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,
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Department of Veterans Affairs, Neuroimmunology Research, and Departments of
Neurology and
Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol were found to significantly reduce the clinical
manifestations of active EAE in both male and female mice. Estriol was
also effective but at doses below those previously established for
pregnancy. The reduction in disease severity was accompanied by a
coincident reduction in the number and size of inflammatory foci in the
CNS of estrogen (17-estradiol or estriol)-treated mice. Recipients
of encephalitogenic T cells from low-dose estrogen-treated mice
developed less severe paralysis than mice receiving T cells from
placebo-treated mice. A modest shift in Th1/Th2 balance suggested that
low dose estrogen therapy could bias the immune reaction toward a
protective anti-inflammatory cytokine response. However, estrogen
treatment at the onset of active EAE failed to reduce disease severity,
a result that is consistent with the hypothesis that naive cells are
more sensitive to sex hormones than differentiated effector cells.
These data suggest that treatment with low doses of estrogen can reduce
the capacity of developing myelin-reactive T cells to initiate disease
and challenges the idea that increased susceptibility to autoimmunity
in females is dependent on homeostatic levels of
estrogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is commonly induced by immunizing susceptible strains of laboratory rodents with myelin proteins or peptides (8). EAE is a useful model that has provided considerable insights into the pathogenesis of MS. The inflammatory CD4+ T cells that mediate EAE secrete proinflammatory Th1 cytokines, and Th2 cells are thought to play a role in disease recovery (9, 10). Gender differences in the susceptibility and severity of EAE can parallel gender differences in MS. For example, female SJL mice develop more severe EAE after immunization with proteolipid protein peptide (PLP) 139-151 or after the adoptive transfer of PLP139-151-specific T cells (11, 12, 13). The increased severity in females is associated with elevated levels of Th1 cytokines, whereas males produce high levels of Th2 cytokines and develop less severe disease (14, 15). Interestingly, sex hormones have been found to alter the disease course in the EAE model system. Pregnancy has been shown to protect animals from EAE (16, 17), and estrogen administered at levels equal or greater than those found during pregnancy have been shown to suppress the clinical and histopathological symptoms of EAE in mice and rats (18, 19). Recent studies have found that estriol (E3), a hormone produced by the placenta during pregnancy, had profound effects on EAE (20), and, based on these results, human trials using E3 to treat MS patients have begun.
A shift toward Th2 cytokine production has been observed during pregnancy (21, 22) that may explain, at least in part, the decrease in cell-mediated immune responses and cell-mediated autoimmunity during this time. High-dose estrogen therapy has been shown by some to mimic this shift in the immune response. Specifically, pregnancy levels of E3 increased IL-10 secretion and IgG1 Ab production with a subsequent reduction in the severity of EAE (20). Other studies have suggested that the response to estrogens may be biphasic, with low levels of estrogen conferring increased Th1 immunity and susceptibility to cell-mediated autoimmune diseases, whereas high levels shift the response toward Th2 immunity and protection from cell-mediated pathology (23). This rationale explains both the increase in the incidence and severity of EAE in females and the protection afforded by increased estrogen levels during pregnancy. However, the concept of biphasic responses to estrogen remains a hypothesis and has yet to be critically tested in the EAE model.
In this study, the efficacy of low-dose estrogen therapy was evaluated
in two different models of EAE. Treatment with low levels of both
17-estradiol (E2) and E3 reduced the severity of EAE caused by
active immunization. Estrogen treatment was equally effective in both
SJL and B10.PL models of disease. No differences in the level of
protection were observed between E2 and E3, and both genders were
equally sensitive to the effects of estrogen. Low-dose estrogen
treatment of donor mice reduced the severity of EAE induced by the
adoptive transfer of PLP139-151-specific T cells, but
treatment of EAE at disease onset had no effect, suggesting that the
priming phase of the immune response was more sensitive to estrogen
regulation. No evidence was found for a biphasic response to estrogen
in EAE. These observations challenge the hypothesis that increased
susceptibility to autoimmune disease in females is dependent on low
levels of estrogen found during estrus or diestrus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Age-matched SJL/J and B10.PL mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). Young adult (
10 wk old) mice
were used for all experiments mentioned in this report. The animals
were housed in the Animal Resource Facility at the Portland Veterans
Affairs Medical Center in accordance with institutional guidelines.
