Receptor α Mediates Estrogen's Immune Protection in Autoimmune Disease Hong-biao Liu, Kyi Kyi Loo, Karen Palaszynski, Judith Ashouri, Dennis B. Lubahn and Rhonda R. Voskuhl This information is current as of September 24, 2021. J Immunol 2003; 171:6936-6940; ; doi: 10.4049/jimmunol.171.12.6936 http://www.jimmunol.org/content/171/12/6936 Downloaded from

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Estrogen Receptor ␣ Mediates Estrogen’s Immune Protection in Autoimmune Disease

Hong-biao Liu,* Kyi Kyi Loo,* Karen Palaszynski,* Judith Ashouri,* Dennis B. Lubahn,† and Rhonda R. Voskuhl2*

Estrogens are known to influence a variety of autoimmune diseases, but it is not known whether their actions are mediated through classic ␣ (ER␣). The presence of a functional ER was demonstrated in secondary lymphoid tissues, then ER␣ expression was shown at both the RNA and protein levels in these tissues. Use of ER␣ knockout mice revealed that both the estrogen-induced disease protection and the estrogen-induced reduction in proinflammatory cytokines were dependent upon ER␣ in the prototypic Th1-mediated autoimmune disease experimental autoimmune encephalomyelitis. These findings are central to the design of selective ER modifiers which aim to target biologic responses in specific organ systems. The Journal of Immunology,

2003, 171: 6936–6940. Downloaded from

strogens have biological activity in numerous organ sys- RA that estrogen treatment may be beneficial, while estrogen treat- tems including the immune system, the reproductive sys- ment of SLE worsens the disease (7–13). E tem, the cardiovascular system, the CNS, bone, breast, may act through genomic or nongenomic mecha- colon, and prostate. An understanding of which estrogen receptor nisms. Genomic mechanisms were originally described and medi- 3

