IFN-gamma-inducing transcription factor, T-bet is upregulated by estrogen in murine splenocytes: role of IL-27 but not IL-12 - PubMed (original) (raw)

IFN-gamma-inducing transcription factor, T-bet is upregulated by estrogen in murine splenocytes: role of IL-27 but not IL-12

Ebru Karpuzoglu et al. Mol Immunol. 2007 Mar.

Abstract

Estrogen is believed to be involved in regulation of the differentiation, survival, or function of diverse immune cells as well as in many autoimmune and inflammatory diseases. However, the mechanisms behind the immunomodulatory effects of estrogen are poorly understood. Previously, we have shown that natural estrogen can upregulate IFN-gamma and IFN-gamma-mediated-inflammatory events (iNOS, nitric oxide, COX-2). Since IFN-gamma is regulated by T-bet, in this study, we investigated whether estrogen induces T-bet expression in primary murine splenocytes. We found that in vivo estrogen treatment primes splenocytes for early upregulation of T-bet upon activation by T cell stimulants, Concanavalin-A (Con-A) or anti-CD3 antibodies. The expression of T-bet protein was not altered by IL-12 while IFN-gamma had partial effects on T-bet in splenocytes from estrogen-treated mice. Notably, T-bet expression increased in Con-A-activated splenocytes from estrogen-treated mice in the presence of IL-27. Together, our studies show that in vivo estrogen exposure primes lymphocytes towards Th1 type development by promoting/upregulating T-bet expression, which is upregulated in part by IFN-gamma and IL-27. Given that T-bet is a potent inducer of IFN-gamma, these studies may lead to new lines of investigation in relation to many female-predominant autoimmune diseases and inflammatory disorders.

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Figures

Figure 1

Figure 1. Estrogen upregulates early production of IFN-γ protein

(A) Intracellular IFN-γ protein was detected in freshly isolated lymphocytes from estrogen and placebo-treated mice. The cells were stained with anti-IFN-γ antibodies and evaluated by flow cytometry. The relative expression of IFN-γ-stained cells was presented as means ± SEM (standard errors of mean) (placebo: n=5 mice, estrogen: n=5 mice; *p<0.05). (B) Representative histograms from placebo and estrogen-treated mice are overlayed on top of each other. The faint unidentified histogram represents background control. (C) The protein levels of IFN-γ were determined in the supernatants from Concanavalin-A (Con-A; 10 μg/ml) -stimulated or unstimulated (media only) splenic lymphocytes from estrogen or placebo-treated mice. Extracellular protein levels of IFN-γ were upregulated as early as 3 hrs (placebo: n=4 mice, estrogen: n=4 mice; *p<0.05). Data are presented as means with standard errors of the mean.There was no detectable IFN-γ in supernatants from unstimulated cultures (media only).

Figure 2

Figure 2. Estrogen upregulates T-bet protein expression in activated splenic lymphocytes in a time-dependent fashion

(A) Nuclear extracts from splenic lymphocytes from estrogen or placebo-treated mice which were Con-A-stimulated for 3, 6, 18, or 24 hrs of culture were analyzed for T-bet protein expression with a Western blot assay. One representative experiment is shown above the graph. The mean relative densitometry data of T-bet protein expression from nuclear extracts from Con-A stimulated splenic lymphocytes from estrogen or placebo-treated mice are shown (placebo (P): n=4 mice, estrogen (E2): n=4 mice; *p<0.05). (B) IFN-γ protein levels in supernatants from Con-A-activated splenocytes cultured for 3, 6, 18, or 24 hrs from estrogen and placebo treated mice were analyzed with an ELISA (placebo: n=4 mice, estrogen: n=4 mice; *p<0.05). (C) T-bet expression is increased in anti-CD3 antibody-stimulated splenocytes from estrogen-treated mice. Nuclear extracts from anti-CD3 antibody (10 μg/ml) stimulated splenic lymphocytes or unstimulated (media only) cells from placebo or estrogen-treated mice incubated for 3 hrs were assayed to determine T-bet protein expression. The top portion of the figure shows one representative experiment (n=4 mice per placebo (P) or estrogen (E2)-treatment; *p<0.05). The bottom portion of the figure depicts the mean relative densitometry data for T-bet protein expression as detected with Western blot analysis. Data are presented as means with standard error bars.

Figure 3

Figure 3. IL-12 does not directly alter T-bet protein expression in activated splenocytes from estrogen-treated mice

Splenocytes from estrogen or placebo-treated mice were stimulated with Con-A (10 μg/ml), Con-A and rIL-12 (20 ng/ml), or Con-A and anti-IL-12 antibodies (3 μg/ml) for 3 hrs and the expression of T-bet protein in the nuclear extracts was determined. (A) One representative experiment of T-bet protein expression in cells stimulated with Con-A or Con-A and rIL-12 is shown in the top portion of the figure. The bottom portion shows the mean relative densitometry data of T-bet protein expression (placebo (P): n=6 mice, estrogen (E2): n=6 mice; **p<0.01). (B) One representative experiment of T-bet protein expression is shown in the top portion of the figure. The bottom portion shows the mean relative densitometry data of T-bet protein expression in nuclear extracts from Con-A or Con-A and anti-IL-12-antibodies-activated splenocytes from estrogen or placebo-treated mice (placebo: n=10 mice, estrogen: n=10 mice; **p<0.01). Data are presented as means with standard error bars.

Figure 4

Figure 4. T-bet expression is partially regulated by IFN-γ in activated splenocytes and purified T cells from estrogen-treated mice

(A) Splenic lymphocytes from placebo or estrogen-treated mice were stimulated with Con-A (10 μg/ml) or Con-A plus various doses of rIFN-γ (100, 1,000, 10,000 pg/ml) for 3 hrs of culture and T-bet expression was explored in nuclear extracts of splenocytes. The top portion shows one representative experiment for T-bet expression. The figure demonstrates the mean relative densitometry data for T-bet protein expression (placebo: n=4 mice, estrogen: n=4 mice; *p<0.05). (B) Purified splenic T cells from estrogen or placebo-treated mice were cultured with Con-A (10 μg/ml) or Con-A and rIFN-γ (10,000 pg/ml) for 3 hrs and T-bet protein expression was explored in nuclear extracts from T cells (placebo: n=4 mice, estrogen: n=4 mice; **p<0.01). One representative experiment is shown at the top of Panel A. (C) IFN-γ protein levels in the supernatants from Con-A-activated T cells from estrogen and placebo-treated mice are shown (placebo: n=4 mice, estrogen: n=4 mice). (D) Intracellular IFN-γ protein in the cytoplasmic extracts from Con-A or Con-A and rIFN-γ-activated T cells was detected with Western blot assay. The mean relative densitometry data and one representative Western blot experiment are shown (placebo: n=4 mice, estrogen: n=4 mice; *p<0.05). Data are presented as means with standard error bars.

Figure 5

Figure 5. The early expression of T-bet is increased in response to deliberate addition of IL-27 to Con-A-activated splenocyte cultures from estrogen-treated mice

Nuclear extracts from Con-A (10 μg/ml), Con-A and rIL-27 (10 ng/ml) cultured splenic lymphocytes from placebo or estrogen-treated mice that were incubated for 3 hrs were assayed to determine T-bet expression. The top portion of the figure shows all 4 pairs of experiments (n=4 mice per placebo or estrogen-treatment). The bottom portion of the figure depicts the mean relative densitometry data for T-bet protein expression as detected with Western blot analysis (n=4 mice per placebo or estrogen-treatment; *p<0.05). Data are presented as means with standard error bars.

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