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The Journal of Immunology, 2004, 173: 2435-2442.
Copyright © 2004 by The American Association of Immunologists

Estrogen Receptor {alpha} Signaling in Inflammatory Leukocytes Is Dispensable for 17{beta}-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis1

Lucile Garidou*, Sophie Laffont*, Victorine Douin-Echinard*, Christiane Coureau*, Andrée Krust{dagger}, Pierre Chambon{dagger} and Jean-Charles Guéry2,*

* Institut National de la Santé et de la Recherche Médicale Unité 563, Centre de Physiopathologie de Toulouse Purpan, Institut Claude de Préval, Hôpital Purpan, Toulouse, France; and {dagger} Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale-Université Louis Pasteur-Collège de France, Illkirch, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17{beta}-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor {alpha} (ER{alpha}) has been unambiguously inactivated, we found that ER{alpha} was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ER{alpha} expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ER{alpha} signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ER{alpha} in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ER{alpha} signaling in blood-derived inflammatory cells, and indicate that ER{alpha} expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS)3 is a chronic inflammatory disease of the CNS characterized by demyelination of axons, which affects women 2 times more often than men (1). MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are considered to be Th1-mediated autoimmune diseases (2) in which neuroantigen-specific T lymphocytes infiltrate the CNS, where they attract macrophages and cause inflammatory lesions, resulting in the destruction of myelin sheath, leading to progressive paralysis (3). Fluctuation of disease activity during pregnancy has suggested that sex hormones could modulate autoimmunity (4, 5, 6). Clinical remissions in MS patients have been preferentialy observed during the last trimester, when estrogens and progesterone concentrations are highest, whereas increased number of exacerbations have been often reported postpartum, when sex hormone levels fall (4, 5, 6). Likewise, a protective effect of pregnancy also has been reported in animal models of MS (7, 8).

Based on these observations, the effect of exogenous administration of estrogens has been evaluated in EAE in mice. It was shown that 17{beta}-estradiol (E2) administration, at both physiological and pharmacological doses, strongly inhibited EAE development (9, 10, 11). The protective effect of E2 was associated with a modest shift toward Th2 cytokine synthesis (10), suggesting that the mechanism of estrogen-mediated protection involved a favorable alteration in cytokine production by pathogenic T cells. Such observation has been reported recently in humans in a pilot clinical trial using estriol (12, 13). Oral estriol therapy was shown to cause a significant improvement in MS patients, associated with a decreased production of TNF-{alpha} and an enhanced synthesis of IL-5 and IL-10 by stimulated PBMC (13). However, the Th1 to Th2 bias hypothesis has been challenged by experiments showing that E2 could effectively suppress EAE in IL-4 or IL-10 knockout mice, suggesting that the E2-mediated protection did not require these two regulatory cytokines (11). It was shown, however, that E2 treatment resulted in a reduced frequency of TNF-{alpha}-producing cells and, to a lesser extent, IFN-{gamma}, in autoantigen-specific CD4 T cells. It was therefore suggested that E2 could mediate its protective effect on EAE through its capacity to selectively down-regulate TNF-{alpha} production by T cells and/or macrophages (11).

Estrogens exert their biological effects through nuclear hormone receptors. Two different estrogen receptors (ERs) have been described: ER{alpha} (14, 15) and ER{beta} (16). ERs have been shown to be expressed in various cell types of the immune system, including T cells (17, 18), macrophages (18, 19), and microglia (20). In support of a role of estrogens in the down-regulation of proinflammatory cytokine production, it has been shown that estrogen deficiency leads to the up-regulation of TNF-producing T cells in vivo (21). Down-regulation of TNF-{alpha} production by E2 has been well documented in other cell types, including macrophages (22, 23, 24), and an E2-inhibitory element has been identified in the promoter region of the TNF-{alpha} gene (25). Furthermore, E2 has been shown to prevent the LPS-induced inflammatory response in microglia, the resident macrophages of the brain, through activation of ER{alpha}, but not ER{beta} (20, 26). Recently, two studies have implicated ER{alpha} in the protective effect of estrogens on EAE development (27, 28). However, the demonstration that the protective effect of E2 on EAE would be mediated through ER{alpha} signaling in inflammatory cells, including macrophages and T cells, was still lacking.