(ER) mediates estrogen’s action in a given organ system is central ated through ER␣, the “classic” ER (14). However, since charac- http://www.jimmunol.org/ to the development of selective ER modifiers (SERMs) which aim terization of ER␣, other ERs have been described including ER␤ to target therapeutic effects and minimize toxic side effects. (15). Most recently, ER␥ has been detected, but thus far only in Gender and hormonal influence play a role in a wide variety of teleosts (16). Nongenomic mechanisms involve membrane effects human autoimmune diseases. Multiple sclerosis (MS), rheumatoid (17) with a putative membrane associated “ER-X” being recently arthritis (RA), uveitis, and thyroiditis improve during pregnancy reported (18). Interestingly, the estrogen-mediated protection in while other diseases such as systemic lupus erythematosis (SLE) CIA was recently shown to possibly be caused by genomic effects tend to worsen (1). An immune shift from Th1 to Th2 that occurs since treatment with ICI 182,780 blocked the protective effect of during pregnancy as the maternal immune system attempts to estrogen on disease when used at a dose known to block ER␣ and avoid fetal rejection has been proposed as a mechanism underlying ER␤, but not membrane effects (19). However, it was not deter- by guest on September 24, 2021 the improvement in Th1-mediated autoimmune diseases during mined which nuclear receptor was involved in the estrogen in- pregnancy (1). duced immune protection in CIA. In this manuscript the prototypic Pregnancy is a complex situation characterized by changes in Th1-mediated autoimmune disease EAE is used to determine numerous factors including a steady increase in levels of estrogens. whether the protective effect of estrogen treatment is mediated Interestingly, increases in estrogens appear to recapitulate the ef- through classic ER␣. fect of pregnancy on autoimmune diseases, at least in part. Estro- gen treatment in mice decreases the severity of experimental au- Materials and Methods toimmune encephalomyelitis (EAE) and collagen-induced arthritis Animals and reagents (CIA), while it worsens SLE (2–5). Immune shifts from Th1 to ␣ Th2 have been observed during estrogen treatment in these animal Homozygous male ER knockout mice were generated by backcrossing the knockout (20) onto the C57BL/6 strain for 16 generations. Littermates models (2, 3, 6). There is also evidence in humans with MS and were used as wild-type (WT) controls. Animals were maintained in accor- dance with guidelines established by the National Institutes of Health and as mandated by the Univeristy of California, Los Angeles Office for the *Department of Neurology, Reed Neurological Research Center, University of Cal- Protection of Research Subjects. Human ovary was purchased from Bio- ifornia, Los Angeles, CA 90095; and †Department of Biochemistry, Child Health and Chain Institute (Hayward, CA). Myelin oligodendrocyte glycoprotein Animal Sciences, University of Missouri, Columbia, MO 65211 (MOG) 35–55 peptide was synthesized to Ͼ98% purity by Chiron Mimo- Received for publication May 29, 2003. Accepted for publication October 10, 2003. topes (San Diego, CA). 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 In vitro estrogen treatment and cytokine analysis with 18 U.S.C. Section 1734 solely to indicate this fact. ␮ C57BL/6 mice were immunized with MOG35–55 peptide (100 g/mouse) 1 This work was supported by the National Institutes of Health Grants AI50839 and and draining lymph node cells were harvested after 10 days. Cells were NS45443 (to R.R.V.) and the National Multiple Sclerosis Society Grant RD3407 (to stimulated in vitro with autoantigen (25 ␮g/ml) in the presence or absence R.R.V.). of (28 ng/ml). After6hofstimulation, TNF-␣ was detected by 2 Address correspondence and reprint requests to Dr. Rhonda Voskuhl, Reed Neuro- intracellular staining with TNF-␣-specific Ab (BD PharMingen, San Di- logical Research Center, Room A145, 710 Westwood Plaza, Los Angeles, CA 90095. ego, CA) and subsequent FACS analysis. E-mail address: [email protected] 3 Abbreviations used in this paper: ER, estrogen receptor; EAE, experimental auto- EMSA immune encephalomyelitis; SERM, selective ER modifier; ERE, estrogen response element; SLE, systemic lupus erythematosis; CIA, collagen-induced arthritis; MOG, Cytoplasmic or nuclear proteins from spleen and brain were prepared using myelin oligodendrocyte glycoprotein; WT, wild type; MS, multiple sclerosis; RA, NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the man- rheumatoid arthritis. ufacturer’s instruction (Pierce, Rockford, IL). Samples were preincubated

