at the peak of disease for untreated mice (12–16 days postimmunization). Pooled LN cells were cultured
with indicated amounts of MOG35–55 and T
cell proliferation was determined as described in Materials and Methods. The data
shown are representative of two to six experiments for each group. B, The expression of
adhesion and memory/activation markers on
LN cells from untreated and E2-treated mice
was analyzed by three-color flow cytometry.
Data are expressed as the frequency of
CD4⫹/V␤8.2⫹ T cells expressing the indicated marker. Student’s t test was used to
determine whether differences between untreated and E2-treated groups were significant (p ⬍ 0.05). KO, Knockout; FASL, Fas
ligand.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Estrogen treatment failed to alter T cell proliferation and the
expression of cell surface adhesion and activation Ags
The Journal of Immunology
549
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 E2treated mice also suggests that these cells also produce lower levels of TNF-␣ compared with the untreated mice. As the number of
V␤8.1/8.2⫹ 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 V␤8.2⫺ 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
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Estrogen treatment significantly reduces the frequency of TNF-␣-producing T cells. C57BL/6 and cytokine knockout (KO) mice were treated
with E2 1 wk before immunization with MOG35–55. At the peak of EAE (13–16 days postimmunization) splenocytes were recovered from representative
animals and cultured for 24 h with MOG35–55. Brefeldin A was added for the last 5 h. The cells were stained first with anti-V␤8.1/8.2 FITC and then
permeabilized and stained with anti-cytokine Abs conjugated to PE. The cells were gated on lymphocytes based on forward and side scatter characteristics,
and 40,000 events were collected. A, Dot plots from representative E2-treated and untreated C57BL/6 mice stained with IFN-␥, TNF-␣, or isotype control
Abs. B, Dual staining of V␤8.2⫹ T lymphocytes from C57BL/6 mice stained with anti-IFN-␥ and anti-TNF-␣. C, The frequency of cytokine-producing
V␤8.2⫹ T lymphocytes from untreated and E2-treated WT and cytokine knockout mice. Each point represents data from an individual mouse; the lines
correspond to the mean frequency of cytokine-producing cells for that group.
550
ESTROGEN REDUCES SEVERITY OF EAE IN CYTOKINE KNOCKOUT MICE
Table II. EAE is less severe in TNF-␣-deficient mice
Incidence
Onset
BL6.129s
8/8
11.8 ⫾ 3.1
TNF-␣ KO 4/7
13.0 ⫾ 0.8
p
0.153a
0.340
Mortality
3/8
0/7
0.244
Peak
CDI
4.9 ⫾ 1.5 78.3 ⫾ 35.8
0.6 ⫾ 0.6 2.6 ⫾ 4.6
⬍0.0001 ⬍0.0001
a
Significant differences in incidence and mortality between untreated and E2treated mice were assessed by ␹2 analysis. Difference in onset was determined using
the two-tailed Student t test. Differences in peak score and cumulative disease index
(CDI) were assessed by the Mann-Whitney test. A value of p ⱕ 0.05 was considered
significant.
(34 –36), and their regulation by E2 provides an important new
insight into the regulatory effects of estrogen.
Discussion
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The results of the current study have revealed two important new
findings regarding the mechanisms by which E2 suppresses the
development of EAE. First, treatment with E2 diminished both the
clinical and histological manifestations of EAE in C57BL/6, IL-4,
IL-10, and IFN-␥ knockout mice. In conjunction with a lack of any
detectable compensatory cytokines, this finding provides strong
evidence 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– 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 E2treated mice. However, E2 also considerably reduced the intracellular staining intensity and percentage of cells producing IFN-␥
and TNF-␣ in both V␤8.2⫹ and V␤8.2⫺ 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, 52–54). 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
The Journal of Immunology
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