Because clinical trials using estrogen are currently ongoing in MS patients (12, 13), it is important to delineate the mechanisms implicated in the protective effect of E2 in EAE, the animal model of MS. In the present study, we investigated the effect of administration of E2 on the development of myelin oligodendrocyte glycoprotein (MOG) 35–55-induced EAE in recently described ER{alpha}-null mutant mice, in which exon 2 of ER{alpha} has been deleted (ER{alpha}-{Delta}2–/–), leading to a complete inactivation of ER{alpha} gene (29, 30). Our data clearly confirm that ER{alpha} plays a pivotal role in the protective effect of E2 on disease development. Using irradiation bone marrow chimeras, we have assessed the role of ER{alpha} signaling in inflammatory cells, including macrophages and T cells. In this study, we present evidence that the E2-mediated protective effect on active EAE induction is largely independent of ER{alpha} expression in blood-derived inflammatory cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and hormonal treatments

C57BL/6 (B6) mice were purchased from the Centre d’Elevage R. Janvier (Le Genest St. Isle, France) and maintained in our animal facilities under pathogen-free conditions. Rag2-deficient B6 mice were obtained from the Centre National de la Recherche Scientifique (Centre de Développement des Techniques Avancées, Orléans, France). Mice with a disrupted ER{alpha} gene have been previously described (29) and backcrossed on B6 background for at least six generations. Because ER{alpha}-deficient female mice have higher levels of circulating E2 than normal mice, animals were castrated at 4 wk of age. Eight- to 10-wk-old mice from either sex were used in all experiments. The incidence and severity of EAE in castrated male and female mice were similar, and there was no difference between castrated male and female in the E2-mediated protection of EAE (data not shown). For estrogen hormone administration, 3-mm pellets (Innovative Research of America, Sarasota, FL) containing varying amounts of E2 were implanted s.c. on the animal back, as indicated. These pellets provide continuous controlled release of a constant level of hormone over a period of 60 days. All of the protocols used have been approved by our institutional review board for animal experimentation.

EAE induction and clinical evaluation

MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide (purity >85%) was purchased from Neosystem (Strasbourg, France). Mice were immunized s.c. in the flanks with 50 µg of MOG35–55 peptide emulsified in IFA supplemented with 4 mg/ml Mycobacterium tuberculosis (H37Ra; Difco Laboratories, Detroit, MI). Mice were injected i.p. with 200 ng of pertussis toxin (Calbiochem, Darmstadt, Germany) at days 0 and 2 and examined daily for clinical signs of disease. The mice were scored as follows: 0, no detectable signs of EAE; 1, complete limp tail; 2, limp tail and hindlimb weakness; 3, severe hindlimb weakness; 4, complete bilateral hindlimb paralysis; 4.5, complete hindlimb paralysis and unilateral forelimb paralysis; 5, total paralysis of both forelimbs and hindlimbs; 6, death.

T cell assays

For cytokine production analysis, splenic CD4 T cells (1.5 x 106 cells/ml) were cultured with irradiated syngeneic splenocytes (2 x 106 cells/ml) in the presence of MOG35–55 in 96- or 24-well culture plates (Costar, Cambridge, MA) in a final volume of 200 µl or 1 ml, respectively. For CD4 T cell enrichment, spleen cells were incubated with anti-B220 RA3-3A1 (American Type Culture Collection (ATCC), Manassas, VA; TIB 146), anti-CD8 KT1.5, anti-class II M5/114 (ATCC; TIB 120), anti-CD11b (ATCC; TIB 128) mAb, anti-NK DX5 mAb (BD Pharmingen, San Diego, CA), anti-Ly-6G RB6-8C5 mAb (BD Pharmingen), and anti-erythroid cells TER-119 mAb (BD Pharmingen). After washing, cells were incubated with sheep anti-rat IgG M-450 Dynabeads (Dynal Biotech, Oslo, Normay) and then selectively depleted using a magnet (BioSource International, Camarillo, CA). Purity of CD4 T cells was always superior to 90%. Cells were cultured in HL-1 synthetic medium (Cambrex, Walkersville, MD) supplemented with 2 mM L-glutamine and 50 µg/ml gentamicin (Sigma-Aldrich, St. Louis, MO). Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air. Supernatants from replicate cultures were collected after 48–72 h and assessed for cytokine concentration. IFN-{gamma} was quantified by two-site sandwich ELISA, as described (31). TNF-{alpha} was measured by ELISA using mAb TN3-19.12 for coating and biotin-labeled polyclonal rabbit anti-TNF-{alpha} Abs for detection, all obtained from BD Pharmingen. The sensitivity of the assays was ≤30 pg/ml for IFN-{gamma} and ≤5 pg/ml for TNF-{alpha} using recombinant cytokines as standard (BD Pharmingen). For T cell proliferation assays, cells were pulsed with 1 µCi of [3H]TdR (40 Ci/nmol; Radiochemical Centre, Amersham, U.K.) for the last 16 h of culture before harvesting on glass fiber filter. Incorporation of [3H]TdR was measured by direct counting using an automated beta plate counter (Matrix 9600; Packard Instrument, Meriden, CT).