Antigens
Mouse PLP139-151 (HCLGKWLGHPDKF) and myelin basic protein (MBP) peptide Ac1-11 (ASQKRPSQRSK) were synthesized using solid-phase chemistry on a Synergy 432A peptide synthesizer (Applied Biosystems, Foster City, CA), and purified prior to use.
Estrogen treatment and measurement of serum estrogen levels
Sixty-day release pellets of E2, E3, and placebo were implanted s.c. in the scapular region behind the neck using a 12-gauge trochar as described by the manufacturer (Innovative Research, Sarasota, FL). The mice were implanted 1 wk before immunization with the appropriate myelin Ag.
Representative animals were bled by cardiac puncture, and the blood was allowed to clot at 4°C overnight. The samples were centrifuged, and the sera were collected and stored at -80°C until hormone analysis was performed. Serum levels of E2 and E3 were determined by radioimmunoassay (RIA) after Sephadex LH-20 column chromatography. All samples were analyzed in a single assay for each hormone.
Induction of EAE
SJL mice were inoculated s.c. in the flanks with 0.2 ml of an emulsion containing 150 µg of PLP139-151 in saline and an equal volume of CFA containing 200 µg of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). B10.PL mice were immunized with an emulsion containing 400 µg of MBP Ac1-11 and 200 µg of Mycobacterium tuberculosis. Disease induction in B10.PL mice required treatment with pertussis toxin on the day of immunization (75 ng/mouse) and 2 days later (200 ng/mouse). For adoptive transfer, SJL mice were implanted with either E2 or E3 pellets and, 1 wk later, immunized as described above. Ten days after the immunization, splenocytes were recovered and stimulated in vitro with 10 µg of PLP139-151 for 72 h. Live cells were isolated by ficoll density centrifugation, and 30 x 106 T cell blasts were injected i.p into naive female SJL hosts. The mice were examined daily for clinical signs of disease and scored according to the following scale: 0, normal; 1, minimal or mild hind limb weakness; 2, moderate hind limb weakness or mild ataxia; 3, moderately severe hind limb weakness; 4, severe hind limb weakness or moderate ataxia; 5, paraplegia with no more than moderate forelimb weakness; 6, paraplegia with severe forelimb weakness or severe ataxia.
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 luxol fast blue-periodic acid schiff-hematoxylin and analyzed by light microscopy. Semiquantitative analysis of inflammation and demyelination was determined by examining at least ten sections from each mouse.
Immunofluorescent staining for flow cytometry
Draining lymph node (DLN) cells were removed during the peak of clinical symptoms and analyzed for the expression of cell surface proteins by fluorescent staining ex vivo. The following fluorochrome-conjugated Abs obtained from PharMingen (San Diego, CA) were used for the direct staining of DLN cells: anti-CD4, anti-CD25, anti-CD69, anti-CD95 ligand (anti-CD95L), anti-CD44, anti-CD62L, anti-CD49d. Two-color immunofluorescent analysis was performed on a FACScan instrument (Becton Dickinson, Mountain View, CA) using CellQuest software. For each experiment the cells were stained with isotype control Abs to establish background staining and to set the quadrants before calculating the percent positive staining cells.
Proliferation assays
DLN cells were recovered from the mice at peak of clinical EAE (days 12–16 postimmunization) as previously described (13). The in vitro proliferative response was determined using a standard microtiter assay (24). Briefly, DLN cells were cultured in 96-well, flat-bottom tissue culture plates at 4 x 105 cells per well in stimulation medium alone (control) or with test Ags (i.e., PLP139-151) and incubated for 3 days at 37°C in 7% CO2. Wells were pulsed for the final 18 h with 0.5 mCi of [3H]methylthymidine (Amersham, Arlington Heights, IL). The cells were harvested onto glass fiber filters, and tritiated thymidine uptake was measured by a liquid scintillation counter. Results were determined from the means of triplicate cultures. Stimulation indices were determined by calculating the ratio of Ag-specific cpm to control cpm.
Cytokine detection by ELISA
DLN cells were cultured at 4 x
106/ml and stimulated with the appropriate Ag in
24-well culture plates. Cell culture supernatants were recovered
between 48 and 72 h and frozen at -70°C until needed for the
cytokine assay. Measurement of cytokines was performed by ELISA using
cytokine-specific capture and detection Abs (PharMingen). Standard
curves for each assay were generated using recombinant mouse cytokines
(PharMingen), and the concentration of cytokines in the cell
supernatants was determined by interpolation from the appropriate
standard curve. IFN-
, TNF-
, and IL-12 were chosen as
representative Th1 cytokines, whereas IL-4 and IL-10 were measured as
representative Th2 cytokines.
