Download and Function by Progesterone TLR4

Differential Modulation of TLR3- and
TLR4-Mediated Dendritic Cell Maturation
and Function by Progesterone
This information is current as
of June 15, 2017.
Leigh A. Jones, Shrook Kreem, Muhannad Shweash,
Andrew Paul, James Alexander and Craig W. Roberts
J Immunol 2010; 185:4525-4534; Prepublished online 15
September 2010;
doi: 10.4049/jimmunol.0901155
http://www.jimmunol.org/content/185/8/4525
References
Subscription
Permissions
Email Alerts
http://www.jimmunol.org/content/suppl/2010/09/13/jimmunol.090115
5.DC1
This article cites 56 articles, 13 of which you can access for free at:
http://www.jimmunol.org/content/185/8/4525.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Supplementary
Material
The Journal of Immunology
Differential Modulation of TLR3- and TLR4-Mediated
Dendritic Cell Maturation and Function by Progesterone
Leigh A. Jones, Shrook Kreem, Muhannad Shweash, Andrew Paul, James Alexander, and
Craig W. Roberts
D
endritic cells (DCs) play a pivotal role in immunity by
linking the innate and adaptive immune systems and are
vital in initiating protective responses to a number of
pathogens (1–3). Ligation of TLRs with pathogen-associated molecular patterns induces maturation of DCs characterized by increased surface expression of CD40, CD80, and CD86 accompanied by the production of a number of cytokines. Following
maturation and Ag uptake, DCs migrate to the lymphoid tissues
to interact with B and T cells and initiate the development of
the adaptive response (1, 4). Depending on the cytokine milieu
created by the innate response to a particular pathogen, DCs can
orchestrate the T cell response to either a Th1 or Th2 phenotype
(1, 5).
The induction of a Th2 response has been demonstrated to occur
in the vicinity of the placenta during mammalian pregnancy, and
disruption of this has been associated with abortion (6, 7). In
addition, during pregnancy, T regulatory cells specific for fetal
allograft Ags are involved in expression of tolerance to the fetus
(8, 9). DCs have been demonstrated around the fetomaternal interface, and studies have shown that in this situation, their function
is altered to favor production of regulatory cytokines, such as
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom
Received for publication April 16, 2009. Accepted for publication July 29, 2010.
L.A.J. was supported by a Caledonian Research Foundation scholarship from the
Carnegie Trust for the Universities of Scotland. S.K. and M.S. are supported by the
Iraqi Ministry of Higher Education.
Address correspondence and reprint requests to Dr. Craig W. Roberts, Strathclyde
Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor
Street, John Arbuthnott Building, Glasgow G4 0NR, United Kingdom. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cell; DC,
dendritic cell; GR, glucocorticoid receptor; IRF, IFN regulatory factor; poly I:C,
polyinosinic-polycytidylic acid; PR, progesterone receptor; SAPK, stress-activated
protein kinase.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901155
IL-10, and reduce secretion of inflammatory cytokines, such as
IL-12 (10, 11). The microenvironment created during pregnancy
differs from the normal physiological situation, particularly with
regard to circulating hormones, and levels of glucocorticoids,
estrogen, and progesterone are all increased. This change in the
hormonal milieu is thought to play a major role in skewing the
maternal immune response to a Th2 and T regulatory phenotype
(12, 13), influenced in part by actions of hormones on a number of
hematopoietic cells including DCs (14, 15).
Glucocorticoids have been reported to influence multiple aspects
of DC function from initial development to T cell stimulation.
Consequently, glucocorticoids have been shown to limit the capacity of monocytes to develop into DCs, reduce Ag uptake, upregulate adhesion molecule expression, downregulate maturation
markers, such as CD40, CD80, CD86, and MHC class II, inhibit
production of proinflammatory cytokines including IL-1a, IL-1b,
IL-6, IL-12p70, IFN-g, and TNF-a, reduce Ag presentation via the
MHC class II pathway, and downmodulate T cell stimulatory
capacity (16–22).
Given the paramount importance of DCs in linking, as well as
driving, the innate and adaptive immune response and the welldocumented ability of progesterone to modulate immune activity, there have been surprisingly few studies investigating how
progesterone specifically modulates DC maturation and function.