Generation of bone marrow chimeras

All recipient mice were castrated at 4 wk of age. To assess the role of ER{alpha} expression in T and B cells, sublethally irradiated (450 rad) B6 Rag2–/– were reconstituted with 15 x 106 bone marrow cells from either ER{alpha}-{Delta}2+/+ or ER{alpha}-{Delta}2–/– mice. To generate chimeras selectively lacking ER{alpha} in host nonhemopoietic tissues and resident microglia, bone marrow cells from wild-type (WT) B6 mice were injected into lethally irradiated (850 rad) ER{alpha}-{Delta}2+/+ or ER{alpha}-{Delta}2–/– B6 mice. Mice were used for the experiments 6 wk after reconstitution. The appropriate genotype of peripheral hemopoietic cells was checked by PCR on splenocyte DNA using primers P1 (5'-TTGCCCGATAACAATAACAT-3'), P2 (5'-ATTGTCTCTTTCTGACAC-3'), and P3 (5'-GGCATTACCACTTCTCCTGGGAGTCT-3'), as described (29). The sizes of P1-P2 and P1-P3 fragments from WT are 390 and 920 bp, respectively, and that of the P1-P3 fragments from ER{alpha}null allele is 380 bp (29).

Flow cytometric analysis of CNS-infiltrating cells

Spinal cords were removed by flushing the spinal cord with ice-cold PBS. It was then homogenized and digested for 1 h with collagenase D (Roche, Indianapolis, IN) and DNase I (Roche) under continuous agitation at 37°C. The cell suspension was then strained through a 100-µm nylon filter (Falcon) and washed with PBS containing 20 mM HEPES. Cells were then resupended in 30% isotonic Percoll and centrifuged at 600 x g for 20 min at room temperature. The pellet, containing mononuclear cells, was resuspended in PBS-HEPES and washed extensively in FACS buffer containing 1% FCS, 5 mM EDTA, and 0.1% NaN3 in PBS. For flow cytometry, cells were stained with biotin-M1/9.3.4 anti-CD45 mAb (TIB 122; ATCC) and FITC-labeled anti-CD11b or anti-CD4 mAbs, all purchased from BD Pharmingen. Data were collected on a FACSCalibur cytometer and analyzed using CellQuest software (BD Biosciences, Mountain View, CA).

Quantitative assessment of transcripts in the CNS

Spinal cords were homogenized in TRIzol reagent (Invitrogen Life Technologies, San Diego, CA), and RNA was extracted following manufacturer’s instructions. RNA samples (1 µg) were treated with DNase I (Invitrogen Life Technologies) and transcribed into cDNA using random primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Reactions were performed in a 25 µl final volume using either the Taqman Universal PCR Master Mix (Eurogentec, Seraing, Belgium) for TCR {beta}-chain transcripts or the SYBR Green PCR Master Mix (Sigma-Aldrich) for most of the other transcripts, according to manufacturer’s recommendations (Applied Biosystems, Foster City, CA). Cycling conditions were 10 min at 90°C, followed by 40 two-temperature cycles (15 s at 90°C and 1 min at 60°C). Data were analyzed using the sequence detection software supplied with the instrument (Applied Biosystems). Cytokine and chemokine transcripts were normalized to hypoxanthine phosphoribosyltransferase (HPRT) transcripts abundance analyzed in parallel. Briefly, each set of sample was normalized using {Delta}CT = ({Delta}CTsample – {Delta}CT HPRT). The calibrator sample ({Delta}CTcalibration) was assigned as the sample with the highest {Delta}CT in each set. Relative mRNA levels were calculated by the expression 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = {Delta}CTsample (n) – {Delta}CTcalibration (n). Each reaction was performed in duplicate. For TCR {beta}-chain transcript determination, primers and probes have been described (32). Reverse and forward primers used for the SYBR Green PCR analysis of the following transcripts have also been described: TNF-{alpha}, IFN-{gamma}, and HPRT (33); lymphotoxin-{alpha} (34); CCL-1, CCL2, CCL3, CCL22, CXCL2, and CXCL9 (35).

Statistical analysis

Statistical significance of differences between groups of continuous variables was analyzed using the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 administration inhibits EAE development without affecting MOG-specific Th1 cell development

It has been shown by others that E2 administration before immunization with MOG35–55 peptide in CFA had a protective effect on EAE development in mice (9, 10, 36). However, in these studies, the doses of E2 used were usually well above the physiological range found during late pregnancy in mice that have been reported to be ~100 pg/ml (37, 38). Such levels of E2 can be achieved by treating animals with 0.25 mg of E2 pellets delivering a constant level of hormone over a 60-day period (39). We first assessed the effect of E2 treatment on EAE development and on the Ag-specific CD4 T cell response in ovariectomized B6 mice. Splenic CD4 T cells were recovered at the peak of the disease and stimulated in vitro with irradiated splenocytes in the presence of MOG35–55 peptide. The data in Fig. 1 show that despite a strong inhibitory effect of E2 on EAE development (Fig. 1A), neither MOG35–55-induced CD4 T cell proliferation (Fig. 1B) nor the production of IFN-{gamma} (Fig. 1C) or TNF-{alpha} (Fig. 1D) was inhibited in E2-treated mice as compared with untreated animals. These data show that E2-mediated protection from EAE can occur despite unaltered autoantigen-induced proliferation and production of proinflammatory cytokines by splenic autoantigen-specific CD4 T cells.



