Selective inhibition and augmentation of alternative macrophage activation by progesterone - PubMed (original) (raw)
Selective inhibition and augmentation of alternative macrophage activation by progesterone
Fiona M Menzies et al. Immunology. 2011 Nov.
Abstract
Progesterone is the female sex hormone necessary for the maintenance of pregnancy, and is known to modulate macrophage activation. However, studies have concentrated exclusively on the ability of progesterone to negatively regulate the innate and classical pathways of activation, associated with nitric oxide (NO) and interleukin (IL)-12 production. Our aim was to examine the ability of progesterone to modulate alternative macrophage activation. Bone marrow cells were isolated and differentiated from male BALB/c mice, exposed to varying concentrations of progesterone and stimulated with lipopolysaccharide (LPS) (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. Our present study demonstrates that progesterone not only down-regulates inducible nitric oxide synthase 2 (iNOS) activity in macrophages but also arginase activity, in a dose-dependent manner, independent of the stimuli, whether it is induced by LPS (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. The ability of progesterone to down-modulate IL-4-induced cell surface expression of the mannose receptor further suggested a negative regulation of alternative macrophage activation by this hormone. Analysis of mRNA expression, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), of genes associated with innate and alternative macrophage activation revealed that progesterone down-regulated LPS-induced macrophage nos2, argI and p40 (IL-12/IL-23) expression and IL-4-induced argI, mrc-1 and fizz1 expression. However, progesterone up-regulated IL-4-induced macrophage expression of ym1, while dectin-1 expression remained unaltered. Following treatment of macrophages with LPS and IL-4 in combination a similar pattern was observed, with the exception that progesterone up-regulated macrophage expression of fizz1 as well as ym1 and did not modify mrc-1 expression. Our data demonstrate for the first time that a hormone has the ability to regulate selectively the expression of different genes associated with alternative macrophage activation.
© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.
Figures
Figure 1
Progesterone down-modulates arginase activity and nitrite production by bone marrow-derived (BMD) macrophages. Arginase activity (a, b, c) and nitric oxide (NO) production (d, e) in progesterone-treated BMD macrophages that have been subsequently stimulated with interleukin (IL)-4 (100 U/ml) for 48 hr (a), lipopolysaccharide (LPS) (200 ng/ml) for 48 hr (b, d) or both IL-4 (100 U/ml) and LPS (200 ng/ml) for 48 hr (c, e). Open bars represent samples that were treated with progesterone at the stated concentration only. Closed bars represent those samples that were preincubated with progesterone for 16 hr prior to the addition of IL-4, LPS or IL-4 + LPS. Solvent vehicle (0·01–0·04% chloroform) did not result in any significant differences from the medium control (data not shown). Results show mean ± standard error (SE) of n = 3 and are representative of three separate experiments. *P < 0·05 in comparison with stimulated control.
Figure 2
The effect of progesterone on argI and nos2 mRNA expression in bone marrow-derived (BMD) macrophages. Cells were left untreated, incubated with 0·04% chloroform (solvent control) or 62·5 μ
m
progesterone for 16 hr prior to the addition of 100 U/ml interleukin 4 (IL-4) (a, b), 200 ng/ml lipopolysaccharide (LPS) (c, d, g, h) or 100 U/ml IL-4 and 200 ng/ml LPS (e, f, i, j) for 6 hr (a, c, e, g, i) or 24 hr (b, d, f, h, j). Control cultures were not treated with IL-4 and/or LPS. Expression levels of argI and nos2 mRNA transcripts were normalized to the housekeeping gene Tbp for each experimental run. The maximum gene expression for each run was designated 100% and all experiments calculated in comparison to this. Data are represented as the mean expression ± standard error (SE) of n = 3. *P < 0·05 in comparison with stimulated controls not treated with hormone or solvent for the same length of time.