Progesterone has been shown to upregulate the differentiation of
DCs as measured by increased endocytic activity from bone
marrow precursors, although following LPS stimulation, an immature phenotype was maintained under the influence of progesterone as demonstrated by decreased MHC class II, CD40,
CD54, and CD80 expression (23, 24). In contrast, a further study
reported that progesterone treatment of resting murine splenic
DCs resulted in increased expression of MHC class II and CD40
and enhanced T cell stimulatory activity (25). Progesterone is a
ligand for the glucocorticoid receptor (GR) as well as the progesterone receptor (PR), and we have already demonstrated that
progesterone can differentially regulate macrophage cytokine production by using individual receptors (26). In the current study, we
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
The role of progesterone in modulating dendritic cell (DC) function following stimulation of different TLRs is relatively unknown.
We compared the ability of progesterone to modulate murine bone marrow-derived DC cytokine production (IL-6 and IL-12) and
costimulatory molecule expression (CD40, CD80, and CD86) induced by either TLR3 or TLR4 ligation and determined whether
activity was via the progesterone receptor (PR) or glucocorticoid receptor (GR) by comparative studies with the PR-specific agonist
norgestrel and the GR agonist dexamethasone. Progesterone was found to downregulate, albeit with different sensitivities, both
TLR3- and TLR4-induced IL-6 production entirely via the GR, but IL-12p40 production via either the GR or PR. Of particular
significance was that progesterone was able to significantly inhibit TLR3- but not TLR4-induced CD40 expression in bone marrowderived DCs. Stimulation of the PR (with progesterone and norgestrel) by pretreatment of DCs was found to sustain IFN regulatory
factor-3 phosphorylation following TLR3 ligation, but not TLR4 ligation. Overall, these studies demonstrate that progesterone can
differentially regulate the signaling pathways employed by TLR3 and TLR4 agonists to affect costimulatory molecule expression
and cytokine production. The Journal of Immunology, 2010, 185: 4525–4534.
4526
therefore determined, using bone marrow-derived DCs (BMDCs),
whether DC TLR-induced cytokine production and maturation
was also GR or PR dependent. In addition, we determined whether
different TLR ligands that used different signaling pathways
were equally subject to progesterone modulation. Our results indicate significant plasticity in progesterone-influenced DC activity, whereby the hormone cannot only use either the PR or GR or
both to mediate effects but also can selectively modulate the activities induced by distinct TLRs.
Materials and Methods
Compounds
DC culture and treatments for cytokine and cell viability assays
Bone marrow cells were flushed from the femurs of 8-wk-old male BALB/c
mice. These cells were cultured to produce BMDCs using GM-CSF–
enriched media obtained from 363 GM-CSF myeloma cells, which allows
differentiation of bone marrow stem cells to immature DCs (27). BMDCs
were grown in 75 cm3 tissue culture flasks (TPP, Trasadingen, Switzerland)
in culture medium containing RPMI 1640 (Life Technologies, Paisley,
U.K.), 10% heat-inactivated FCS (Harlan Sera-Lab, Loughborough, U.K.),
10% GM-CSF–enriched medium, and 2 mM L-glutamine (GIBCO, Invitrogen, Paisley, U.K.), and incubated at 37˚C and 5% CO2 for 8 d. This
method produces a large number of CD11c+ DCs largely free from contamination with other cell types as previously described (27). BMDCs
were then seeded at 5 3 105 cells/ml on 96-well microtiter tissue culture
plates at 100 ml volumes and incubated under standard conditions for 48 h.
BMDCs were then activated with either LPS (50 ng/ml or 800 ng/ml) or
poly I:C (50 ng/ml) in 50 ml volumes.
Cells were simultaneously treated with medium alone, medium containing solvent vehicle controls (either chloroform or ethanol accordingly),
or hormones. Progesterone and norgestrel were added in 50 ml volumes at
0.122–62.5 mM. Dexamethasone was added in 50 ml volumes at 0.122–
62.5 nM. Cultures were incubated at 37˚C and 5% CO2 for 72 h. After this
time, supernatants were collected from each triplicate well and stored at
220˚C for subsequent determination of IL-6, IL-12p40, and IL-12p70.
For cell viability assays, 20 ml alamarBlue (Biosource, Nivelle, Belgium)
was added to the culture 48 h before experiment termination.
