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¶
From the Department of Neurology,* Oregon Health and Science University, Portland, Oregon; Neuroimmunology Research, Veterans Affairs Medical Center, Portland, Oregon; the Department of Food Hygiene, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland; the Oregon Regional Primate Research Center, Beaverton, Oregon; the Division of Reproductive Science,¶ Oregon Health and Science University, Portland, Oregon; the Department of Microbiology and Immunology,|| Drexel University College of Medicine, Philadelphia, Pennsylvania; the Department of Medicine,** University of Vermont, Burlington, Vermont; and the Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon
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Abstract |
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(Esr1) and -ß (Esr2) genes. We demonstrate that the protective effect of E2 is abrogated in B6.129-Esr1tm1Unc mice (Esr1-/-) but not in B6.129-Esr2tm1Unc mice (Esr2-/-). The loss of E2-mediated protection from EAE in Esr1-/- mice immunized with the encephalitogenic MOG-35–55 peptide was manifested phenotypically by the development of severe acute clinical signs and histopathological lesions even in the presence of moderately high serum E2 levels. This is in contrast to C57BL/6 wild-type (WT) mice and Esr2-/- mice in which E2 treatment resulted in comparable serum levels and markedly suppressed clinical signs of EAE and abolished inflammatory lesions in the CNS. This pattern showing a lack of E2-dependent inhibition of EAE in Esr1-/- mice was mirrored by an enhanced rather than a reduced secretion of TNF-, IFN-
, and interleukin (IL)-6 in MOG-specific splenocytes and a lack of inhibition of message for inflammatory cytokines, chemokines and chemokine receptors in CNS tissue. These results indicate that the immunomodulatory effects of E2 in EAE are dependent on Esr1 and not Esr2 signaling.
We demonstrated previously that administration of low doses of 17ß-estradiol reduced the severity of experimental autoimmune encephalomyelitis (EAE) in several different inbred strains of female mice.6-8
The exact E2-sensitive immune-related target genes have not been fully characterized. Our data from DNA microarray studies in BV8S2 transgenic mice suggests that E2 down-regulates several members of the TNF gene family including TNF- and Pgrp, and the chemokine RANTES, and up-regulates the expression of several cytokines, chemokines/receptors, adhesion molecules, and activation markers.9
The effects of E2 on gene expression in pathogenic, recruited, and regulatory cells in EAE are likely to be the result of estrogen receptor-mediated signaling.
Although E2 can have a protective effect on susceptibility to EAE, much remains to be learned about the molecular mechanisms underlying its immunomodulatory activity and regulation of the sexual dimorphism and pathogenesis of autoimmune diseases.6-11
A major question is the nature of the signaling pathways underlying these effects. There is considerable controversy as to whether or not E2 exerts its biological effects directly or indirectly on immune cells and whether or not they are the result of genomic responses. To elicit a classical genomic response, and to be a direct target for E2, the cells of the immune system must express the cognate receptors. The first E2 receptor to be cloned was the E2 receptor- gene (Esr1).12
Among cells relevant to the functioning of the immune system, Esr1 is expressed by thymocytes,5,6
thymic epithelial cells,5
T cells,7,8
B lymphocytes and their precursors,9,10
and non-hematopoietic bone marrow cells.9,11
The existence of a second iER, estrogen receptor-ß (Esr2), was identified12
and has been shown to be expressed by non-hematopoietic bone marrow cells13
and in the human thymus and spleen.14,15
The precise roles of these two receptors in regulating cell-specific responses are still not well defined. However, it is becoming evident that Esr1 and Esr2 may be responsible for regulating different biological functions based on their expression patterns, localization profiles, and protein structures.14 For example, Esr1 and Esr2 differ in their C-terminal ligand-binding domains and in their N-terminal transactivator domains. In addition to these two cytoplasmic receptors, there is also a membrane-associated E2 receptor (mER) that may play a role in mediating the non-genomic actions of E2.15 However, mER regulation of gene expression cannot be fully excluded since some phenotypes elicited by steroid hormones appear to be the result of membrane receptor activation of G-protein-coupled second messenger pathways.16 In the current study, we use B6.129-ESR1 mice lacking Esr1 and B6.129-Esr2tm1Unc mice lacking Esr2 to address for the first time which ER mediates the protective effect of E2 on EAE.