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  		FIGURE 1. Lack of correlation between E2-mediated protection of EAE and inhibition of autoantigen-specific Th1 development. Ovariectomized B6 mice were implanted with E2 pellets (0.25 mg/60 days) at the time of immunization with MOG35–55 in CFA. Control mice were ovariectomized, but received no pellet. Mice were assessed daily for clinical signs of disease (A). CD4 T cells were purified from the spleens recovered at the peak of the disease (day 17). Recall CD4 T cell proliferation (B), IFN-{gamma} (C), and TNF-{alpha} (D) secretion in response to MOG35–55 presented by irradiated syngeneic APCs were assessed in vitro. Data are expressed as mean ± SEM of four to five mice per group. Results are from one representative experiment of two performed.

 
ER{alpha} mediates the protective effect of E2 on EAE

We next evaluated whether the protective effect of E2 was mediated through ER{alpha}. To this end, we used ER{alpha}-deficient mice in which exon 2 has been deleted (ER{alpha}-{Delta}2–/–). Castrated ER{alpha}-deficient B6 mice and their ER{alpha}-{Delta}2+/+ littermate controls were implanted with E2 or placebo pellets and simultaneously immunized with MOG35–55 peptide in adjuvant. Animals were then monitored daily for clinical signs of EAE. As shown in Fig. 2, A and B, E2 administration strongly inhibits the development of EAE as compared with WT B6 mice treated with placebo control pellets. In contrast, in ER{alpha}-{Delta}2–/– mice treated with E2 at physiological (0.25 mg) or pharmacological (2.5 mg) doses, the disease onset and severity were similar to the placebo-treated control group (Fig. 2, A and B). As previously reported (10), no differences in EAE induction and estrogen-mediated protection on EAE were detected between castrated males and females (data not shown).



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  		FIGURE 2. The protective effect of physiological and pharmacological doses of E2 on active EAE and CNS inflammation is mediated by ER{alpha}. Male or female ER{alpha}-{Delta}2–/– B6 mice or WT littermate controls (four to six mice per group) were castrated at 4 wk of age. At week 8, mice were implanted with E2 pellets (0.25 or 2.5 mg, 60 days release) the day of immunization with MOG35–55. A, Mice were assessed daily for clinical signs of disease. B, Cumulative disease index (CDI), defined as the mean (± SD) of the sum of the daily disease scores for the indicated period of time. C and D, RNA were isolated, as described, from spinal cords 14 days after immunization. mRNA levels for TCR {beta}-chains, cytokines (C), and chemokines (D) were determined by real-time PCR analysis of spinal cord samples from individual mice. Transcript levels were normalized to HPRT transcripts abundance. Mean ± SD mRNA expression levels are represented as fold induction over basal (nonimmunized spinal cord) mRNA levels. Statistical significance of difference between groups was analyzed by using the Mann-Whitney U test.

 
We next focused on the CNS to analyze differences in the cytokine and chemokine expression profiles in E2-treated ER{alpha}-deficient and WT mice. At day 14 postimmunization, spinal cords were removed, and expression of cytokine and chemokine transcripts was determined by real-time PCR. The data in Fig. 2C show that the mRNA levels for TCR{beta}, IFN-{gamma}, TNF-{alpha}, and lymphotoxin-{alpha} were strongly up-regulated within the spinal cord of ER{alpha}-{Delta}2–/– mice and their WT littermate controls. Interestingly, while E2 treatment abolished the expression of these transcripts to nearly basal levels in ER{alpha}-{Delta}2+/+ mice, it had no effect in ER{alpha}-{Delta}2–/– mice for both doses of E2 tested.

Expression levels of chemokines such as CCL1 (TCA-3), CCL2 (MCP-1), and CCL6 (C10) have been shown to be dependent on hemopoietically derived TNF-{alpha} production (40). As compared with noninflamed tissue, these chemokine mRNA levels were strongly up-regulated in the spinal cord of placebo-treated WT mice, but remained at basal level in E2-treated WT animals (Fig. 2D). By contrast, chemokine transcripts were strongly expressed in the CNS of E2-treated ER{alpha}-{Delta}2–/– mice, at levels similar to those measured in WT or ER{alpha}-deficient placebo-treated mice. Examination of other chemokines, such as CXCL2 (MIP-2), CCL5 (RANTES), CXCL9 (monokine induced by IFN-{gamma}), CCL3 (MIP-1{alpha}), and CCL22 (macrophage-derived chemokine) (40), gave similar results (Fig. 2D).