DC culture and treatments for maturation marker assays
Bone marrow cells were collected from BALB/c femurs as previously
described and centrifuged at 200 3 g for 10 min at 4˚C. Cell numbers were
counted and diluted to 2.5 3 105 cells/ml preseeding at 2 ml volumes onto
six-well plates (Nunc, Roskilde, Denmark). This procedure allows minimal
disruption of DCs that could otherwise induce the nonspecific upregulation
of maturation markers prior to experimental treatments. Following 7 d of
culture, BMDCs were pretreated with progesterone (62.5 mM), norgestrel
(62.5 mM), or dexamethasone (62.5 nM) for 24 h preaddition of LPS (100
ng/ml) or poly I:C (10 mg/ml). BMDCs were then left to incubate for 16 h
at 37˚C and 5% CO2.
Abs
For ELISAs, the following capture Abs were used: anti-mouse mAb IL-6
(IgG1, clone MP5-20F3; catalog number 554400), anti-mouse mAb IL-10
(IgG1, clone JES5-2A5; catalog number 551215), anti-mouse mAb IL12p40 (IgG1, clone 15.6; catalog number 551219), and anti-mouse mAb
IL-12p70 (IgG2b, clone 9AG; catalog number 554658). Abs used for detection were biotin-labeled rat anti-mouse anti-cytokine mAbs for IL-6
(IgG2a, clone MP5-32C11; catalog number 554402), IL-10 (IgM, clone
SXC-1; catalog number 554423), and IL-12p40 (IgG2a, clone C17.8; catalog
number 554476). For flow cytometry analysis, the anti-mouse Abs used were
PE-labeled anti-CD11c (IgG2b, clone S-HCL-3; catalog number 333149),
FITC-labeled anti-CD40 (IgG2a, clone 3/23; catalog number 553790),
FITC-labeled anti-CD80 (IgG2, clone 16-10A1; catalog number 553768),
and FITC-labeled anti-CD86 (IgG2a, clone GL-1; catalog number 553691).
All Abs were purchased from BD Pharmingen (San Diego, CA).
Cytokine assays
The concentration of IL-6, IL-10, IL-12p40, and IL-12p70 present in cell
culture supernatants was assayed by sandwich ELISA. Plates were coated
with capture anti-mouse mAbs IL-6, IL-10, IL-12p40, and IL-12p70 as
described above. Recombinant murine cytokines IL-10, IL-12p40, and IL12p70 were purchased from BD Pharmingen, whereas recombinant murine IL-6 was purchased from Bender MedSystems (Vienna, Austria). The
detecting Abs used were biotin-labeled rat anti-mouse mAbs (BD Pharmingen). The conjugate used for IL-6, IL-10, and IL-12p40 detection was
streptavidin-alkaline phosphatase (BD Pharmingen). IL-12p70 was detected by addition of streptavidin-HRP (BD Pharmingen). IL-6, IL-10, and
IL-12p40 ELISAs were completed by addition of p-nitrophenyl phosphate
(Sigma-Aldrich) in glycine buffer and absorbances measured at 450 nm
using a SpectraMax 190 microtiter plate spectrophotometer (Molecular
Devices, Sunnyvale, CA) and SoftMax Pro 3.0 software (Molecular
Devices). The substrate used for IL-12p70 detection was 1% 3, 39, 5, 59tetramethylbenzidine (Sigma-Aldrich) in sodium acetate buffer at pH 5.5
containing 0.0075% hydrogen peroxide (BDH, Toronto, Ontario, Canada).
This reaction was stopped by addition of 10% sulphuric acid.
Surface marker staining
Postincubation, cells were washed and stained with the Pan marker antiCD11c and incubated with the appropriate surface marker at 4˚C for 1 h. Abs
used are as described above. Cells were washed, and fluorescence analysis
was performed using the BD FACSCanto flow cytometer (BD Biosciences,
San Jose, CA). Positive cells were gated on obtained dot plots and data
analyzed for mean fluorescence intensity of CD11c+ cells expressing the
chosen maturation marker using FACSDiva software (BD Biosciences).
Isotype-matched negative controls were used to confirm staining specificity.
Assessment of cell viability
BMDCs plated in triplicate at 5 3 105 cells/ml and treated with hormones/
stimulants as previously were tested for cell viability using alamarBlue
assay (Biosource). Reagent was added at 10% of the total well volume.
