Alex Zamora

Estrogen Treatment Down-Regulates TNF-α Production and Reduces the Severity of Experimental Autoimmune Encephalomyelitis in Cytokine Knockout Mice

A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis. However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease suppression was also seen in IFN-γ-deficient mice. The decrease in disease severity was accompanied by a concomitant reduction in the number of proinflammatory cytokine- and chemokine-producing cells in the CNS. Although there was no apparent increase in compensatory Th2 cytokine production in cytokine-deficient mice, there was a profound decrease in the frequency of TNF-α-producing cells in the CNS and the periphery. Therefore, we propose that one mechanism by which estrogen protects females from the development of cell-mediated autoimmunity is through a hormone-dependent regulation of TNF-α production.

The Protective Effect of 17β-Estradiol on Experimental Autoimmune Encephalomyelitis Is Mediated through Estrogen Receptor-α

Low-dose estrogen (E2) treatment significantly inhibits the clinical signs and histopathological lesions of experimental autoimmune encephalomyelitis (EAE), and is being used in clinical trials to treat multiple sclerosis. To assess the role of intracytoplasmic estrogen receptors in mediating suppression of EAE, we studied mice with disrupted estrogen receptor-α (Esr1) and -β (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-α, IFN-γ, 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.

Estrogen inhibition of EAE involves effects on dendritic cell function

Estrogen has been found to have suppressive effects on the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. We have investigated the effects of 17β‐estradiol (E2) treatment on dendritic cells (DCs) in two different mouse models of EAE. The frequency of CD11b+/CD11c+ DCs was significantly decreased in the brain of mice protected from EAE induction by E2 treatment. In addition, the frequency of CD11c+/CD8α+ DCs producing tumor necrosis factor (TNF)α and interferon (IFN)γ in the spleen of E2‐treated mice was dramatically decreased compared to that in control mice with EAE, demonstrating an effect of E2 on DC function. In order to examine E2 effects on DCs in more detail, splenic DCs were cultured in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4to promote maturation. E2 pretreatment was found to suppress the ability of cultured DCs bearing a mature phenotype to present Ag to myelin basic protein (MBP)‐specific T cells. Analysis of cytokine production demonstrated that E2 decreased TNFα, IFNγ and IL‐12 production in mature DCs. In addition, MBP‐specific T cells cocultured with E2‐pretreated mature DCs in the presence of antigen demonstrated a shift towards production of Th2 cytokines IL‐4 and IL‐10 and a concomitant decrease in the production of Th1 cytokines TNFα and IFNγ. Thus, E2 treatment appears to have multiple effects on the DC population, which may contribute to a down‐regulation or block in the activation of Th1 cells involved in the induction of EAE.

17β‐estradiol inhibits cytokine, chemokine, and chemokine receptor mRNA expression in the central nervous system of female mice with experimental autoimmune encephalomyelitis

Cytokines and chemokines govern leukocyte trafficking, thus regulating inflammatory responses. In this study, the anti‐inflammatory effects of low dose 17β‐estradiol were evaluated on chemokine, chemokine receptor, and cytokine expression in the spinal cords (SC) of BV8S2 transgenic female mice during acute and recovery phases of experimental autoimmune encephalomyelitis (EAE). In EAE protected mice, 17β‐estradiol strongly inhibited mRNA expression of the chemokines RANTES, MIP‐1α, MIP‐2, IP‐10, and MCP‐1, and of the chemokine receptors CCR1, CCR2 and CCR5 at both time points. Conversely, ovariectomy, which abrogated basal 17β‐estradiol levels and increased the severity of EAE, enhanced the expression of MIP‐1α and MIP‐2 that were over‐expressed by inflammatory mononuclear cells in SC. 17β‐estradiol inhibited expression of LT‐β, TNF‐α, and IFN‐γ in SC, but had no effect on IL‐4 or IL‐10, indicating reduced inflammation but no deviation toward a Th2 response. Interestingly, elevated expression of CCR1 and CCR5 by lymph node cells was also inhibited in 17β‐estradiol treated mice with EAE. Low doses of 17β‐estradiol added in vitro to lymphocyte cultures had no direct effect on the activation of MBP‐Ac1‐11 specific T cells, and only at high doses diminished production of IFN‐γ, but not IL‐12 or IL‐10. These results suggest that the beneficial effects of 17β‐estradiol are mediated in part by strong inhibition of recruited inflammatory cells, resulting in reduced production of inflammatory chemokines and cytokines in CNS, with modest effects on encephalitogenic T cells that seem to be relatively 17β‐estradiol insensitive. J. Neurosci. Res. 65:529–542, 2001.