Figure 3
The effect of progesterone on mrc-1 and p40 [interleukin 12 (IL-12)/IL-23] mRNA expression in bone marrow-derived (BMD) macrophages. Cells were left untreated, incubated with 0·04% chloroform (solvent control) or 62·5 μ
m
progesterone for 16 hr prior to the addition of 100 U/ml IL-4 (a, b), 200 ng/ml lipopolysaccharide (LPS) (e, f) or 100 U/ml IL-4 and 200 ng/ml LPS (c, d, g, h) for 6 hr (a, c, e, g) or 24 hr (b, d, f, h). Control cultures were not treated with IL-4, LPS or LPS and IL-4. Expression levels of mrc-1 and p40 (IL-12/IL-23) mRNA transcripts were normalized to the housekeeping gene Tbp for each experimental run. The maximum gene expression for each run was designated 100% and all experiments calculated in comparison to this. Data are represented as the mean expression ± standard error (SE) of n = 3. *P < 0·05 in comparison with stimulated controls not treated with hormone or solvent for the same length of time.
Figure 4
Influence of progesterone on interleukin 4 (IL-4) induced expression of mannose receptor (MR) in bone marrow-derived (BMD) macrophages. Cells were preincubated with medium (open bar), 62·5 μ
m
progesterone (black bar), 0·04% chloroform (grey bar) for 16 hr followed by a 48-hr stimulation with IL-4 (100 U/ml). Unstimulated cells received medium. Results are the mean fluorescence intensity ± standard error (SE) of n = 3 samples and are representative of at least two experimental runs. *P < 0·05 compared with medium controls (open bar).
Figure 5
The effect of progesterone on dectin-1 mRNA expression in bone marrow-derived (BMD) macrophages. Cells were left untreated, incubated with 0·04% chloroform (solvent control) or 62·5 μ
m
progesterone for 16 hr prior to the addition of 100 U/ml interleukin 4 (IL-4) (a, b) or 100 U/ml IL-4 and 200 ng/ml lipopolysaccharide (LPS) (c, d) for 6 hr (a, c) or 24 hr (b, d). Control cultures were not treated with IL-4 or LPS and IL-4. Expression levels of dectin-1 mRNA transcripts were normalized to the housekeeping gene Tbp for each experimental run. The maximum gene expression for each run was designated 100% and all experiments calculated in comparison to this. Data are represented as the mean expression ± standard error (SE) of n = 3. *P < 0·05 in comparison with stimulated controls not treated with hormone or solvent for the same length of time.
Figure 6
The effect of progesterone on fizz1 mRNA expression in bone marrow-derived (BMD) macrophages. Cells were left untreated, incubated with 0·04% chloroform (solvent control) or 62·5 μ
m
progesterone for 16 hr prior to the addition of 100 U/ml interleukin 4 (IL-4) (a, b) or 100 U/ml IL-4 and 200 ng/ml lipopolysaccharide (LPS) (c, d) for 6 hr (a, c) or 24 hr (b, d). Control cultures were not treated with IL-4 or LPS and IL-4. Expression levels of fizz1 mRNA transcripts were normalized to the housekeeping gene Tbp for each experimental run. The maximum gene expression for each run was designated 100% and all experiments calculated in comparison to this. Data are represented as the mean expression ± standard error (SE) of n = 3. *P < 0·05 in comparison with stimulated controls not treated with hormone or solvent for the same length of time.
Figure 7
The effect of progesterone on ym1 mRNA expression in bone marrow-derived (BMD) macrophages. Cells were left untreated, incubated with 0·04% chloroform (solvent control) or 62·5 μ
m
progesterone for 16 hr prior to the addition of 100 U/ml interleukin 4 (IL-4) (a, b) or 100 U/ml IL-4 and 200 ng/ml lipopolysaccharide (LPS) (c, d) for 6 hr (a, c) or 24 hr (b, d). Control cultures were not treated with IL-4 or LPS and IL-4. Expression levels of ym1 mRNA transcripts were normalised to the housekeeping gene Tbp for each experimental run. The maximum gene expression for each run was designated 100% and all experiments calculated in comparison to this. Data are represented as the mean expression ± standard error (SE) of n = 3. *P < 0·05 in comparison with stimulated controls not treated with hormone or solvent for the same length of time.
References
- Stout RD, Jiang C, Matta B, Tietzel I, Watkins SK, Suttles J. Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences. J Immunol. 2005;175:342–9. - PubMed
- Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3:23–35. - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Miscellaneous