T lymphocytes do not directly mediate the protective effect of estrogen on experimental autoimmune encephalomyelitis

Abstract

Gender influences mediated by 17 beta-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-alpha (Esr1) is crucial for the protective effect of 17 beta-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wild-type Esr1+/+ or Esr1 knockout (Esr1-/-) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1+/+ disease-inducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in EAE.

Figure 1

E2 treatment decreased the frequency of encephalitogenic T cells in the CNS of Esr1+/+ WT and Esr1+/+ SCID recipients. MNCs were isolated from brain and spinal cord harvested 10 days after disease onset from five mice. Cells were stained with anti-mouse CD3 and anti-mouse CD4 and anti-mouse CD8. Data presented are mean from two independent experiments and indicate the percentage of total gated cells that were dual-positive for CD3 and CD4 or CD8. Significance between control and experimental groups were determined by Student’s t-test (*, P < 0.05).

Figure 2

E2 treatment decreased the frequency of adhesion molecules on encephalitogenic T cells in the CNS of C57BL/6 and C57BL/6 SCID recipients. MNCs were isolated from brain and spinal cord harvested at 10 days after onset from five mice. Cells were stained with anti-mouse CD3 and anti-mouse VLA-4 and anti-mouse LFA-1 to identify expression of these adhesion molecules on T cells. Data presented are percentage of total gated cells that were dual-positive for CD3 and VLA-4 or LFA-1. Significance between control and experimental groups were determined by Student’s t-test (*, P < 0.05).

Figure 3

Fixed paraffin-embedded spinal cord sections from control (A–F) or E2-treated (G–L) recipients. Spinal cord from control Esr1+/+ T cells → Esr1+/+ WT (A, B), Esr1+/+ T cells → Esr1+/+ SCID (C, D), and Esr1−/− T cells → Esr1+/+ SCID recipients showed dense mononuclear infiltration with apparent loss of myelin (blue stain, luxol fast blue) in the surrounding myelinated tissue. Spinal cord from E2-treated control Esr1+/+ T cells → Esr1+/+ WT (G, H), Esr1+/+ T cells → Esr1+/+ SCID (I, J) showed no visible signs of inflammation whereas in spinal cord of Esr1−/− T cells → Esr1+/+ SCID (K, L) single mononuclear cells were present without visible demyelination. Original magnification: ×50 (A, C, E, G, I, K); ×150 (B, D, F, H, J, L).

Figure 4

CD45 expression of microglia/CNS-associated macrophages from control and E2-treated recipients. Microglia/CNS-associated macrophages were gated as CD11b⁺CD11c⁻ cells (R2) to exclude the potential dendritic cells. All cells from R2 express CD45 and subdivide into two main populations: CD45^intr (R3) and CD45^high (R4).