Estrogen potentiates treatment with T-cell receptor protein of female mice with experimental encephalomyelitis

Transgenic mice expressing the BV8S2 chain, which is specific for the myelin basic protein determinant Ac1-11, possess a naturally induced set of regulatory T cells directed against BV8S2. Further activation of anti-BV8S2 T cells in male mice with recombinant BV8S2 protein can inhibit IFN-gamma release by Ac1-11-specific T cells through a cytokine-driven mechanism and prevent induction of experimental autoimmune encephalomyelitis (EAE). In contrast, naive female mice possess fewer anti-BV8S2-reactive T cells, and treatment with BV8S2 delayed but did not prevent EAE. We here demonstrate that combining T-cell receptor (TCR) vaccination with supplemental estrus doses of estrogen potentiated IL-10 production by anti-BV8S2-reactive T cells and induced Ac1-11-specific T cells to produce IL-10 and TGF-beta. This combined treatment resulted in full protection against EAE, which was not observed with either therapy alone. These findings imply that supplemental estrogen can enhance the efficacy of TCR-based immunotherapy for autoimmune diseases that predominate in females.

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

There is much interest in the possible ameliorating effects of estrogen on various autoimmune diseases. We previously established the protective effects of 17β-estradiol (E2) on experimental autoimmune encephalomyelitis (EAE). In the current study we investigated the effectiveness of oral treatment with ethinyl estradiol (EE) on EAE and the mechanisms involved. Ethinyl estradiol is a semisynthetic estrogen compound found in birth control pills, and its chemical structure allows this compound to retain activity when given orally. We found that oral EE, like E2, drastically suppressed EAE induced by proteolipid protein 139–151 peptide when given at initiation of EAE. However, unlike E2, EE reduced clinical severity when given after the onset of clinical signs. Treatment with EE significantly decreased the secretion of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) by activated T cells as well as the expression of a key matrix metalloproteinase, disease-mediating chemokines/receptors, and IgG2a levels, but increased the expression of TGF-β3 in the CNS. The absence of infiltrating lymphocytes together with the suppression of cytokines, matrix metalloproteinase, and chemokines/receptors suggests that EE, like E2, protects mice from EAE by inhibiting the recruitment of T cells and macrophages into the CNS. These results suggest that oral ethinyl estradiol might be a successful candidate as therapy for multiple sclerosis.

Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis

A number of clinical and experimental observations have been made relating elevated estrogen levels with the amelioration of autoimmune diseases, yet questions remain about the levels required for efficacy as well as the mechanism of disease inhibition. Using the collagen-induced arthritis (CIA) model, we have studied the effects of physiological, sustained levels of 17β-estradiol in preventing the development of autoimmune arthritis and analyzed the changes in the autoimmune response. Using time-release pellets of 17β-estradiol, arthritis development was significantly inhibited in three different strains of CIA-susceptible mice compared with the effect of placebo treatment, and serum estradiol levels similar to those of mice in estrus were found to be equally effective as higher estradiol concentrations. Analysis of the autoimmune response in the estradiol-treated mice indicated that T cell production of IFN-γ was markedly decreased, and significant decreases were also observed in levels of IL-10 and GM-CSF produced by lymph nodes cells from estradiol-treated mice. Although the total IgG anti-CII response was only minimally affected by estrogen treatment, a significant reduction in the levels of IgG2a anti-CII Abs and an increase in the levels of IgG1 anti-CII Abs were observed in estradiol-treated mice. These data indicate that estradiol treatment altered the Th profile of the autoimmune T cell response, which, in turn, altered the production of IgG Abs to an isotype that is poor at fixing complement, an important component in the immunopathogenesis of CIA.

Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice

In a previous study, we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide could induce severe chronic experimental autoimmune encephalomyelitis (EAE) in HLA-DR2+ transgenic mice lacking all mouse MHC class II genes. We used this model to evaluate clinical efficacy and mechanism of action of a novel recombinant TCR ligand (RTL) comprised of the α1 and β1 domains of DR2 (DRB1*1501) covalently linked to the encephalitogenic MOG-35-55 peptide (VG312). We found that the MOG/DR2 VG312 RTL could induce long-term tolerance to MOG-35-55 peptide and reverse clinical and histological signs of EAE in a dose- and peptide-dependent manner. Some mice treated with lower doses of VG312 relapsed after cessation of daily treatment, but the mice could be successfully re-treated with a higher dose of VG312. Treatment with VG312 strongly reduced secretion of Th1 cytokines (TNF-α and IFN-γ) produced in response to MOG-35-55 peptide, and to a lesser degree purified protein derivative and Con A, but had no inhibitory effect on serum Ab levels to MOG-35-55 peptide. Abs specific for both the peptide and MHC moieties of the RTLs were also present after treatment with EAE, but these Abs had only a minor enhancing effect on T cell activation in vitro. These data demonstrate the powerful tolerance-inducing therapeutic effects of VG312 on MOG peptide-induced EAE in transgenic DR2 mice and support the potential of this approach to inhibit myelin Ag-specific responses in multiple sclerosis patients.

Evaluation of the effects of 17β-estradiol (17β-E2) on gene expression in experimental autoimmune encephalomyelitis using DNA microarray

The aim of this study was to identify immune-related genes affected by treatment with 17-estradiol (17-E2) that contribute to protection of T cell antigen receptor double transgenic mice from experimental autoimmune encephalomyelitis (EAE). The Affymetrix microarray system was used to screen more than 12,000 genes from E2-treated mice protected from EAE vs. control mice with severe EAE. In general, E2 treatment affected about 10% of the genes tested, but only 18 cytokine, chemokine/receptor, adhesion molecule, or activation genes were up- or down-regulated more than 2.4-fold by E2 treatment. Down-regulated genes included TNF (an important proinflammatory cytokine in EAE); peptidoglycan recognition proteins (Pgrp); regulated on activation, normal T cell expressed and secreted (RANTES); and neural cell adhesion molecule (MCP-1). Up-regulated genes included cytotoxic T lymphocyte antigen-4 (CTLA-4; known to inhibit T cell activation), TGF3, IL-18, and two interferon--induced genes, the chemokines: monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1), vascular cell adhesion molecule (VCAM), and disintegrin metalloprotease (thought to regulate TNF production). These results implicate a limited set of known and previously unsuspected E2-sensitive genes that may be crucial for inhibition of EAE and potentially the human disease, multiple sclerosis. (Endocrinology 143: 313–319, 2002)

Myelin oligodendrocyte glycoprotein-35–55 peptide induces severe chronic experimental autoimmune encephalomyelitis in HLA-DR2-transgenic mice

The use of HLA class II‐transgenic (Tg) mice has facilitated identification of antigenic T cell epitopes that may contribute to inflammation in T cell‐mediated diseases such as rheumatoid arthritis and multiple sclerosis (MS). In this study, we compared the encephalitogenic activity of three DR2‐restricted myelin determinants [mouse (m) myelin oligodendrocyte glycoprotein (MOG)‐35–55, human (h)MOG‐35–55 and myelin basic protein (MBP)‐87–99] in Tg mice expressing the MS‐associated DR2 allele, DRB1*1501. We found that mMOG‐35–55 peptide was strongly immunogenic and induced moderatelysevere chronic experimental autoimmune encephalomyelitis (EAE) with white matter lesions after a single injection in Freunds complete adjuvant followed by pertussis toxin. hMOG‐35–55 peptide,which differs from mMOG‐35–55 peptide by a proline for serine substitution at position 42, was also immunogenic, but not encephalitogenic, and was only partially cross‐reactive with mMOG‐35–55. In contrast, MBP‐87–99, which can induce EAE in double‐Tg mice expressing both HLA‐DR2 and a human MBP‐specific TCR, was completely non‐encephalitogenic in HLA‐DR2‐Tg mice lacking the human TCR transgene. These findings demonstrate potent encephalitogenic activity of the mMOG‐35–55 peptide in association with HLA‐DR2, thus providing a strong rationale for further study of hMOG‐35–55 peptide as a potential pathogenic determinant in humans.