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6937 with biotin-conjugated estrogen response element (ERE; Panomics, Red- wood City, CA) then subjected to electrophoresis. Recombinant ER␣ and ER␤ receptors (Affinity, Golden, CO) were used as positive controls. RT-PCR Total RNA (2 ␮g) was reverse-transcribed to cDNA using the Reverse Transcription System (Applied Bioscience, Branchburg, NJ). For amplifi- cation, the outer primer set corresponded to nucleotides 461–480 (sense) and 1918–1937 (anti-sense) of the ER␣ gene, while with the inner primer set corresponded to 659–679 (sense) and 981-1003 (anti-sense) as previ- ously numbered (21). The primer sequences are as follows: mER␣ outer set sense 461–480 5Ј-GCC GCC TTC AGT GCC AAC AG-3Ј; mER␣ inner set sense 659–679 5Ј-AAT TCT GAC AAT CGA CGC CAG-3Ј; mER␣ inner set anti-sense 1003–981 5Ј-GTG CTT CAA CAT TCT CCC TCC TC-3Ј; mER␣ outer set anti-sense 1937–1918 5Ј-AGG AAT GTG CTG AAG TGG AG-3Ј. Western blots Intracellular proteins were prepared using NE-PER Nuclear and Cytoplas- mic Extraction Reagents (as above) and Western blots run as described previously (22). To detect ER␣ (67 kDa) a rabbit ER␣-specific primary Ab (Santa Cruz Biotechnology, Santa Cruz, CA) was used with a biotin-la- Downloaded from beled goat anti-rabbit IgG secondary Ab. Alternatively, westerns were probed with E2-BSA (Sigma-Aldrich, St. Louis, MO) labeled with biotin using the EZ-link Sulfo-NHS-LC-Biotinylation kit (Pierce). In vivo hormone treatment, induction of active EAE, and cytokine analysis FIGURE 1. There are functional ERs expressed in secondary lymphoid http://www.jimmunol.org/ C57BL/6 male mice were treated with estriol (5 mg s.c. 90-day release tissues. A, Estrogen treatment was shown to have direct effects on cytokine pellets) or with placebo pellets (Innovative Research of America, Sarasota, production by immune cells. MOG peptide-specific lymph node cells FL) as described (2). One week later, active EAE was induced by immu- 35–55 were harvested from MOG35–55-immunized C57BL/6 mice. Cells were nizing with MOG35–55 peptide in complete Freund’s adjuvant as described (23). Mice were monitored daily for signs of EAE on a standard EAE stimulated in vitro with autoantigen in the presence or absence of estriol at grading scale of 0–5 (0, unaffected; 5, moribund). Mice in each group were a dose physiologic with levels that occur during pregnancy (28 ng/ml) (46). sacrificed at days 16–24 during EAE, splenocytes were harvested and stim- TNF-␣ was detected by intracellular staining and subsequent FACS anal- ␮ ␣ ulated with autoantigen (MOG35–55 peptide at 25 g/ml) as described (2). ysis. Data are expressed as percent change of TNF- -positive staining in Supernatants were collected after 48 h and levels of TNF-␣, IFN-␥, IL-2, placebo or estriol treated, each as compared with placebo treated. Data are and IL-5 were determined by cytometric bead array (BD PharMingen). pooled from four separate experiments. Error bars indicate variation be- p Ͻ 0.05 when comparing estriol vs placebo treated. by guest on September 24, 2021 ,ء .tween experiments Results In vitro estriol treatment had no effect on cell viability as assessed by 72 h Functional ER are present in peripheral immune tissues proliferation assays (data not shown). B, Splenic ER was shown to bind the estrogen response element (ERE). Cytoplasmic or nuclear proteins from We first examined immune cells from secondary lymphoid tissues spleen were prepared and samples were preincubated with biotin-conju- of mice for the presence of a functional ER. Lymph node cells gated ERE. Splenic nuclear (N) and cytoplasmic (C) proteins were shown from MOG peptide 35–55-immunized C57BL/6 mice were stim- to bind ERE as demonstrated by retardation of migration of bands in ulated with the autoantigen in the presence or absence of estriol in EMSA. Recombinant human ER␣, recombinant human ER␤, and murine vitro, and a significant decrease in TNF ␣ (TNF-␣) was observed, brain nuclear (N) and cytoplasmic (C) proteins served as positive controls. p ϭ 0.036 (Fig. 1A). This reduction in TNF-␣ production during an Biotin-EAE alone served as the negative control. autoantigen-specific immune response had been previously re- ported during in vivo estrogen treatment of mice with EAE (3). These in vitro data indicated that estrogen’s effect can be mediated by a direct effect of an estrogen on murine immune cells during an transcripts in peripheral immune tissues have been reported in autoantigen-specific immune response. Next, we assessed murine mice, there has been no sequencing of these products (29, 30). splenic tissue proteins for their ability to bind to the ERE. In the Furthermore, expression of ER␣ at the protein level in the periph- nuclear fraction, binding was evidenced by retardation of migra- eral immune system has remained poorly characterized since tion of bands in EMSA (Fig. 1B). The lack of well delineated Western blot data were negative for specific bands of the correct bands in lanes containing tissue proteins, with clear single bands in size (31) while flow cytometry of immune cells was positive (29, lanes containing recombinant proteins, is likely due to heteroge- 32, 33). Together these previous reports raised the question of neity in ER types within tissues. This is consistent with the fact whether the classic ER␣, vs another ER variant (24, 25), was de- that either ER␣ or ER␤ could bind the ERE and that variants of tected in immune cells by flow cytometry. Thus, we characterized ERs have been demonstrated in nonimmune tissues previously (24, ␣ 25). Together the alteration in cytokine production during in vitro ER expression in secondary lymphoid tissues of mice at the RNA estriol treatment of immune cells and the binding of splenic protein level by both nested RT-PCR and sequencing and at the protein to ERE indicated that functional ERs are expressed in murine sec- level by Western blot using both Ab and ligand binding. Nested ␣ ondary lymphoid tissues. RT-PCR using ER -specific primers revealed bands of the ex- pected size in both spleen (Fig. 2A) and lymph node (data not ER␣ expression at the RNA and protein levels shown). The product from the inner primer set was then sequenced ER␣ expression has been reported in bone marrow and thymus and confirmed to be ER␣. Western blots of splenic tissue using an (26–28), but direct evidence of expression in murine peripheral ER␣-specific Ab detected expression at the protein level at the immune tissues is lacking. While RT-PCR data detecting ER␣ expected size of 67 kDa (Fig. 2B). ER␣ expression at the protein 6938 ER␣ MEDIATES ESTROGENЈS IMMUNE PROTECTION IN AUTOIMMUNE DISEASE