Wells containing medium only were used as a blank. After 48 h, well
contents were assayed for alamarBlue reduction by measuring the absorbance of the wells at 570 and 600 nm on a SpectroMax 190 plate reader
(Molecular Devices) and the percentage reduction of alamarBlue calculated as previously described (28).
Western blotting
Abs used in Western blotting studies, including phospho–stress-activated
protein kinase (SAPK)/JNK (Thr183/Tyr185; catalog number 9251S),
phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; catalog number
4377S), p44/42 MAPK (ERK1/2; catalog number 4695), phospho-IFN
regulatory factor (IRF)-3 (Ser396; catalog number 4947S), IRF-3 (catalog
number 4962), and phospho–NF-kB (p65; Ser536; catalog number 3031L),
were purchased from Cell Signaling Technology (Beverly, MA). Abs
raised against IkBa (Santa Cruz Biotechnology, Santa Cruz, CA; catalog
number 2607) and p65 (Santa Cruz Biotechnology; catalog number L0607)
were purchased from Insight Biotechnology, Middlesex, U.K., and anti–
phospho-p38 (catalog number 44-684G) was purchased from Invitrogen.
All other materials used during gel electrophoresis and Western blotting were of the highest commercial grade available and purchased from
Sigma-Aldrich.
Analysis of protein expression and/or phosphorylation in
BMDCs
BMDCs (1 3 106 cells; 1 ml) prepared on 12-well plates were exposed to
vehicles, hormones, LPS, or poly I:C as appropriate and whole cell lysates
prepared for Western blotting as described previously (29).
Data analysis, scanning densitometry, and statistical analyses
Scanning densitometry was performed on Western blots using an Epson
Perfection 164054 Scan jet and Epson twain 55.52 (32.32) Scanjet Picture
software (Epson, Long Beach, CA). All values are mean 6 SE of n = 3
experiments. Statistical significance of differences was calculated by the
one-way ANOVA followed by a Newman-Keuls test using GraphPad
Prism Version 4.0 software (GraphPad, San Diego, CA). A p value ,0.05
was accepted as significant. All other sets of data were analyzed using
a Mann–Whitney U test using Statview software (SAS Institute, Marlow,
U.K.) with a value of p , 0.05 taken as significant.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
LPS from Escherichia coli 055:B5, polyinosinic-polycytidylic acid (poly
I:C), progesterone, norgestrel, and dexamethasone were all obtained from
Sigma-Aldrich (Dorset, U.K.). Progesterone and norgestrel were initially
dissolved in 100% chloroform (BDH Laboratory Supplies, Poole, U.K.) at
a concentration of 50 mg/ml. This stock solution was further diluted with
complete medium to give a 250 mM solution for experimental use.
Dexamethasone was initially dissolved in 100% ethanol to give a 50 mM
solution. This stock solution was further diluted with complete medium to
give a 250 nM solution for experimental use.
PROGESTERONE MODULATION OF DCs
The Journal of Immunology
Results
Progesterone uses the GR to inhibit TLR3- and TLR4-induced
IL-6 production by BMDCs
2 nM; Fig. 1F), again indicating that the TLR engaged determines
sensitivity to hormone treatment and activity via the GR.
Progesterone uses both the GR and PR to inhibit TLR3- and
TLR4-induced total IL-12 production (p40) by BMDCs
The ability of BMDCs to produce total IL-12 (p40) following
stimulation with LPS or poly I:C was first confirmed using a simple
dose-response experiment (0.012–25 mg/ml LPS/6.25–100 mg/ml
poly I:C; data not shown). Totals of 50 ng/ml LPS and 50 mg/ml
poly I:C were identified as the most suitable stimulant concentrations for use in studies with hormones because they allowed
both potentiation and inhibition of IL-12 (p40) production to
be assessed.
Progesterone induced a significant reduction of IL-12p40 production by LPS-stimulated BMDCs at all concentrations tested and
had an IC50 of 1–2 mM (Fig. 2A). Progesterone also significantly
reduced IL-12p40 production from poly I:C-stimulated BMDCs at
concentrations .1 mM and had an IC50 of 0.5–1 mM. (Fig. 2D).