Flow cytometric analysis of CNS mononuclear cells (MNC) was then performed at peak of clinical disease (Fig. 3). Within the CNS-infiltrating cells, CD4 T cells and macrophages are characterized by high CD45 expression, whereas resident brain macrophages (microglia) are CD45lowCD11b+. There were no overt differences in the cellular composition of the infiltrates between control WT (Fig. 3A) and ER{alpha}-{Delta}2–/– (Fig. 3B) B6 mice treated or not with E2. By contrast, in WT mice treated with E2, the few CNS MNC recovered were mainly composed of microglia (>60%), with <3% of CD4 T cells and 23% of CD11b+ macrophages (Fig. 3A). Results shown were obtained in mice treated with 2.5 mg of E2 pellets, but similar results were observed with the lower dose (0.25 mg; data not shown). The absence of inflammatory cell infiltrates in the CNS of estrogen-treated WT mice is consistent with the lack of up-regulation of TCR{beta} transcript levels (Fig. 2C).



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  		FIGURE 3. E2 prevents the recruitment of inflammatory infiltrates in the CNS in WT, but not in ER{alpha}-deficient mice. WT littermate controls (A) or ER{alpha}-{Delta}2–/– B6 (B) mice were treated and immunized, as described in legend for Fig. 1. Mice were sacrified at day 14 postimmunization with MOG35–55/CFA, and spinal cord inflammation was assessed by flow cytometry. Infiltrates obtained from pooled spinal cords were stained with Abs against CD45, CD11b, and CD4. Analysis of the frequency of brain macrophages (CD45low/CD11b+) and infiltrating macrophages (CD11b+/CD45high) and CD4 T cells (CD4+/CD45high) was performed on CD45+ MNC infiltrates.

 
Taken together, our data demonstrate that E2, at both physiological and pharmacological doses, inhibits EAE by preventing the infiltration of proinflammatory cells in the CNS through ER{alpha} signaling. The inhibitory effect of E2 on EAE was completely prevented in ER{alpha}-{Delta}2–/– B6 mice, strongly suggesting that ER{beta} plays no additional role.

In the absence of ER{alpha} expression in inflammatory T lymphocytes E2 still induced protection on EAE

To evaluate whether the protective effect of physiological doses of E2 on EAE was dependent on the expression of ER{alpha} in blood-derived hemopoietic cells, including encephalitogenic T cells, radiation bone marrow chimeras were generated. Irradiated B6 Rag2–/– recipients were reconstituted with bone marrow cells from ER{alpha}-deficient mice (ER{alpha}-{Delta}2–/–->B6Rag2–/–) or WT littermate controls (ER{alpha}-{Delta}2+/+->B6Rag2–/–). In ER{alpha}-{Delta}2–/–->B6Rag2–/– mice, T and B cells are devoid of functional ER{alpha}. However, because animals were only sublethally irradiated, a small proportion of radiation-resistant leukocytes, such as macrophages and NK cells, expresses the host WT genotype. As shown in Fig. 4A, a strong protective effect of E2 was still observed in mice lacking ER{alpha} expression in T and B cells. The protective effect of E2 was associated with reduced inflammatory T cell infiltrates, as shown by the expression of TCR{beta} and Th1 cytokine transcripts in the CNS (Fig. 4B). Indeed, cytofluorometric analysis of the CNS-infiltrating cells at the peak of disease showed a strong reduction in E2-treated mice of both CD4 T cells and blood-derived macrophages (Fig. 5 and data not shown). As assessed by measuring the transcripts in the CNS coding for TCR {beta}-chains and cytokines (Fig. 4B) as well as chemokines (Fig. 4C), the E2-mediated protective effect on CNS inflammation was similar between ER{alpha}-{Delta}2+/+->B6Rag2–/– and ER{alpha}-{Delta}2–/–->B6Rag2–/– radiation bone marrow chimeras. The cytokine secretion profile of splenic CD4 T cells recovered from mice at the peak of EAE was tested in response to MOG35–55 in presence of syngeneic APCs in vitro. In agreement with data in Fig. 1, the levels of both IFN-{gamma} (Fig. 6, A and C) and TNF-{alpha} (Fig. 6, B and D) were not reduced in E2-treated ER{alpha}-{Delta}2+/+->B6Rag2–/– and ER{alpha}-{Delta}2–/–->B6Rag2–/– chimeras as compared with the placebo control groups. Taken together, these data show that ER{alpha} expression in T lymphocytes, including MOG35–55-specific CD4 T cells, is dispensable for the E2-mediated protective effect on active EAE.