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Materials and Methods |
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Female B6.129-Esr1tm1Unc (Esr1-/-), B6.129-Esr2tm1Unc (Esr2-/-), and C57BL/6 wild-type control littermate mice were purchased from Taconic Laboratory (Germantown, NY). The mice were housed and cared for in the Animal Resource Facility at the Portland VA Medical Center according to institutional guidelines.
Hormone Treatment
For treatment of mice with 17ß-estradiol, 3-mm pellets containing 2.5 mg of E2 (Innovative Research of America, Sarasota, FL), providing a constant continuous controlled release of hormone over a period of 60 days, were implanted subcutaneously on the dorsal back of each mouse 7 days before immunization for induction of EAE. Control animals were sham-operated and implanted with pellets containing saline.
Induction of Active EAE
Mice were immunized with 400 µg of MOG-35–55 peptide in complete Freund’s adjuvant (CFA) containing 200 µg of Mycobacterium tuberculosis H37Ra by subcutaneous injection over four sites on the flank, with additional injections of Pertussis toxin (Ptx) on days 0 and 2.7 Mice were examined daily for clinical signs of EAE according to the following scale: 0, no signs; 1, limp tail; 1.5, moderate hind limb weakness with difficulty in righting; 2, moderate hind limb weakness without ability to right itself; 2.5, moderate hind limb weakness (waddling gait) without ability to right itself; 3, moderately severe hind limb weakness with the ability to walk upright for only a few steps; 3.5, moderately severe hind limb weakness with paralysis of one limb; 4, severe hind limb weakness; 4.5, severe hind limb weakness with mild forelimb weakness; 5, paraplegia with no more than moderate forelimb weakness; 5.5, paraplegia with severe forelimb weakness (quadriplegia); and 6, moribund condition.
IL-6 Quantification
Splenic cells were cultured at 4 x 106/ml and stimulated with the appropriate Ag in 24-well culture plates. Cell culture supernatants were recovered after 72 hours. Measurement of cytokines was performed by ELISA using cytokine-specific capture and detection Abs (BD PharMingen, San Diego, CA). A standard curve for IL-6 was generated using recombinant mouse cytokines (BD PharMingen), and the concentration of cytokine in the cell supernatants was determined by interpolation from the appropriate standard curve.
Cytometric Bead Array (CBA)
TNF-, IFN-, IL-5, IL-4, and IL-2 were detected using the mouse inflammation cytokine CBA kit from BD Biosciences (San Jose, CA). Fifty µl of cell culture supernatant was mixed with 50 µl of the mixed capture beads and 50 µl of the mouse phycoerythrin detection reagent. The tubes were incubated at room temperature for 2 hours in the dark, followed by a wash step. The samples were then resuspended in 400 µl of wash buffer before acquisition on the FACScan. The data were analyzed using CBA software. Standard curves were generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the cell supernatant was determined by interpolation from the appropriate standard curve.
RNase Protection Assay (RPA)
Total RNA was extracted from spinal cords (SC) using the STAT-60 reagent (Tel-Test, Friendswood, TX). Chemokine expression was determined using the RiboQuant RPA kit to assess the expression of chemokines and chemokines receptors (PharMingen) according to manufacturer’s instructions.6
Histology
Representative mice were selected from the E2 and placebo-treated control group. Spinal cords were isolated and fixed in 10% paraformaldehyde. Transverse sections of the spinal cords were stained with Luxol Fast Blue-Periodic acid or with hematoxylin and eosin (H&E) and analyzed by light microscopy.
E2 and Testosterone Serum Levels
Serum E2 and testosterone levels were measured using a radioimmunoassay as described previously.6
Statistical Analysis
Differences in the incidence of EAE in the respective mouse strains was tested using 
2. A one-way analysis of variance and Student-Newman-Keuls multiple comparisons test was used to evaluate significant differences in quantitative EAE trait values (day of onset, E2 levels, T4 levels, thymic index and uterine index). The Kruskal-Wallis test was used to evaluate non-parametric EAE trait values (peak score and CDI score). In all tests, P values 
0.05 were used as the significance threshold.