Norgestrel, the PR agonist, also downmodulated LPS-induced
IL-12p40 production at all concentrations tested and had an IC50
of 0.5–1 mM (Fig. 2B). Norgestrel also significantly inhibited
IL-12p40 production by poly I:C-stimulated BMDCs .31.3 mM
(Fig. 2E), this time indicating that engagement of different TLRs
is subject to different hormonal sensitivity via PR. Dexamethasone
inhibited LPS-induced IL-12p40 levels from BMDCs, with significant downregulation occurring at concentrations .2 nM, demonstrating an IC50 of 1–2 nM (Fig. 2C). Dexamethasone also
FIGURE 1. The influence of progesterone, norgestrel, and dexamethasone on IL-6 production by BMDCs stimulated with 800 ng/ml LPS or 50 mg/ml
poly I:C for 72 h. Cells were cultured with LPS or poly I:C in the presence of progesterone (A, D), norgestrel (B, E), or dexamethasone (C, F). Unstimulated
BMDCs failed to produce detectable levels of IL-6 (nd). Unstimulated cells treated with the solvent vehicle of 0.04% chloroform (progesterone and
norgestrel) or 0.0001% ethanol (dexamethasone) produced undetectable or very low levels of IL-6 (data not shown). Stimulated control cell cultures were
treated with 800 ng/ml LPS or 50 mg/ml poly I:C (closed bars). Stimulated control cell cultures treated with 800 ng/ml LPS or 50 mg/ml poly I:C and either
0.04% chloroform or 0.0001% ethanol produced levels of IL-6 that were comparable to stimulated cells treated with LPS or poly I:C 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. pp , 0.05 compared with stimulated controls using a Mann–Whitney U test.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
The ability of BMDCs to produce IL-6 following stimulation with
LPS or poly I:C was first confirmed using a simple dose-response
experiment (0.012–25 mg/ml LPS/6.25–100 mg/ml poly I:C; data
not shown). Totals of 800 ng/ml LPS and 50 mg/ml poly I:C were
identified as the most suitable stimulant concentrations for use in
studies with hormones because they allowed both potentiation and
inhibition of IL-6 production to be assessed.
Once the assays had been standardized, we determined whether
progesterone, norgestrel, and dexamethasone could modulate LPSor poly I:C-induced IL-6 production by BMDCs. Over a number of
experiments (n = 3), progesterone caused a significant inhibition
of LPS-induced IL-6 production at 62.5 mM and 31.25 mM (Fig.
1A). Progesterone also significantly inhibited poly I:C-induced
IL-6 production, but at lower concentrations .0.5 mM (IC50 of
0.5–1 nM; Fig. 1D), indicating that IL-6 production via TLR3
ligation was more sensitive to progesterone treatment than TLR4
ligation. Whereas norgestrel, the PR agonist, did not modulate
IL-6 production by LPS- or poly I:C-stimulated BMDCs (Fig. 1B,
1E), the GR agonist dexamethasone demonstrated a greater capacity to reduce IL-6 levels compared with progesterone. Dexamethasone treatment using concentrations above 2 nM resulted in
significant downregulation of LPS-induced IL-6 (IC50 of 7.8–15.7
nM; Fig. 1C), and at all concentrations tested, dexamethasone
reduced poly I:C-induced IL-6 production by BMDCs (IC50 of 1–
4527
4528
PROGESTERONE MODULATION OF DCs
caused significant downregulation of poly I:C-induced IL-12p40
production at all concentrations tested and had an IC50 of 0.2–0.5
nM (Fig. 2F).
Progesterone, norgestrel, and dexamethasone have no effect on
the viability of stimulated BMDCs
alamarBlue, a nonspecific stable dye that is used to assess cell
viability and cell proliferation by measuring the reducing environment of the proliferating cell, was employed to determine
whether the effects of hormones on BMDC cytokine production
were due to a specific effect on signaling pathways or a nonspecific
mechanism caused by cell death. Although stimulation with LPS
and poly I:C significantly upregulated mitochondrial activity as
compared with control cultures treated with medium alone, treatment with hormones had no effect on cell viability or proliferation
as compared with LPS- or poly I:C-stimulated controls (data not
shown). In addition, BMDCs stimulated with LPS or poly I:C and
treated with progesterone, norgestrel, or dexamethasone failed to
produce detectable levels of IL-10 (not shown).