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  		FIGURE 4. ER{alpha} expression in T and B lymphocytes is dispensable for the E2-mediated protection on EAE. Recipient B6 Rag2–/– mice were castrated at 4 wk of age. Bone marrow chimeras were generated by injecting bone marrow cells (15 x 106 cells/mouse i.v.) from WT littermate control or ER{alpha}-deficient B6 mice into 6-wk-old irradiated (450 rad) B6Rag2–/– recipients. Five to six weeks after bone marrow reconstitution, mice (four to five per group) were implanted with placebo control or E2 pellets (0.25 mg) and immunized with MOG peptide in CFA to induce EAE. A, Mice were assessed daily for clinical signs of disease. B and C, RNA was isolated from spinal cords recovered at the peak of disease. Real-time PCR analysis of the indicated mRNA transcripts was performed, as in Fig. 2. Results are from one representative experiment of three performed.

 


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  		FIGURE 5. Lack of inflammatory infiltrates in E2-treated mice in the absence of ER{alpha} expression in T cells. ER{alpha}-{Delta}2+/+->B6Rag2–/– (A) and ER{alpha}-{Delta}2–/–->B6Rag2–/– (B) irradiation bone marrow chimeras treated and immunized, as described in legend for Fig. 4, were assessed for spinal cord inflammation by flow cytometry, as in Fig. 3. Infiltrates obtained from pooled spinal cords were stained with Abs against CD45 and CD4. Analysis of the frequency of infiltrating CD4 T cells (CD4+CD45high) was performed on CD45+ MNC infiltrates. Results are from one representative experiment of two performed.

 


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  		FIGURE 6. IFN-{gamma} and TNF-{alpha} production by Ag-specific ER{alpha}-deficient or WT CD4 T cells is not inhibited by E2 treatment. CD4 T cells were purified from the spleens of ER{alpha}-{Delta}2+/+->B6Rag2–/– (A and B) and ER{alpha}-{Delta}2–/–->B6Rag2–/– mice (C and D) at the peak of disease, and stimulated in vitro with MOG35–55 in the presence of irradiated syngeneic splenocytes. {square}, Medium alone; {blacksquare}, 10 µM MOG35–55. IFN-{gamma} (A and C) and TNF-{alpha} (B and D) production were tested in 48-h culture supernatants. Results are expressed as mean ± SD of triplicate cultures. Results are from one representative experiment of two performed.

 
Lack of E2-mediated protective effect on EAE in mice that selectively express ER{alpha} in the peripheral hemopoietic compartment

To assess whether the protective effect of E2 was essentially due to ER{alpha}-mediated signaling outside the sytemic immune compartment, WT->ER{alpha}-{Delta}2–/– radiation bone marrow chimeras were generated. Six weeks after reconstitution, mice were immunized to induce EAE and treated or not with low dose E2. The data in Fig. 7, A and B, show that incidence and severity of clinical disease were similar in placebo-treated WT->ER{alpha}-{Delta}2–/– mice and WT->ER{alpha}-{Delta}2+/+ mice. Interestingly, while treatment with E2 strongly inhibited EAE development in control WT->ER{alpha}-{Delta}2+/+ mice, EAE incidence and severity in E2-treated WT->ER{alpha}-{Delta}2–/– were indistinguishable from placebo-treated WT->ER{alpha}-{Delta}2–/– mice (Fig. 7, A and B). The genotype of the peripheral immune compartment was assessed by PCR analysis of spleen cell DNA. As shown in Fig. 7C, the targeted allele was not detected in the splenocytes of WT->ER{alpha}-{Delta}2–/– mice, demonstrating that most of the peripheral immune compartment possessed a functional ER{alpha} gene.