which had previously been shown to induce estriol levels in sera of mice which were physiologic with natural murine pregnancy (2). Homozygous ER␣ KO mice were not protected from disease dur- ing estriol treatment, while WT mice were protected as evidenced by a significant decrease in disease severity in estriol vs placebo treated, p Ͻ 0.0001 (Fig. 3). These data demonstrate that the pro- tective effect of estriol in EAE is dependent upon the presence of an intact ER␣.

The estrogen-mediated immune modulation in EAE is dependent upon ER␣ Significant reductions in proinflammatory cytokine production (TNF-␣, p Ͻ 0.005; IFN-␥, p Ͻ 0.005; and IL-2, p Ͻ 0.05) were observed during autoantigen-specific immune responses of WT mice treated with estriol as compared with placebo (Fig. 4). Im- portantly, these reductions did not occur in ER␣ knockout mice treated with estriol. Since not all cytokines were reduced by estriol treatment, apoptosis was an unlikely explanation for the decrease in the above cytokines in estriol-treated WT mice. Levels of the Downloaded from Th2 cytokine IL-5 were increased in estriol-treated WT mice as compared with placebo-treated WT mice. This estriol-induced in- crease in IL-5 production was less dramatic but still present in ER␣ KO mice. Together these data demonstrate that both the disease pro- tection and the reduction in proinflammatory cytokine production

induced by estriol treatment in EAE are mediated through ER␣. http://www.jimmunol.org/

Discussion Recently ER␣ was shown to be critical in estrogen-induced pro- tection against brain injury during stroke (34). Direct estrogen- induced, ER␣-mediated protective effects on the CNS remain pos- sible in EAE as well. However, the fact that estrogen treatment ␣ FIGURE 2. ER is expressed at the RNA and protein levels in mouse influences a wide variety of autoimmune diseases which do not spleen. A, ER␣ gene expression was detected in murine spleen by nested have the CNS as a target organ indicates that the protective effects

RT-PCR. RNA from murine tissue served as a positive control. No by guest on September 24, 2021 RNA served as a negative control. The inner primer PCR product was of estrogen are clearly not limited to direct effects on the CNS. confirmed by sequencing. B, ER␣ protein expression was demonstrated in This conclusion is consistent with the finding in this report that the spleen. Western blots of tissue proteins were probed with ER␣-specific primary Ab. A protein of the expected size for ER␣ (67 kDa) was detected in spleen. Human ovary and murine brain served as positive control tissues. Rabbit IgG served as a primary Ab negative control. The amounts of pro- tein loaded per lane were 100 ␮g for spleen and brain and 60 ␮g for ovary. C, ER␣ expression at the protein level is confirmed by probing Western blots with -BSA. Western blots of tissue proteins were probed with E2-BSA-biotin instead of ER␣-specific Ab. A protein of the expected size for ER␣ (67 kDa) was detected in spleen. Human ovary and murine brain served as positive control tissues. Probing with BSA-biotin alone served as a negative control.