PLP139-151-specific Ab ELISA
Nunc-Immuno 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with PLP139-151 at 4 µg/ml in PBS overnight at 4°C. The plates were washed and blocked before the addition of serum at the indicated dilution in triplicate. The samples were incubated overnight at 4°C, and the plate was washed before the addition of an affinity-purified, biotinylated goat anti-mouse Ig (diluted 1:10,000) detecting Ab (Accurate Chemical and Scientific, Westbury, NY). The plates were incubated for 1 h at room temperature before they were washed. A 1:400 dilution of avidin-peroxidase conjugate (Sigma, St. Louis, MO) was added to each well, and the plates were incubated for an additional 45 min. After the final wash, a peroxidase substrate (3,3',5,5'-tetramethylbenzidine; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the wells, and the reaction was stopped by the addition of 0.18 M sulfuric acid. The plates were read in a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm. Wells coated with an irrelevant peptide (myelin oligodendrocyte glycoprotein 35-55) acted as a negative control.
Anexin V staining
Female SJL mice were treated with estrogen pellets and immunized with PLP139-151 as described earlier. Ten days after immunization, DLN and splenocytes were recovered and an aliquot of cells was stained with anti CD4-FITC, annexin V-PE, and 7-amino-actinomycin D as per the manufacturer’s protocol (PharMingen). The remaining cells were stimulated with PLP139-151 for 72 h, washed, and stained as above. CD4+ T cells were gated on acquisition, and three-color immunofluorescent analysis of apoptosis was performed using a FACScan instrument and CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously established that pregnancy can ameliorate
cell-mediated autoimmune diseases such as rheumatoid arthritis
(25, 26) and MS (5, 6, 7). Estrogens and other
sex hormones are found at high levels in the serum during pregnancy and
are thought to be an important factor mediating this protection. As a
result, high-dose estrogen therapy is being considered for the
treatment of these diseases in humans. The evaluation of low-dose
estrogen therapy as an alternative to high-dose therapy has not been
fully investigated. The purpose of this study was to determine whether
low doses of estrogen could provide protection against EAE an animal
model for MS. Female SJL mice were implanted with 60-day release
tablets (Innovative Research, Sarasota, FL) containing E2 1 wk before
the active induction of EAE by immunization with
PLP139-151. The dose of E2 chosen for these studies (Table I
) was intended to mimic the levels of E2
found during pregnancy, estrus, or diestrus phases of the hormone cycle
(27) (Table II). E2 levels
were measured in representative animals and were determined to be
equivalent to those reported by the manufacturer (data not shown).
Pregnancy levels of E2 reduced the incidence and severity of clinical
disease in a manner similar to that reported previously (Fig. 1). More importantly, low levels of E2
profoundly reduced the clinical manifestations of disease. Pellets
releasing as little as 25–50 pg of E2 per ml of serum significantly
diminished the severity of paralysis when compared with placebo
controls (Fig. 1). Estrus levels of E2 also delayed the onset of
clinical disease achieving statistical significance in animals
implanted with 0.36 mg E2 pellets. A significant reduction in the
incidence of disease was only seen in animals implanted with pregnancy
levels of E2. In addition, pathological examination revealed a dramatic
reduction in mononuclear cell infiltration and demyelination in the
spinal cords of E2-protected mice when compared with placebo-treated
mice (Fig. 2).
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The diminution of MS-related symptoms during pregnancy appear to
be most profound during the third trimester (7). E3 is a
hormone produced by the placenta and is at its highest levels during
the third trimester. Recently, high-dose E3 treatment has been shown to
reduce the severity of EAE induced by the adoptive transfer of
myelin-reactive T cells (20). In this study, high-dose E3
therapy effectively reduced the incidence and severity of EAE induced
by active immunization of female SJL mice with PLP139-151
(Fig. 3). Treatment of female mice with
1.5 mg E3 pellets resulted in serum hormone levels that were one-half
to one-third of that known to result from pregnancy (Table I
). Disease
severity was significantly reduced in mice receiving low-dose E3
treatment as reflected by a significant decrease in mean peak disease
score and cumulative disease index (CDI; Fig. 3). The diminution in
clinical disease score was accompanied by a substantial reduction in
inflammation and demyelination upon pathological examination (Fig. 2).
The direct comparison of E2 and E3 in the same animal model also
allowed us to determine whether one form of estrogen was more or less
potent than the other form. No statistically significant differences
(as determined by the Fisher exact test) in the incidence or severity
of EAE were found, indicating that E2 and E3 were equally
protective.