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  		FIGURE 7. Selective ER{alpha} expression in blood-derived inflammatory cells is not sufficient to mediate the E2-driven protection of EAE. Recipient mice were castrated at 4 wk of age. Bone marrow chimeras were generated by injecting bone marrow cells (20 x 106 cells/mouse i.v.) from WT or ER{alpha}-deficient B6 mice into 5-wk-old irradiated (850 rad) B6 recipients. Six weeks after bone marrow reconstitution, mice were implanted with placebo control or E2 pellets (0.25 mg) and immunized with MOG peptide for EAE induction. A, Mice (four to five per group) were assessed daily for clinical signs of disease. B, Cumulative disease index (CDI), defined as the mean ± SD of the sum of the daily disease scores for the indicated period of time. C, PCR genotyping on DNA purified from the splenocytes of bone marrow chimeras, showing absence of the targeted allele in the WT->ER{alpha}-{Delta}2–/–B6 mice (+->–). Statistical significance of difference between groups was analyzed by using the Mann-Whitney U test (*, p < 0.01). Results are from one representative experiment of three performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogen treatment has been shown to exert a protective effect on clinical signs and inflammatory lesions in EAE, a Th1-mediated autoimmune disease (9, 10, 11). It has been suggested that estrogens could suppress the development of inflammatory Th1-type responses in vivo, and particularly TNF-{alpha} production, through a direct action on immunocompetent cells such as T cells or macrophages (11, 41). The goal of our study was to characterize: 1) the role of ER{alpha} signaling in the E2-mediated protection of EAE, and 2) the role of ER{alpha} expression in the blood-derived inflammatory cells in this protective effect. We report that in the complete absence of functional ER{alpha}, E2 treatment had no inhibitory effect on disease onset and severity. Consistent with the clinical findings, cellular infiltrates and production of inflammatory cytokines and chemokines in the CNS were not affected with E2 treatment in ER{alpha}-{Delta}2–/– B6 mice. To test whether ER{alpha} expression in hemopoietic cells was essential for the protective effect of E2 on EAE, we generated radiation bone marrow chimeras using WT and ER{alpha}-{Delta}2–/– B6 mice. Two series of chimeras and their appropriate controls were generated: 1) mice that lack ER{alpha} expression in inflammatory T cells, but express ER{alpha} in nonhemopoietic tissues, including most resident CNS cells (ER{alpha}-{Delta}2–/–->Rag2–/–), and 2) the reciprocal chimeras (WT->ER{alpha}-{Delta}2–/–) that selectively expressed ER{alpha} in the systemic hemopoietic compartment. Our data firmly establish that ER{alpha}-mediated signaling plays a pivotal role in the protective effect of E2 on EAE development and CNS inflammation, and demonstrate that this effect of E2 does not require ER{alpha} expression in blood-derived inflammatory cells.

TNF-{alpha} has been shown to play an important role in the pathogenesis of EAE, and TNF-{alpha}-deficient mice exhibited a delayed onset of EAE (42, 43). It has been proposed that TNF-{alpha} production by hemopoietic cells, such as macrophages and T cells, may drive chemokine production by resident stromal cells in the CNS, thereby initiating the autoimmune inflammation of the brain (40, 44). Despite observations by others showing reduced numbers of TNF-{alpha}-producing autoantigen-specific CD4 T cells in E2-treated mice (10, 11), direct evidence for an ER{alpha}-mediated inhibitory effect of E2 on the production of Th1 cytokines by T cells is still lacking. Our data provide several lines of evidence supporting the conclusion that the protective effect of E2 on EAE is not mediated through inhibition of TNF-{alpha} production by hemopoietic cells. We demonstrate in this work that the E2 protection against EAE does not require ER{alpha} in T cells and is not correlated with a reduced production of IFN-{gamma} and TNF-{alpha} by Ag-specific CD4 T cells in vivo. This observation contrasts with data showing reduced production of TNF-{alpha}, and to a lesser extent, IFN-{gamma} by MOG35–55-specific T cells in the spleen of E2-treated mice (10, 11, 27). These discrepancies could be due to differences in doses and timing of E2 administration, as well as the use of normal rather than castrated mice. Whatever the mechanisms implicated in these contrasting effects of E2 on cytokine production by T cells, our data show that E2-mediated protection against EAE can occur not only in the absence of ER{alpha} signaling in T cells, but also in the absence of inhibition of effector/memory autoantigen-specific Th1 cell development. Finally, a role for an E2-mediated inhibitory effect on inflammatory macrophage function through ER{alpha} is also unlikely, because E2 could not prevent EAE development in WT->ER{alpha}-{Delta}2–/– bone marrow chimeras. In these mice, systemic inflammatory cells, such as macrophages and T cells, are derived from the donor bone marrow cells, whereas CNS-resident microglial cells remain of host origin (40, 45). Although our data unambiguously demonstrate that ER{alpha} signaling in systemic hemopoietic cells is not necessary for the E2-mediated protection of EAE, resident microglia may still play a role in this effect. Indeed, E2 have been recently shown to inhibit microglial reactivity in vitro and in vivo through ER{alpha} (20, 26).

In T cells, E2 has been shown to enhance the activity of the IFN-{gamma} promoter that contains sequences ressembling estrogen response element (46), and to up-regulate IFN-{gamma} secretion by T cells through IL-12/STAT-4-mediated signaling (47). Although the respective role of ER{alpha} vs ER{beta} was not completely clarified, E2 has recently been shown to be required for the in vitro differentiation of dendritic cells from bone marrow precursors (48). These observations suggest that ER{alpha} signaling in hemopoietic cells, such as T cells and dendritic cells, might enhance rather than suppress immunity. Indeed, we have recently shown that administration of physiological doses of E2 can enhance Ag-specific Th1 responses in draining lymph node through ER{alpha} expression in hemopoietic cells (49). This is in agreement with our observation that MOG-specific CD4 T cells from E2-treated mice have a tendency to exhibit increased production of type 1 cytokines.

