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. 2003 Sep 2;100(18):10405-10.
doi: 10.1073/pnas.1533207100. Epub 2003 Aug 15.

Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator

Affiliations

Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator

Simone Cenci et al. Proc Natl Acad Sci U S A. .

Abstract

Expansion of the pool of tumor necrosis factor (TNF)-alpha-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ovariectomy up-regulates IFN-gamma-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-gamma by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are prevented in vivo by both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-gamma receptor signaling. Thus, increased IFN-gamma-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.

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Figures

Fig. 1.
Fig. 1.
In vivo analysis of T cell proliferation, apoptosis, and Ag presentation in ovx mice. (a) Analysis of BrdUrd incorporation and total DNA content (7-amino-actinomycin, 7-AAD) in spleen CD3+ T cells from sham-operated mice (Left) and ovx mice (Right). Proliferation is indicated as percentage of T cells in S phase. Percentage of apoptotic T cells is represented by the population in SubG0/G1 phase. (b) FACS analysis of T cell activation in the bone marrow. Data are expressed as percentage (mean ± SEM) of CD3+ T cells expressing CD25 or CD69. (c) APC activity of spleen cells and purified subpopulations of professional APCs from sham and ovx mice, as assessed by incorporation of [3H]thymidine by target T cells (data expressed as mean ± SEM of disintegrations per minute). (d) FACS analysis of MHCII expression in peritoneal Mϕ from sham and ovx mice. Data are expressed as percentage (mean ± SEM) of Mϕ expressing MHCII. (e) FACS analysis of activation- and irradiation-induced apoptosis in spleen CD4+ T cells. Data are expressed as percentage (mean ± SEM) of live apoptotic cells assessed by binding of annexin V. (f) FACS analysis of activation- and irradiation-induced FasL expression in spleen CD4+ T cells. Isotype control is indicated by the dotted line. All data are representative of at least three independent experiments. *, P < 0.05 compared with the corresponding sham group. (g) In vivo role of Ag presentation in ovx-induced bone loss. Shown is the effect of ovx on femoral BMD of DO11.10 mice (n = 6 per group). Data are expressed as percentage change (mean ± SEM) from baseline. *, P < 0.05 compared with both baseline and intact mice.
Fig. 2.
Fig. 2.
Ovx increases IFN-γ-induced CIITA in APCs and T cells. (a) Reverse transcriptase real-time PCR analysis of CIITA mRNA levels in BM Mϕ and T cells from WT and IFN-γR-/- mice of identical genetic background. (b) FACS analysis of IFN-γ levels in gated CD4+ T cells in BM (Left), and lymph nodes (Right) from sham and ovx mice. Isotype control is indicated by the dotted line. (c) ELISA for IFN-γ concentration in culture supernatants of purified BM CD90+ T cells, unstimulated or treated with cyclosporin A or SB203580. Data are expressed as mean ± SEM of six replicates. *, P < 0.05 compared with all undetectable groups by Fisher exact test. †, P < 0.05 compared with ovx, SB203580-treated groups. The dashed line indicates assay detection threshold. (d) ELISA for IL-12 and IL-18 levels in culture supernatants of purified CD11b+ BM Mϕ, treated with 17β estradiol (E2) or left untreated. Data are expressed as mean ± SEM of six replicates. *, P < 0.05 compared with all undetectable groups by Fisher exact test. †, P < 0.05 compared with ovx, E2-treated groups. The dashed line indicates assay detection threshold. All data are representative of at least three independent experiments.
Fig. 3.
Fig. 3.
IFN-γR-/- mice are protected from ovx-induced immune alterations, T cell expansion, and bone loss. (a) Basal and IFN-γ-stimulated MHCII expression by Mϕ from sham and ovx WT and IFN-γR-/- mice. Data are expressed as percentage (mean ± SEM) of Mϕ expressing MHCII. (b) FACS analysis of AICD in TH cells from sham and ovx WT and IFN-γR-/- mice. Percentage (mean ± SEM) of live apoptotic cells assessed by binding of annexin V is shown. All data are representative of at least three independent experiments. (c and d) Levels of TNF and IFN-γ in supernatants from purified unstimulated TH cells from sham and ovx WT and IFN-γR-/- mice. Data are expressed as average (±SEM) of six replicate samples. *, P < 0.05 compared with all other groups. The dashed line indicates assay detection threshold. (e) Analysis of BMD in sham and ovx WT and IFN-γR-/- mice (n = 6 per group). Data are expressed as percentage change (mean ± SEM) from baseline. *, P < 0.05 compared with baseline. (f) Histology of proximal tibia of sham and ovx WT and IFN-γR-/- mice. Representative samples from one mouse per group are shown. WT ovx mice, but not IFN-γR-/- mice, exhibit decreased trabecular bone and increased tartrate-resistant acid phosphatase positive osteoclasts (red staining) as compared with sham controls.

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