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Results |
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We sought to determine whether there were differences between Esr1 and Esr2 in mediating the inhibitory effects of E2 on EAE. Thus, B6.129-Esr1tm1Unc (Esr1-/-) mice, B6.129-Esr2tm1Unc (Esr2-/-) mice, and their C57BL/6 WT littermate controls were immunized with MOG-35–55 peptide + CFA + Ptx in the presence of implanted E2 or placebo pellets, and monitored for clinical and histological signs EAE (Table 1
and Figures 1 and 2
). All of the WT C57BL/6 mice treated with placebo pellets developed severe EAE, and as expected from our previous studies,6-9
treatment of the WT mice with E2 pellets markedly reduced the incidence and severity of clinical and histological disease. Interestingly, both Esr1-/- and Esr2-/- mice treated with placebo pellets developed EAE that was nearly indistinguishable from WT controls. Of critical importance, however, treatment with E2 pellets had no inhibitory effect on the severity of clinical or histological disease in Esr1-/- mice, although onset of EAE was delayed by 4 days. In contrast, the identical treatment with E2 almost completely inhibited clinical and histological EAE in Esr2-/- mice (Table 1
and Figure 1
).
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. Moreover, treatment with E2 pellets produced comparable levels of serum E2 in all three mouse strains, indicating that the lack of functional estrogen receptors did not effect E2 degradation. As expected, Esr1-/- mice had no significant increase in uterine index [expressed as uterine weight (mg) divided by body weight (g)] compared to WT controls and Esr2-/- mice. However, E2 treatment significantly increased the uterine index nearly three-fold in WT and Esr2-/- mice but not Esr1-/- mice. In contrast, lack of either Esr1 or Esr2 resulted in a moderate decrease in thymic index compared to WT controls, and treatment with E2 strongly diminished thymus weight in all three mouse strains. Testosterone levels were low but detectable in WT and Esr-2-/- mice, but were markedly increased in Esr1-/- mice, consistent with a previous report.17
Taken together, these results are consistent with previous findings regarding the role of Esr1 and Esr2 signaling, and serve as internal controls for the observed effects on EAE.18-22 Cytokine, Chemokine, and Chemokine Receptor Production by Esr1-/- and Esr2-/- Mice
In accordance with our previous studies,6-9
we observed that E2 treatment significantly reduced the secretion of TNF- and IFN- by MOG-35–55 peptide-stimulated spleen cells recovered from WT mice at the peak of EAE (Figure 3)
. A similar result occurred in Esr2-/- mice, although levels of both cytokines were reduced compared to WT placebo-treated mice. In contrast, E2 treatment significantly enhanced secretion of TNF- and IFN- in Esr1-/- mice with EAE compared to treatment with placebo (Figure 3)
. IL-2, IL-4, and IL-5 levels were very low and were not affected by E2 treatment in any mice. Since E2 has been purported to up-regulate serum IL-6 levels through Esr1 and not Esr2,23
and elevated IL-6 levels have been implicated in mediating suppression of delayed-type hypersensitivity responses in post-burn patients and experimental animals,24
we compared the levels of IL-6 secretion by splenic cells in E2- versus placebo-treated Esr1-/- and Esr2-/- mice. As is shown in Figure 3
, IL-6 production was significantly reduced in E2-treated WT mice without EAE, but was significantly increased in E2-treated versus placebo-treated Esr1-/- mice with EAE. These data indicate that the up-regulation of IL-6 is not restricted to Esr1 signaling and that increased levels of IL-6 do not account for suppression of clinical and histological EAE.
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, and IFN-), chemokines (RANTES, MIP-1, MIP-2, IP-10, and MCP-1) and chemokine receptors (CCR1, CCR2, and CCR5) were detectable at similar levels in the spinal cords of placebo-treated WT, Esr1-/-, and Esr2-/- mice with clinical signs of EAE (Figure 4)
. Treatment of EAE with E2 significantly reduced the expression of all detected cytokines, chemokines, and chemokine receptors in WT C57BL/6 mice and Esr2-/- mice, but did not significantly change the level or pattern of expression in CNS tissue from E2-treated Esr1-/- mice that developed EAE (Figures 4, A to C)
. Thus, the inhibitory effect of E2 on inflammatory factors in CNS that was highly significant in C57BL/6 and Esr2-/- mice was lost in Esr1-/- mice.
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Discussion |
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, IFN-, and IL-6 in MOG-specific splenocytes and a lack of inhibition of message for inflammatory cytokines, chemokines, and chemokine receptors in CNS tissue.
