Treatment with selective estrogen receptor modulators regulates myelin specific T-cells and suppresses experimental autoimmune encephalomyelitis

Abstract

Steroidal estrogens can regulate inflammatory immune responses and may be involved in the suppression of multiple sclerosis (MS) during pregnancy. However, the risks and side effects associated with steroidal estrogens may limit their usefulness for long-term MS therapy. Selective estrogen receptor modulators (SERMs) could provide an alternative therapeutic strategy, because they behave as estrogen agonists in some tissues, but are either inert or behave like estrogen antagonists in other tissues. In this study, we investigated the ability of two commercially available SERMs (tamoxifen and raloxifene) to regulate myelin specific immunity and experimental autoimmune encephalomyelitis (EAE) in mice. Both tamoxifen and raloxifene suppressed myelin antigen specific T-cell proliferation. However, tamoxifen was more effective in this regard. Tamoxifen treatment reduced the induction of major histocompatibility complex II by lipopolysaccharide stimulated dendritic cells and decreased their ability to activate myelin specific T-cells. At lower doses, tamoxifen was found to increase the levels of Th2 transcription factors and induce a Th2 bias in cultures of myelin-specific splenocytes. EAE symptoms and the degree of demyelination were less severe in mice treated with tamoxifen than in control mice. These findings support the notion that tamoxifen or related SERMs are potential agents that could be used in the treatment of inflammatory autoimmune disorders that affect the central nervous system.

Figures

Figure 1. Treatment with SERMs inhibits proliferation of myelin-specific splenocytes

Splenocytes from naïve female and male Vβ8.2/Vα4 TCR transgenic mice were stimulated with 2 μg/ml of MBP Ac1-11 peptide and treated with the indicated amounts of tamoxifen, raloxifene, 17β-estradiol, or 17α-estradiol for 72hrs. The data are presented as the percent change in proliferation (as measured by [3H]-thymidine incorporation) compared to vehicle treated control cultures (see Supplemental Fig. 1). The means and standard deviations of three separate experiments are shown.

Figure 2. Treatment with SERMs inhibits proliferation of ER-α deficient splenocytes

Splenocytes from naïve female estrogen receptor-alpha knockout (ERKO-alpha) mice were stimulated with plate-bound anti-CD3/anti-CD28 and treated with the indicated amount of tamoxifen, raloxifene, 17β-estradiol, or 17α-estradiol for 72hrs. [3H]-thymidine was added for the last 18 hrs of culture and the incorporation of radioactive thymidine by the cells was quantified as a measure of cellular proliferation. The data are presented as mean counts per minute from triplicate cultures.

Figure 3. Tamoxifen suppresses the induction of MHC II expression on dendritic cells and their ability to activate myelin-specific T-cells

Dendritic cells were selected from the bone marrow by growth in media containing GM-CSF for 8 days. The cells were subsequently matured by culture with LPS (0.1 μg/ml) in the presence or absence of tamoxifen (10-7M) for 48 hrs prior to staining with fluorochrome labeled antibodies and analysis by flow cytometry. (A) Induction of MHC II on dendritic cells by treatment with LPS, (2) comparison of MHC II expression between vehicle (90% ethanol, 10% DMSO) and tamoxifen treated dendritic cells, (C) Induction of CD86 (B72) on dendritic cells by treatment with LPS, (D) comparison of CD86 expression between vehicle and tamoxifen treated dendritic cells. *Statistical differences between control and treated cultures were determined using a two-tailed student t-test (p<0.01). (E) The immature dendritic cells were pulsed with MBP Ac1-11 peptide in the presence of vehicle or the indicated dose of tamoxifen for 2hrs. LPS (0.1 μg/ml) was then added to the antigen pulsed dendritic cells for 2hrs as a maturation trigger. The cells were washed and used as APCs to stimulate CD4+, MBP Ac1-11 specific transgenic T-cells. The cultures were incubated for 7hrs. [3H]-thymidine was added for the last 18 hrs of culture and the incorporation of radioactive thymidine by the cells was quantified as a measure of cellular proliferation.

Figure 4. Treatment with tamoxifen induces a Th2 bias in myelin specific T-cells derived from female TCR transgenic mice

Splenocytes from naïve female TCR transgenic mice were stimulated with 2 μg/ml of MBP Ac1-11 peptide and treated with 100 nM of tamoxifen for 5 days. The cells were re-stimulated with PMA and ionomycin in the presence of the Golgi inhibitor brefeldin A for 5 hrs before staining with FITC-labeled anti-Vβ8.2 and PE-Cy3 labeled anti-CD4 antibodies. The cells were fixed and permeabilized prior to staining with PE-labeled anti-cytokine antibodies. The cells were gated on lymphocytes based on forward and side scatter characteristics and staining with anti-Vβ8.2 using a FACScan flow cytometer. The frequency of Vβ8.2+/CD4+ helper T cells expressing the indicated cytokine was determined by setting quadrants based on staining with a PE-labeled isotype control antibody. Each line in the figure represents data from an individual mouse splenocyte culture. The controls include data from either untreated or vehicle treated cultures. Statistical differences between control and treated cultures were determined using a two-tailed Student’s t-test.

Figure 5. Tamoxifen treatment inhibits relapsing-remitting EAE in female SJL mice

(A, B) Eight SJL mice were implanted with time-release pellets containing 25 mg of tamoxifen and 8 mice were treated with placebo pellets one week prior to immunization with PLP 139-151 and CFA. The mice were monitored daily and scored for EAE disability. The mean daily EAE score for each group is presented in panel A. The cumulative disease index (CDI) for each group show in panel B. (C) The CDI from B10.PL mice implanted with time-release tamoxifen or 17β-estradiol (E2) pellets (at 0.1 mg or 2.5 mg) or placebo and immunized with MBP Ac1-11 and CFA. (D) Spinal cord sections from the mice in the experiment described in panel C were immunolabeled with an anti-MBP antibody. Note the reduction in myelin staining in the white matter (arrows) of placebo-treated mice compared to mice treated with either 17β-estradiol or tamoxifen. *Statistically significant difference in the cumulative disease index (CDI) between tamoxifen treated and placebo, p

Figure 6. Treatment with tamoxifen increases the frequency of Treg cells in naïve female SJL mice

Tamoxifen pellets (25 mg) were implanted into 9 female SJL mice and placebo pellets were implanted into 10 female SJL mice one week prior to immunization with PLP 139-151 in CFA. The spleens were harvested from all the mice 3 weeks later and stained with FITC-labeled anti-CD3, PE-Cy3 labeled anti-CTLA-4 antibodies, and PE-labeled CD25. The cells were gated on CD3+ lymphocytes and the frequency of CTLA-4+/CD25+ regulatory cells was determined by flow cytometry. The data are presented as the mean and standard deviation. The differences between tamoxifen and placebo treated mice were determined to be significant in spleen, thymus and lymph node cells as using a Student’s t-test (p<= 0.01).