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The sensitivity of the immune response to estrogens may be linked
to genetic factors both within and outside of the immune response loci.
B10.PL mice are genetically distinct from SJL mice and respond to a
different dominant myelin Ag, MBP Ac1-11. The sensitivity of these mice
to estrogen therapy was tested by treating the mice with
estrogen-containing pellets before immunization. Pregnancy levels of E2
profoundly lowered the incidence, delayed the onset, and reduced
clinical paralysis (Table III). Low-level
E2 treatment also significantly delayed the onset and reduced the
severity of EAE induced by the immunization with MBP Ac1-11 peptide
(Table III). When the cumulative disease indices and peak disease
scores were compared (Fisher’s exact test), no significant differences
in E2 sensitivity between SJL and B10.PL mice at low E2 levels were
found (data not shown); however, B10.PL mice appeared to be more
sensitive to high-dose E2 treatment. Strain differences in peak disease
score and CDI were significant in mice receiving 2.5 mg E2 pellets
(p = 0.005). E3 also delayed the onset and
reduced the severity of disease in B10.PL mice (Table IV), but no differences in sensitivity to
E3 were detected between these mice and SJL mice as determined by the
Fisher exact test (data not shown).
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The direct influence of estrogen on cells of the immune system is
supported by evidence demonstrating estrogen receptors (ERs) in
immunocompetent cells (28, 29). Furthermore, we have found
that ER expression is not limited to female cells, but is also present
in male cells (B. F. Bebo and H. Offner, unpublished observations).
Because male cells are potentially sensitive to estrogens, estrogen
therapy was performed on male SJL mice. Male mice were treated with E2-
and E3-containing pellets as described previously and 1 wk later they
were immunized with PLP139-151. Treatment with either E2 or
E3 delayed the onset and reduced the severity of clinical disease, even
at doses equivalent to estrus levels (150–200 pg/ml) in females (Fig. 4). No significant differences in
estrogen sensitivity (as determined by the Fisher exact test) were
detected between males and females (data not shown).
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Estrogen has been shown to directly regulate immunocompetent cell
function by several investigators (29, 30). However, it is
unclear whether estrogen influences the induction and/or effector phase
of the immune response. To address this question, female SJL mice were
treated with either E2- or E3-containing pellets at the indicated doses
1 wk before immunization with PLP 139-151. Splenocytes were recovered
10 days after immunization and stimulated in vitro with
PLP139-151 for 72 h before transfer of 30 x
106 T cell blasts into naive female hosts. The
adoptive transfer of placebo-treated splenocytes resulted in the rapid
development of severe paralysis, whereas the onset of disease was
delayed and significantly less severe in mice receiving splenocytes
from estrogen-treated mice (Table V). No
differences in protection were observed between equivalent doses of E2
and E3, but splenocytes from high-dose E3-treated mice were the least
encephalitogenic. These data support the hypothesis that estrogen has
profound effects on the development of the nascent immune response and
is consistent with the hypothesis that sex hormones in the lymphoid
microenvironment during the initial contact with Ag play a critical
role in determining the nature of the response.
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The ability of low-dose estrogen therapy to treat established
disease was also investigated. Female SJL mice were immunized with
PLP139-151 and treated with low-dose E2- and E3-containing
pellets at the onset of clinical disease (day 11). Treatment with low-
dose E2 failed to alter the severity of established disease (Table VI), and although there was some
indication that E3 at higher doses could reduce severity of disease,
these results failed to achieve statistical significance. Estrogen
treatment at the onset of paralysis in the B10.PL model also failed to
significantly alter the disease course (Table VI). These results show
that established disease is relatively resistant to regulation by
estrogens and suggest that naive cells are more sensitive to the
effects of sex hormones.
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Given that estrogen therapy reduced the capacity of myelin-reactive T cells to transfer disease, our attention focused on investigating estrogen-regulated properties of T cells. mAbs specific for adhesion and activation markers were used to assess whether estrogen therapy altered their expression of T cell adhesion or activation markers. DLN cells were recovered from mice during the peak of clinical EAE and incubated with the indicated fluorochrome-conjugated mAbs, and surface expression was measured by FACS. Approximately 50% (42–49%) of the DLN cells were CD4+, and no differences between placebo- and estrogen-treated mice were found (data not shown). Approximately 5% of the CD4+ T cells in the DLN had an activated phenotype (CD25+, CD69+, FasL+) but, again, no differences between placebo- and estrogen-treated mice were noted (data not shown). In addition, no differences in adhesion molecule expression (CD44, CD62L, CD49d) were observed (data not shown). These data indicate that estrogen therapy had no apparent effect on the phenotype of T lymphocytes in the lymph nodes draining the site of immunization.