Although the severity of clinical and histological EAE was not diminished in E2-treated Esr1-/- mice, these mice did exhibit a 4-day delay in disease onset compared to C57BL/6 controls or Esr2-/- mice. This may be the result of residual Esr2 signaling,25
which leads to less efficient E2 protection. MOG-specific splenocytes from placebo-treated Esr1-/- and Esr2-/- mice had strongly reduced levels of secreted TNF- and IFN- compared to WT controls, suggesting that lack of Esr1 or Esr2 might limit production of these cytokines. However, treatment with E2 strongly increased the secretion of these cytokines to WT levels in Esr1-/- mice, suggesting a possible enhancing effect of E2 mediated through the residual Esr2 gene. E2 treatment also significantly enhanced secretion of IL-6 in MOG peptide-stimulated splenocytes from Esr1-/- mice, indicating that E2 up-regulation of IL-6 is not restricted to Esr1 signaling and that increased levels of IL-6 do not account for suppression of EAE, as proposed previously.23,24
In addition, E2 treatment of Esr1-/- mice did not change the expression profile in the spinal cord of the cytokines lymphotoxin, TNF-, and IFN-; the chemokines RANTES, MIP-1, MIP-2, IP-10, and MCP-1; and the chemokine receptors CCR1, CCR2, and CCR5, in contrast to C57BL/6 WT and Esr2-/- mice in which E2 treatment reduced the levels of all of these inflammatory factors. In this regard, it is pertinent that Wang et al26
reported that the two-ligand/receptor pairs, CCL5/CCR5 and CXCL10/CXCR3, may play a role during active MS and serve as predictors of future disease activity. Also Eikelenboom et al27
showed an increase in CCR5+ and CXCR3+ T cells in both the blood and cerebrospinal fluid of MS patients.
Based on incomplete development of the mouse reproductive tract (ovaries) in B6.129-Esr1-/- mice, it has been suggested that the action of Esr2 is directly dependent on the presence of a functional Esr1 gene.28 On the other hand, E2 continues to provide protection against vascular injury in mice in which either Esr1 or Esr2 has been disrupted, suggesting that each of the two known E2 receptors is sufficient to mediate vascular protection.29 In ischemic injury within the brain, E2 can use either ER subtype,30 or alternatively, protection may be receptor-independent.31 Similarly, in our study, lack of Esr1 or Esr2 resulted in a moderate decrease in thymus weight compared to WT controls, and treatment with E2 strongly diminished thymus weight in all three mouse strains. E2 and estrogen receptor are known to effect thymic development and function,20-22 and our results support a role for either Esr1 or Esr2. The spectrum of biological activities mediated by E2 receptor subtypes in diverse disease settings may be explained by: 1) the differences in tissue distribution of the subtypes, 2) the complexity of E2 receptor co-activators which may be different for each cell type, or 3) the existing complexities in the nature of E2 protective mechanisms, which include classical (genomic) and nonclassical (nongenomic) action.32
Our data strongly implicate the classical (genomic) action of E2 in protection against EAE, because we observed a pronounced E2-dependent regulation of cytokine secretion in spleen and inhibition of message for cytokines, chemokines, and chemokine receptors in the spinal cord of immunized mice. Our study demonstrates for the first time that these E2-regulated immunomodulatory effects are mediated by selective signaling through Esr1. Preliminary studies indicate that iERs are present on CD4+ T cells and monocytes, and it remains plausible that Esr1 on immunocytes may directly mediate E2 effects on EAE.
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Footnotes |
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Supported by grants AI42376, NS23221, and NS23444 from the National Institutes of Health (NIH), and grants from the National Multiple Sclerosis Society (NMSS), the Nancy Davis Center Without Walls, and the Department of Veterans Affairs, as well as by NIH Grants AI45105 & AI41747 (to C. T.), NS36526 and NMSS RG3129 (to C. T. and E. P. B.). This investigation was also supported in part by a postdoctoral fellowship (to Agata Matejuk) from the National Multiple Sclerosis Society.
Accepted for publication July 3, 2003.
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References |
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production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine deficient knockout mice. J Immunol 2001, 167:542-552 but not Erß. Mol Endocrinol 2003, 17:1039-1053 and ß in immune organ development and in oestrogen-mediated effects on thymus. Immunol 2001, 103:17-25[Medline]
signaling pathway. Endocrine 2000, 12:207-213[Medline]
is necessary in thymic development and estradiol-induced thymic alterations. J Immunol 1999, 163:4168-4174, but not estrogen receptor ß, is involved in the regulation of the OPG/RANKL (osteoprotegerin/receptor activator of NF-
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