level was then confirmed by probing Western blots with 17␤ es- tradiol-BSA whereby a band was identified in spleen which colo- calized to the size of the band detected with ER␣-specific Ab prob- ing (Fig. 2C). Together these data are conclusive evidence of the expression of classic ER␣ in murine peripheral immune tissues.

The estrogen-mediated protection in EAE is dependent upon ER␣ FIGURE 3. The estrogen induced immune protection in EAE is medi- While an estrogen-mediated protective effect had been previously ated through ER␣. A reduction in mean clinical EAE scores occurred in ␣ reported in EAE and in CIA, the mechanism of the immune pro- estriol-treated WT, but not ER KO, mice. C57BL/6 male mice were treated with estriol or with placebo (sustained release pellets). One week tection has remained unknown. Therefore, we used the EAE model later, active EAE was induced by immunizing with MOG35–55 peptide. to determine whether the estrogen induced immune protection in There were a total of 40 mice, with 10 mice in each of four groups: WT this prototypic Th1 autoimmune disease was mediated through treated with placebo (WT Placebo), WT treated with estriol (WT Estriol), ER␣. Since mechanisms of action of estrogens have been shown to homozygous ER␣ knockout treated with placebo (KO Placebo), and ho- differ depending upon whether physiologic or pharmacologic mozygous ER␣ knockout treated with estriol (KO Estriol). Error bars rep- doses are used (34), we used a dose of estriol for EAE treatment resent variation in scores between mice within each group. The Journal of Immunology 6939

estriol was used in our studies while estradiol was used in the previous study. Estriol and estradiol are clearly not the same. Es- tradiol has been shown to lead to hypertension, increased coagu- lation and to protect from more than estriol (36–41). They can even have opposing effects. For example, estriol can protect from estradiol-mediated breast cancer (42, 43). However, since reports by three groups have shown that both estradiol and estriol are protective in both EAE and CIA (2, 3, 44, 45), there do not appear to be major differences between the two estrogen types in immune effects on disease. Indeed, the finding that estradiol treatment increases Th1 cytokine production during OVA-specific immune responses (35) is in direct contrast to previous reports by several groups that estradiol treatment ameliorates the prototypic Th1-mediated disease EAE. Perhaps differences between the effect estradiol treatment on OVA-specific responses in healthy mice and autoantigen-specific responses in EAE mice relates to either the

strain or the age of the mice treated. F5 generation C57BL/6 mice were used in the former study while F16 C57BL/6 mice were used

in our study. However, such strain differences also appear to be an Downloaded from unlikely explanation since estradiol has been shown to ameliorate EAE in numerous strains (C57BL/6, SJL, B10.PL, B10RIII). The age of the mice treated remains a consideration since the former report involved treatment of 4-wk-old mice, while all the EAE studies involved treatment of adult mice at least 8 wk of age.