Proliferation of DLN T cells from placebo- and estrogen-treated mice
was measured to determine whether estrogen therapy altered the ability
of these cells to respond to Ag. DLN T cells were removed from
representative animals during the peak of clinical EAE and stimulated
with Ag in vitro, and proliferation was measured using a standard
[3H]thymidine incorporation assay. A modest
decrease in proliferation to PLP139-151 was consistently
observed in DLN cells isolated from estrogen-treated mice (Fig. 5). However, in all cases the reduction
in Ag-specific proliferation failed to achieve statistical significance
(p > 0.10). No consistent differences in
background or mitogen-induced proliferation were observed (data not
shown). A similar modest but insignificant reduction in Ag-specific
proliferation was also observed in the B10.PL model (data not
shown).
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, IL-12, and TNF- were used as
representative Th1 cytokines, whereas IL-4 and IL-10 were used as
representative Th2 cytokines. Even though the secretion of IFN- was
consistently lower in E2- and E3-treated groups of mice (Fig. 6A), the reduction in IFN-
levels fell short of being statistically significant
(p > 0.10). The decrease in IFN- secretion
was accompanied by a modest increase in IL-10 (Fig. 6B) and
a small decrease in IL-12 (Fig. 6E). Despite the lack of
statistical significance, the trend toward higher Th1 and lower Th2
cytokines points toward a subtle shift in the Th1/Th2 balance. The
shift can be seen more clearly when the cytokine response of each
individual mouse is plotted as a ratio of IFN- to IL-10 (Fig. 6C). There was a marked decrease in the frequency of high
Th1 responder mice in the E2- and E3-treated groups when compared with
placebo animals that approached significance (p
= 0.09 for the 5.0 mg E3-treated mice). We failed to detect any
informative trends in IL-4 secretion (Fig. 6D), and TNF-
secretion was very often below the limits of detection for the assay
(<31.25 pg/ml). Modest changes in cytokine responses induced by
low-dose estrogen therapy were also observed in the B10.PL model and
were consistent with the data described for the SJL model (data not
shown). Finally, the level of cytokines produced by splenocytes was
measured in cultures before the adoptive transfer of EAE (organs
harvested 10 days postimmunization). The influence of E2 and E3 on
cytokine production in splenocytes was comparable to DLN cells (data
not shown).
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An increase in humoral immunity has been reported during pregnancy and
during high-dose estrogen therapy (20, 21, 22). Consequently,
the humoral immune response in low-dose estrogen-treated animals was
compared with placebo controls. Serum was collected from individual
mice at the peak of clinical disease and
PLP139-151-specific Ig levels were measured using a
standard ELISA. Although a considerable amount of
anti-PLP139-151 activity could be measured, no
significant differences (p 
0.180) in
PLP139-151-specific Ab production were observed
between placebo- and estrogen-treated groups (data not shown).
These data suggest that the modest shift toward Th2 cytokine
production in estrogen-treated mice was insufficient to enhance humoral
immunity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alterations in the lymphoid microenvironment during priming with Ag are
thought to influence the outcome of the subsequent immune response
(40, 41). A reduction in the capacity of lymphocytes from
estrogen-treated mice to transfer EAE was observed, and is consistent
with the hypothesis that estrogen in the lymphoid microenvironment can
alter T cell function. Estrogen has been shown to alter the function of
various immunocompetent cells including B cells (42, 43),
T cells (30), macrophages (44, 45), NK cells
(46), endothelial cells (47, 48), and stromal
cells (49). Much attention has been focused on the ability
of estrogen to regulate T cell cytokine secretion. It is generally
accepted that EAE and other models of organ-specific autoimmunity are
mediated by Th1 cells secreting proinflammatory cytokines (IFN-,
TNF-, LT, IL-2), whereas Th2 cells produce anti-inflammatory
cytokines (IL-4, IL-5, IL-10, TGF-) and act to regulate disease
(9, 10). High-dose estrogen therapy has been shown to bias
cytokine responses toward Th2 (20). However, the skewing
of T cell activation from a Th1 to Th2 phenotype is most likely not the
full explanation of how low-dose estrogen protects against EAE. Despite
the profound effects of estrogen treatment on the clinical course of
active EAE, only a modest shift toward Th2 cytokine production was
measured, an effect that is consistent with previous studies by our
group (39). Furthermore, estrogen therapy failed to
enhance the sensitivity of lymphocytes to apoptotic stimuli. It is
likely that estrogen therapy alters the expression of many genes
related to encephalitogenicity, and the discovery of estrogen-regulated
genes is a subject of investigation in our laboratory.