Our data support the conclusion that ER{alpha}-mediated signaling outside the sytemic immune compartment is necessary and sufficient to inhibit EAE. In this model, CNS inflammation is initiated by autoantigen-specific T lymphocytes that invade the spinal cord white matter through the endothelium of the blood brain barrier (BBB) (3). VLA-4 expression on activated T cells is essential in this process by promoting binding to VCAM-1 on inflamed endothelium, followed by extravasation (50, 51). Upon arrival in the CNS parenchyma, effector T cells are reactivated following encounter of the autoantigen presented by local APCs (52). Activation of T cells in the CNS perivascular space initiates local production of inflammatory cytokines and chemokines by T cells and macrophages. This inflammatory gene expression enhances the secondary recruitment of blood-derived leukocytes, resulting in full-blown CNS inflammation (53). We propose that E2 may regulate the cellular and molecular mechanisms that selectively guide activated encephalitogenic T cells into the CNS, rather than T cell activation and differentiation in the periphery. Indeed, we showed that the MOG-specific Th1 cell response was not affected by E2 treatment, despite a dramatic inhibition of disease development and of CNS infiltration by blood-derived inflammatory leukocytes. These data suggest that priming of effector/memory T cells in the draining lymph nodes was not inhibited by E2 treatment; neither was their capacity to recirculate and to home to the spleen. Rather, it seems that E2 selectively impairs encephalitogenic T cell homing to the target tissues. Blockade of T cell movement into the CNS may take place at various levels. First, the E2-mediated inhibition of CNS inflammation could be caused by action on vascular endothelium of the BBB, resulting in the inhibition of T cell adhesion. Indeed, estrogens have been shown to elicit various biological responses in endothelial cells (54) and to inhibit the expression of cellular adhesion molecules, such as VCAM-1 (55). Some of these effects have been clearly attributed to ER{alpha} signaling (30, 55). Second, E2 could also regulate the production of chemokines, such as CCL19 and CCL21, that have been shown to be produced by endothelial cells at the BBB (56). Such chemokines could be critically involved in the initiation and chronic maintenance of CNS inflammation during EAE, by promoting permanent T cell adhesion to the endothelium, allowing efficient diapedesis of effector T lymphocytes into the spinal cord white matter (51, 56). Finally, E2 may target resident cells in the CNS, particularly microglia, by inhibiting chemokine production at the parenchymal-perivascular interface, thereby preventing inflammatory cell movement into the CNS (40).

Taken together, this study provides the first evidence that the protective effect of E2 on EAE does not involve ER{alpha}-mediated signaling in hemopoietically derived CNS-infiltrating inflammatory cells. We propose that E2 may act on nonhemopoietic tissues, such as endothelium, or on CNS resident cells, such as microglia, to inhibit the migration, recruitment, and/or extravasation of pathogenic effector/memory T cells in the CNS, thereby preventing the inflammatory process. Whether the E2 inhibitory effect on EAE requires ER{alpha} expression in the vascular endothelium, and/or in microglia, remains therefore to be determined. Further understanding as to how E2 inhibits EAE pathogenesis may allow for the rational design of therapeutic strategies not only in the treatment of MS, but also of other inflammatory autoimmune diseases, such as rheumatoid arthritis.


   Acknowledgments
 
The skillful technical assistance of M. Calise and S. Pilipenko is greatly acknowledged. We thank L. Pelletier, A. Saoudi, E. Joly, and J. van Meerwijk for helpful comments on the manuscript, and P. Cavailles for advice on quantitative-PCR analysis.


   Footnotes
 
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 Institut National de la Santé et de la Recherche Médicale, and by grants from Association pour la Recherche sur la Sclérose en Plaques, Association pour la Recherche contre le Cancer, European Community (QLG1-CT2001-01918), and Ministère délégué à la recherche et aux nouvelles technologies (ACI 2003). The work at Institut de Génétique et de Biologie Moléculaire et Cellulaire was supported by a grant from the Association pour la Recherche sur le Cancer and the Fondation pour la Recherche Médicale. L.G. was supported by a fellowship from Ligue Française contre la Sclérose en Plaques. Back

2 Address correspondence and reprint requests to Dr. Jean-Charles Guéry, Institut National de la Santé et de la Recherche Médicale Unité 563, Centre Hospitalo-Universitaire Purpan, 31300 Toulouse, France. E-mail address: Jean-Charles.Guery{at}toulouse.inserm.fr Back

3 Abbreviations used in this paper: MS, multiple sclerosis; BBB, blood brain barrier; CT, cycle threshold; E2, 17{beta}-estradiol; EAE, experimental autoimmune encephalomyelitis; ER, estrogen receptor; HPRT, hypoxanthine phosphoribosyltransferase; MNC, mononuclear cell; MOG, myelin oligodendrocyte glycoprotein; WT, wild type. Back

Received for publication March 29, 2004. Accepted for publication June 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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