Differences in the nature of the immune response to OVA vs au- http://www.jimmunol.org/ toantigen is also a consideration. While both estadiol and estriol have been shown to ameliorate EAE, we have focused considerable attention on estriol treatment of EAE for two reasons. First, unlike estradiol which is present during the menstrual cycle of the nonpregnant state, estriol is not present in appreciable amounts in the nonpregnant state. The non- pregnant female state is not a time of protection from autoimmune diseases such as MS and RA, since nonpregnant females are by guest on September 24, 2021 FIGURE 4. The estrogen-mediated immune modulation in EAE is de- known to be highly susceptible. Estriol becomes detectable only pendent upon ER␣. A reduction in proinflammatory cytokine production during the first trimester of pregnancy and increases progressively occurred in estriol-treated WT, but not ER␣ KO, mice. EAE mice in each throughout gestation, and pregnancy is a state of relative protection group were sacrificed at days 16–24 during EAE, and splenocytes were from MS and RA. Thus, there a correlation between disease pro- harvested and stimulated with MOG35–55 peptide. Supernatants were col- tection and the presence of estriol, while the relationship between ␣ ␥ lected and levels of TNF- , IFN- , IL-2, and IL-5 determined by cyto- disease protection and the presence of estradiol appears more com- metric bead array. Data are expressed as percent change in two to six plex. Second, we focused on estriol treatment of EAE as a trans- pooled experiments for indicated cytokine in supernatants from estriol or lational research strategy for MS in light of the fact that estriol is placebo-treated EAE mice, each as compared with the placebo-treated EAE. Homozygous ER␣ knockout ϭ KO. Error bars indicate variation known to have less toxicity in humans than estradiol (36–38). The p Ͻ 0.005 when comparing cytokine ultimate goal of the findings in this manuscript are to define the ,ءء ;p Ͻ 0.05 ,ء .between experiments levels in estriol vs placebo treated. Cytokine values in picograms per mil- precise mechanism of action of the therapeutic efficacy of estriol liliter from a representative experiment are as follows: TNF-␣: WT Pla- treatment in EAE. These findings will then be contrasted to find- cebo ϭ 862.7 Ϯ 198.5, WT Estriol ϭ 71.1 Ϯ 387.4, KO Placebo ϭ ings of the precise mechanism of action of estrogen related toxic- 497.7 Ϯ 142.0, KO Estriol ϭ 497.7 Ϯ 32.9; IFN-␥: WT Placebo ϭ ities such as increased risk of breast cancer, , 1545.9 Ϯ 92.0, WT Estriol ϭ 152.5 Ϯ 160.4, KO Placebo ϭ 1320.4 Ϯ and vascular disease. Our findings suggest that therapeutic efficacy ϭ Ϯ ϭ Ϯ 16.3, KO Estriol 1118.8 265.2; IL-2: WT Placebo 137.8 74.5, may be achieved through stimulation of ER␣. If it is ultimately ϭ Ϯ ϭ Ϯ ϭ WT Estriol 10.9 1.7, KO Placebo 94.1 23.7, KO Estriol shown that some or all toxicities are mediated through ER␤ or 95.7 Ϯ 12.7; IL-5: WT Estriol ϭ 97.0 Ϯ 12.2, WT Placebo ϭ 29.4 Ϯ 5.5, nongenomic effects, then a window of selectivity exists whereby KO Estriol ϭ 39.8 Ϯ 18.8, KO Placebo ϭ 18.2 Ϯ 2.9 one could treat disease with an ER␣ agonist or other SERM to achieve efficacy and minimize toxicity. In contrast, if it is ulti- estrogen induced alterations in cytokine production during autoan- mately shown that all toxicities are mediated through ER␣, then tigen-specific immune responses were mediated through ER␣. selectivity will not be possible at the level of the ER. However, Regarding the effects of estrogen treatment on cytokine produc- selectivity may still be achievable through modulation of tissue- tion, it is interesting to contrast our findings with those in a pre- specific cofactors involved in ER␣ action. To pursue the latter, one vious report in which estradiol was given in vivo and primary T must first define the ER used in a given tissue for a given biologic cell responses to OVA were shown to be enhanced for Th1 cyto- effect. Then one may begin to identify tissue-specific differences in kine production, with this effect dependent upon ER␣ (35). While cofactors for each of the biologic effects. both studies agree that ER␣ is important for immune efffects, the In conclusion, the protective effect of estrogen treatment on au- studies differ in that Th1 cytokine production was decreased by toantigen-specific immune responses in a prototypic Th1-mediated estrogen treatment in our paper while being increased in the pre- autoimmune disease is mediated through ER␣. This does not how- vious paper. 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