The effector phase of the immune response is also sensitive to regulation by estrogen. Treatment of host mice has been shown to reduce the severity of EAE induced by the adoptive transfer of encephalitogenic lymphocytes (20). In this report, a significant reduction in the number of inflammatory and demyelinating lesions was observed in the CNS of low-dose estrogen-treated mice. This is consistent with a role for estrogen in regulating leukocyte homing and tissue damage in the CNS. This concept is supported by the observation that estrogen can regulate the expression of endothelial cell adhesion molecules such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin and alter the binding of leukocytes to cultured endothelial cell monolayers (47). Estrogen replacement therapy is currently being considered as a potential treatment for a number of neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. This is based on the known ability of estrogen to protect against oxidative injury to neural tissue (50). Consequently, low-dose estrogen therapy may serve to regulate leukocyte entry and reduce oxidative injury in the CNS.
Sex hormones can act on the immune system in a variety of ways. A number of studies have demonstrated that estrogens can directly alter immunocompetent cell function (36, 51), a hypothesis that is supported by evidence demonstrating ERs in Ag-responsive cell types (14, 29). In contrast, estrogen may also act indirectly or mediate interactions with immunomodulatory factors. For example, the influence of estrogen upon the hypothalamic-pituitary-adrenal axis (HPA) is well recognized. Females have an enhanced HPA response to endotoxin compared with males, which is reversed after ovariectomy and is enhanced with estrogen treatment (52, 53). The regulation of glucocorticoid production by estrogen is one likely pathway by which estrogen therapy protects mice from EAE. However, female animals have been shown be more sensitive to estrogen-induced regulation of the HPA than males (54). Estrogen therapy was equally effective in protecting both male and female mice from EAE, suggesting that estrogen regulates the immune response directly or through some other pathway distinct from the HPA.
There are a variety of sex hormones in females that have potential
immunomodulatory functions. E2, E3, and progesterone have received the
most attention as of late because they are secreted at high levels
during pregnancy, a time marked by a significant reduction in the
clinical symptoms of MS (7). A recent comparison of E3 and
progesterone demonstrated that although E3 was quite effective at
reducing the clinical signs of EAE, progesterone failed to have any
significant affect (20). Others have claimed that E3 might
be a more potent treatment for EAE than E2 (19), but the
small number of animals used and the relatively modest difference in
the incidence of clinical disease make it difficult to draw any
substantive conclusions. In this study a direct comparison between E2
and E3 revealed no significant differences in the ability of these
hormones to reduce the incidence or severity of EAE. These observations
were made in a large cohort of mice and in two substantially different
EAE models. Because E2 has a higher affinity for both ERs (10-fold
higher for ER- and 5-fold higher for ER-), and a significantly
longer serum half-life than E3 (55), it would seem to be a
logical choice as a potential therapy for human autoimmune
diseases.
The challenge of using sex hormone-based therapy for autoimmune disease is to maximize the effects of this treatment on the immune system and minimize the effects on other systems, including the reproductive and circulatory systems. The results of this study demonstrate that estrogen dramatically reduces severity of EAE at doses significantly lower than those previously established. These results suggest that estrogen may be an effective treatment for autoimmune disorders at doses that minimize the adverse effects of hormone therapy. Moreover, supplemental estrogen in combination with TCR peptide therapy, or other methods of immunotherapy, may very well be complimentary and could potentially be used together as an effective treatment for MS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce F. Bebo, Jr., Neurological Sciences Institute, OHSU West Campus, Building no. 1, 505 Northwest 185th Avenue, Beaverton, OR 97006.
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; E2, 17-estradiol; E3, estriol; MBP, myelin basic protein; DLN, draining lymph node; L, ligand; PLP, proteolipid protein peptide; ER, estrogen receptor; HPA, hypothalamic-pituitary-adrenal axis; CDI, cumulative disease index.
Received for publication April 21, 2000. Accepted for publication November 8, 2000.