The ability of progesterone to downmodulate CD40, CD80,
and CD86 expression depends on the TLR engaged
The ability of BMDCs to express the costimulatory molecules
CD40, CD80, and CD86 following stimulation with LPS or poly
I:C was first confirmed using a simple dose-response experiment
(62.5–1000 ng/ml LPS/6.25–100 mg/ml poly I:C; data not shown).
Totals of 100 ng/ml LPS and 10 mg/ml poly I:C were identified as
the most suitable stimulant concentrations for use in studies with
hormones because they allowed both potentiation and inhibition of
CD40, CD80, and CD86 expression to be assessed.
BMDCs were pretreated with progesterone, norgestrel, or dexamethasone for 24 h to investigate whether these hormones had
any effect on expression of CD40, CD80, and CD86 (Fig. 3)
following stimulation with LPS (Fig. 3) or poly I:C (Fig. 4).
Progesterone, norgestrel, and dexamethasone did not have an effect on LPS-induced expression of CD40 by BMDCs (Fig. 3A).
However, progesterone had a slight but significant inhibitory effect
on BMDC expression of CD80 and CD86 in comparison with
LPS-stimulated control cultures (Fig. 3B, 3C). Norgestrel had
no significant effect on LPS-induced BMDC expression of CD40
(Fig. 3A), CD80 (Fig. 3B), or CD86 (Fig. 3C). Dexamethasone, in
contrast, induced a significant reduction in expression of CD86
(Fig. 3C) in comparison with LPS controls but not CD40 or CD80
(Fig. 3A, 3B). By comparison with TLR4 ligation, progesterone
demonstrated significant downregulation of poly I:C-induced
BMDC expression of CD40, CD80, and CD86 (Fig. 4). Dexamethasone also inhibited poly I:C-induced BMDC’s CD40, CD80,
and CD86 (Fig. 4) expression in comparison with poly I:C-treated
controls. Norgestrel had no significant effect on poly I:C-induced
expression of CD40 (Fig. 4A). In contrast to TLR4 ligation, it did
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 2. The influence of progesterone, norgestrel, and dexamethasone on IL-12p40 production by BMDCs stimulated with 50 ng/ml LPS or 50 mg/
ml poly I:C for 72 h. Cells were cultured with LPS or poly I:C in the presence of progesterone (A, D), norgestrel (B, E), or dexamethasone (C, F).
Unstimulated BMDCs produced low levels of IL-12p40 (open bars). Unstimulated cells treated with the solvent vehicle of 0.04% chloroform (progesterone
and norgestrel) or 0.0001% ethanol (dexamethasone) produced undetectable or very low levels of IL-12p40 (data not shown). Stimulated control cell
cultures were treated with 50 ng/ml LPS or 50 mg/ml poly I:C (closed bars). Stimulated control cell cultures treated with 50 ng/ml LPS or 50 mg/ml poly I:C
and either 0.04% chloroform or 0.0001% ethanol produced levels of IL-12p40 that were comparable to stimulated cells treated with LPS or poly I:C 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. pp , 0.05 compared with stimulated controls using a Mann–Whitney U test.
The Journal of Immunology
4529
significantly downregulate expression of CD80 and CD86 (Fig.
4B, 4C). Plots showing the mean fluorescence intensities for Figs.
3 and 4 are shown in Supplemental Fig. 1.
Ligation of the PR sustains IRF-3 phosphorylation following
TLR3 ligation
TLR3- and TLR4-stimulated cellular responses are well recognized
as being mediated via recruitment of a number of signaling cascades: classical p42/44 MAPK, p38 MAPK, JNK, NF-kB, and
IRF-3/7 pathways. To examine further the effects of steroid hormones on TLR3- and TLR4-mediated signaling events that may
precede cytokine expression, experiments were constructed to measure the expression and/or phosphorylation of key protein components of these cascades following exposure of cells to LPS or
poly I:C over an acute time frame of 0–8 h. Although both LPS and
poly I:C were effective activators (5–120 min) of the p42/44
MAPK, p38 MAPK, and JNK phosphorylation and parallel IkBa
degradation and p65 RelA phosphorylation in BMDCs, cells pretreated (1 h) with the steroid hormones progesterone, norgestrel,
and dexamethasone did not influence phosphorylation of any of the
MAPK/SAPKs as assessed by Western blotting (data not shown).
Furthermore, no modulation of p65 phosphorylation and IkBa degradation within the NF-kB cascade were observed in cells pre-
treated with the same hormones and exposed to poly I:C or LPS for
5–120 min (data not shown).
