Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors

Abstract

Macrophage function has been demonstrated to be subject to modulation by progesterone. However, as this steroid hormone can act through the glucocorticoid receptor as well as the progesterone receptor, the mechanism of action has not been precisely characterized. To determine the mode of action, we compared the ability of progesterone, norgestrel (a synthetic progesterone-receptor-specific agonist) and dexamethasone (a synthetic glucocorticoid receptor agonist) to modulate macrophage function following stimulation of the Toll-like receptor-4 (TLR-4) ligand lipopolysaccharide (LPS). The results demonstrate that following stimulation of TLR-4 with LPS and cotreatment with either progesterone or dexamethasone, but not norgestrel, there is a significant reduction in nitric oxide (NO) production, indicating that this progesterone-mediated effect is through ligation of the glucocorticoid receptor. In contrast, LPS-induced interleukin-12 (IL-12) production could be downregulated by all three steroids, indicating that ligation by progesterone of either the glucocorticoid or the progesterone receptors or both could mediate this effect. While progesterone downmodulated NO-mediated killing of Leishmania donovani by activated macrophages in vitro, most probably via the glucocorticoid receptor, it had little effect on Toxoplasma gondii growth in these cells. This would suggest that progesterone-mediated increased susceptibility to T. gondii during pregnancy is more likely to be related to the ability of the hormone to downregulate IL-12 production and a type-1 response utilizing the progesterone as well as the glucocorticoid receptors.

Figure 1

The influence of progesterone, norgestrel and dexamethasone on nitric oxide (NO) production by bone-marrow-derived macrophages (BMMs) stimulated with 3·125 μg/ml lipopolysaccharide (LPS) for 72 hr. Cells were cultured with LPS in the presence of progesterone (a), norgestrel (b) or dexamethasone (c). Unstimulated BMMs failed to produce detectable levels of NO (nd). Unstimulated BMMs treated with the solvent vehicle of 0·04% chloroform (progesterone and norgestrel) or 0·0001% ethanol (dexamethasone) failed to produce detectable levels of NO (data not shown). Stimulated control cell cultures were treated with 3·125 μg/ml LPS (closed bars). Stimulated control cell cultures treated with 3·125 μg/ml LPS and either 0·04% chloroform or 0·0001% ethanol produced levels of NO which were comparable to those produced by stimulated cells treated with LPS alone (data not shown). Results are the mean of n = 3 treatments from one of three representative experiments. Where SEs are not evident it is because they are too small to be visible. *P < 0·05 compared with stimulated controls using a Mann–Whitney U-test.

Figure 2

The influence of progesterone, norgestrel and dexamethasone on interleukin-12 p40 (IL-12p40) production by bone-marrow-derived macrophages (BMMs) stimulated with 12·5 μg/ml lipopolysaccharide (LPS) for 72 hr. Cells were cultured with LPS in the presence of progesterone (a), norgestrel (b) or dexamethasone (c). Unstimulated cells produced undetectable (nd) or very low levels of IL-12p40. Unstimulated cells treated with the solvent vehicle of 0·04% chloroform (progesterone and norgestrel) or 0·0001% ethanol (dexamethasone) failed to produce detectable levels of IL-12p40 (data not shown). Stimulated control cell cultures were treated with 12·5 μg/ml LPS (closed bars). Stimulated control cell cultures treated with 12·5 μg/ml LPS and either 0·04% chloroform or 0·0001% ethanol produced levels of IL-12p40 which were comparable to those produced by stimulated cells treated with LPS alone (data not shown). Results are the mean of n = 3 treatments from one of three representative experiments. Where SEs are not evident it is because they are too small to be visible. *P < 0·05 compared with stimulated controls using a Mann–Whitney U-test.

Figure 3

The influence of progesterone, norgestrel and dexamethasone on cell viability and proliferation of bone-marrow-derived macrophages (BMMs) stimulated with 12·5 μg/ml lipopolysaccharide (LPS) for 72 hr. Cells were cultured in the presence or absence of progesterone (a), norgestrel (b) and dexamethasone (c). The alamarBlue™ was added in 20-μl volumes after 48 hr. Unstimulated BMMs were treated with medium alone (open bars). Unstimulated BMMs treated with the solvent vehicle of 0·04% chloroform (progesterone and norgestrel) or 0·0001% ethanol (dexamethasone) reduced alamarBlue™ to the same extent as unstimulated controls receiving medium alone (data not shown). Stimulated control cells were treated with 12·5 μg/ml LPS (closed bars). Stimulated controls treated with 12·5 μg/ml LPS and either 0·04% chloroform or 0·0001% ethanol reduced alamarBlue™ to the same extent as stimulated cells receiving LPS alone (not shown). Where SEs are not evident it is because they are too small to be visible. Results are the mean ± SE of n = 3 treatments from two representative experiments.

Figure 4

The influence of progesterone, norgestrel and dexamethasone on interleukin-10 (IL-10) production by bone-marrow-derived macrophages (BMMs) stimulated with 12·5 μg/ml lipopolysaccharide (LPS) for 72 hr. Cells were cultured with LPS in the presence of progesterone (a), norgestrel (b) or dexamethasone (c). Unstimulated BMMs treated with medium alone did not produce IL-10 (nd). Unstimulated BMMs treated with the solvent vehicle of 0·04% chloroform (progesterone and norgestrel) or 0·0001% ethanol (dexamethasone) did not induce detectable levels of IL-10 (data not shown). Stimulated control cell cultures were treated with 12·5 μg/ml LPS (closed bars). Stimulated control cell cultures treated with 12·5 μg/ml LPS and either 0·04% chloroform or 0·0001% ethanol produced levels of IL-10 which were comparable to those produced by stimulated cells treated with LPS alone (data not shown). Results are the mean of n = 3 treatments from two representative experiments. Where SEs are not evident it is because they are too small to be visible. *P < 0·05 compared with stimulated controls using a Mann–Whitney U-test.

Figure 5

The effect of progesterone on the ability of activated bone-marrow-derived macrophages (BMMs) to kill Toxoplasma gondii and Leishmania donovani. The BMMs were infected with T. gondii tachyzoites of the RH strain at a parasite : host cell ratio of 1 : 1 and treated with 3·125 μg/ml lipopolysaccharide (LPS) in the presence or absence of progesterone (62·5, 15·7 or 4 μm) for 72 hr. Tachyzoite proliferation was measured by uptake of ³H-labelled uracil added at 1 μCi after 48 hr (a). BMMs treated with medium alone = open bars; infected BMMs = shaded bars; infected cells stimulated with LPS = closed bars. Beta emissions are shown as mean counts per minute ± SE. Results are the mean of n = 3 treatments and are representative of three separate experiments. BMMs infected with L. donovani promastigotes at a parasite : host cell ratio of 1 : 20, were treated 24 hr postinfection with 100 U interferon-γ (IFN-γ) and 100 ng/ml LPS in the presence or absence of progesterone (62·5, 15·7 or 4 μm) for a further 72 hr. The mean infection (% ± SE) of cells is shown as assessed by Giemsa staining and microscopical examination (b). Uninfected BMMs = open bars; infected BMMs = shaded bars; infected cells stimulated with IFN-γ/LPS = closed bars. Results are the mean of n = 4 samples from three separate treatments. Where SEs are not evident it is because they are too small to be visible. *P < 0·03 compared with IFN-γ/LPS activated controls.