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V. Douin-Echinard, S. Laffont, C. Seillet, L. Delpy, A. Krust, P. Chambon, P. Gourdy, J.-F. Arnal, and J.-C. Guery Estrogen Receptor {alpha}, but Not {beta}, Is Required for Optimal Dendritic Cell Differentiation and CD40-Induced Cytokine Production J. Immunol., March 15, 2008; 180(6): 3661 - 3669. [Abstract] [Full Text] [PDF]
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 C. Wang, B. Dehghani, I. J. Magrisso, E. A. Rick, E. Bonhomme, D. B. Cody, L. A. Elenich, S. Subramanian, S. J. Murphy, M. J. Kelly, et al. GPR30 Contributes to Estrogen-Induced Thymic Atrophy Mol. Endocrinol., March 1, 2008; 22(3): 636 - 648. [Abstract] [Full Text] [PDF]
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 D. Inman, K. Kawana, D. Schust, R. Lininger, and S. Young Cyclic Regulation of T-Bet and GATA-3 in Human Endometrium Reproductive Sciences, January 1, 2008; 15(1): 83 - 90. [Abstract] [PDF]
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M. A. McClain, N. N. Gatson, N. D. Powell, T. L. Papenfuss, I. E. Gienapp, F. Song, T. M. Shawler, A. Kithcart, and C. C. Whitacre Pregnancy Suppresses Experimental Autoimmune Encephalomyelitis through Immunoregulatory Cytokine Production J. Immunol., December 15, 2007; 179(12): 8146 - 8152. [Abstract] [Full Text] [PDF]
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 R. H. Straub The Complex Role of Estrogens in Inflammation Endocr. Rev., August 1, 2007; 28(5): 521 - 574. [Abstract] [Full Text] [PDF]
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 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]
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 S. Hirano, D. Furutama, and T. Hanafusa Physiologically high concentrations of 17beta-estradiol enhance NF-{kappa}B activity in human T cells Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1465 - R1471. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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L. Arruvito, M. Sanz, A. H. Banham, and L. Fainboim Expansion of CD4+CD25+and FOXP3+ Regulatory T Cells during the Follicular Phase of the Menstrual Cycle: Implications for Human Reproduction J. Immunol., February 15, 2007; 178(4): 2572 - 2578. [Abstract] [Full Text] [PDF]
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 W-H Zhu, C-Z Lu, Y-M Huang, H Link, and B-G Xiao A putative mechanism on remission of multiple sclerosis during pregnancy: estrogen-induced indoleamine 2,3-dioxygenase by dendritic cells Multiple Sclerosis, January 1, 2007; 13(1): 33 - 40. [Abstract] [PDF]
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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]
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A. L. Zoller and G. J. Kersh Estrogen Induces Thymic Atrophy by Eliminating Early Thymic Progenitors and Inhibiting Proliferation of beta-Selected Thymocytes. J. Immunol., June 15, 2006; 176(12): 7371 - 7378. [Abstract] [Full Text] [PDF]
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 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]
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J. Reddy, H. Waldner, X. Zhang, Z. Illes, K. W. Wucherpfennig, R. A. Sobel, and V. K. Kuchroo Cutting Edge: CD4+CD25+ Regulatory T Cells Contribute to Gender Differences in Susceptibility to Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2005; 175(9): 5591 - 5595. [Abstract] [Full Text] [PDF]
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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]
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L. Delpy, V. Douin-Echinard, L. Garidou, C. Bruand, A. Saoudi, and J.-C. Guery Estrogen Enhances Susceptibility to Experimental Autoimmune Myasthenia Gravis by Promoting Type 1-Polarized Immune Responses J. Immunol., October 15, 2005; 175(8): 5050 - 5057. [Abstract] [Full Text] [PDF]
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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]
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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]
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R. Mo, J. Chen, A. Grolleau-Julius, H. S. Murphy, B. C. Richardson, and R. L. Yung Estrogen Regulates CCR Gene Expression and Function in T Lymphocytes J. Immunol., May 15, 2005; 174(10): 6023 - 6029. [Abstract] [Full Text] [PDF]
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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]
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 J. E. Murase, K. K. Chan, T. J. Garite, D. M. Cooper, and G. D. Weinstein Hormonal Effect on Psoriasis in Pregnancy and Post Partum Arch Dermatol, May 1, 2005; 141(5): 601 - 606. [Abstract] [Full Text] [PDF]
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 S. Ghisletti, C. Meda, A. Maggi, and E. Vegeto 17{beta}-Estradiol Inhibits Inflammatory Gene Expression by Controlling NF-{kappa}B Intracellular Localization Mol. Cell. Biol., April 15, 2005; 25(8): 2957 - 2968. [Abstract] [Full Text] [PDF]
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 P. Gourdy, L. M. Araujo, R. Zhu, B. Garmy-Susini, S. Diem, H. Laurell, M. Leite-de-Moraes, M. Dy, J. F. Arnal, F. Bayard, et al. Relevance of sexual dimorphism to regulatory T cells: estradiol promotes IFN-{gamma} production by invariant natural killer T cells Blood, March 15, 2005; 105(6): 2415 - 2420. [Abstract] [Full Text] [PDF]
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M. J. Polanczyk, R. E. Jones, S. Subramanian, M. Afentoulis, C. Rich, M. Zakroczymski, P. Cooke, A. A. Vandenbark, and H. Offner T Lymphocytes Do Not Directly Mediate the Protective Effect of Estrogen on Experimental Autoimmune Encephalomyelitis Am. J. Pathol., December 1, 2004; 165(6): 2069 - 2077. [Abstract] [Full Text] [PDF]
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M. J. Polanczyk, B. D. Carson, S. Subramanian, M. Afentoulis, A. A. Vandenbark, S. F. Ziegler, and H. Offner Cutting Edge: Estrogen Drives Expansion of the CD4+CD25+ Regulatory T Cell Compartment J. Immunol., August 15, 2004; 173(4): 2227 - 2230. [Abstract] [Full Text] [PDF]
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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]
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M. Polanczyk, S. Yellayi, A. Zamora, S. Subramanian, M. Tovey, A. A. Vandenbark, H. Offner, J. F. Zachary, P. D. Fillmore, E. P. Blankenhorn, et al. Estrogen Receptor-1 (Esr1) and -2 (Esr2) Regulate the Severity of Clinical Experimental Allergic Encephalomyelitis in Male Mice Am. J. Pathol., June 1, 2004; 164(6): 1915 - 1924. [Abstract] [Full Text] [PDF]
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J. Huan, S. Subramanian, R. Jones, C. Rich, J. Link, J. Mooney, D. N. Bourdette, A. A. Vandenbark, G. G. Burrows, and H. Offner Monomeric Recombinant TCR Ligand Reduces Relapse Rate and Severity of Experimental Autoimmune Encephalomyelitis in SJL/J Mice through Cytokine Switch J. Immunol., April 1, 2004; 172(7): 4556 - 4566. [Abstract] [Full Text] [PDF]
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V. Paharkova-Vatchkova, R. Maldonado, and S. Kovats Estrogen Preferentially Promotes the Differentiation of CD11c+ CD11bintermediate Dendritic Cells from Bone Marrow Precursors J. Immunol., February 1, 2004; 172(3): 1426 - 1436. [Abstract] [Full Text] [PDF]
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 D. Xing, A. Miller, L. Novak, R. Rocha, Y.-F. Chen, and S. Oparil Estradiol and Progestins Differentially Modulate Leukocyte Infiltration After Vascular Injury Circulation, January 20, 2004; 109(2): 234 - 241. [Abstract] [Full Text] [PDF]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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A. Langer-Gould, H. Garren, A. Slansky, P. J. Ruiz, and L. Steinman Late Pregnancy Suppresses Relapses in Experimental Autoimmune Encephalomyelitis: Evidence for a Suppressive Pregancy-Related Serum Factor J. Immunol., July 15, 2002; 169(2): 1084 - 1091. [Abstract] [Full Text] [PDF]
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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]
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 J. Matsuda, M. T. Vanier, Y. Saito, K. Suzuki, and K. Suzuki Dramatic phenotypic improvement during pregnancy in a genetic leukodystrophy: estrogen appears to be a critical factor Hum. Mol. Genet., November 1, 2001; 10(23): 2709 - 2715. [Abstract] [Full Text] [PDF]
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A. Ito, B. F. Bebo Jr., A. Matejuk, A. Zamora, M. Silverman, A. Fyfe-Johnson, and H. Offner Estrogen Treatment Down-Regulates TNF-{{alpha}} Production and Reduces the Severity of Experimental Autoimmune Encephalomyelitis in Cytokine Knockout Mice J. Immunol., July 1, 2001; 167(1): 542 - 552. [Abstract] [Full Text] [PDF]
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2 analysis and significant differences in the day of
onset, severity at peak, and CDI were calculated by using the
two-tailed Student t test.