It was observed that both poly I:C and LPS were also able to
activate, in parallel to the pathways detailed above, the IRF-3
cascade, as measured by Western blotting using antisera specific
for the phosphorylation of Ser396 in the C terminus of IRF-3.
Pretreatment of cells with progesterone or norgestrel for 1 h
prior to addition of agonist prolonged poly I:C-stimulated IRF-3
phosphorylation in BMDCs significantly at 2 h postexposure
to poly I:C (Fig. 5). Pretreatment of cells with dexamethasone,
in contrast, had minimal effect upon poly I:C-stimulated IRF-3
phosphorylation over a similar time course but inhibited agoniststimulated IRF-3 phosphorylation at later time points (4 and 8 h;
Fig. 5). Pretreatment (1 h) with progesterone, norgestrel, or dexamethasone did not influence LPS-stimulated IRF-3 phosphorylation in BMDCs, again, as assessed by Western blotting (Fig. 6).
Discussion
It is well documented that much of the immunomodulation occurring
during pregnancy is due to alterations in sex hormone levels,
particularly progesterone (6, 30). Given the critical role that DCs
play in orchestrating immune responses and their significant pre-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 3. LPS-induced BMDC expression of CD40
(A), CD80 (B), and CD86 (C) preceded by preincubation
with the hormones progesterone, norgestrel, and dexamethasone. Cells were preincubated in the presence or
absence of progesterone, norgestrel, and dexamethasone
for 24 h followed by a 16-h stimulation with LPS.
Unstimulated cells received medium alone. Unstimulated cells treated with the solvent vehicle of 0.04%
chloroform (progesterone and norgestrel) or 0.001%
ethanol (dexamethasone) produced levels of CD40,
CD80, or CD86 that were comparable to medium controls (not shown). Stimulated control cells were treated
with 100 ng/ml LPS. Stimulated controls receiving 100
ng/ml LPS and either 0.04% chloroform or 0.0001%
ethanol produced equivalent levels of CD40, CD80, and
CD86 compared with stimulated cells receiving LPS
alone. pp , 0.05 compared with stimulated controls
using a Mann–Whitney U test.
4530
PROGESTERONE MODULATION OF DCs
sence at the fetal–maternal interface (11, 31), the current study
investigated the role of progesterone in modulating DC function. As
a result, a number of key findings have arisen from our investigations: first, that the ability of progesterone to downregulate
BMDC inflammatory cytokine production and costimulatory molecule expression depends in large part on what TLR is engaged;
second, progesterone downmodulates some BMDC TLR-induced
inflammatory mediators via the GR alone and others via the PR
and the GR; and third, PR agonists sustain IRF-3 phosphorylation
following TLR3 ligation but not TLR4 ligation.
In the current study, we demonstrate that TLR3- but not TLR4induced CD40 expression can be downmodulated by progesterone.
In addition, the induction of IL-6 via TLR3 ligation is an order of
magnitude more sensitive to inhibition by progesterone than IL-6
produced via TLR4 ligation. That DC TLR-induced activity can be
differentially sensitive to progesterone depending on the TLR
engaged is likely a result of the subtly different signaling pathways
associated with TLR4 and TLR3 (32). Thus, although both TLRs
can use MyD88-independent pathways, TLR3-induced NF-kB
activation is entirely TRIF dependent (33), and, unlike other
TLRs that use IL-1R–associated kinase to activate NF-kB, TLR3induced NF-kB activation is receptor interacting protein kinase
dependent (34). In addition, the TRIF-related adaptor molecule
TRAM is an essential part of and restricted to the TLR4 MyD88independent pathway (35). Recent studies have demonstrated that
not only does TLR3 mediate a more potent antiviral response than
TLR4 (33), but also that TLR3 contributes critically to the debilitating effects of a damaging host inflammatory response as a
result of influenza A infection (36), and, despite a higher viral
production in the lungs, mice deficient in TLR3 had a significant
survival advantage. Consequently, with regard to the well being
of the fetus, there could well be an evolutionary advantage in
the apparent enhanced sensitivity of TLR3-induced inflammatory
processes to downmodulation by progesterone.
It has been reported that women who use the progesterone
contraceptive depot medroxyprogesterone acetate are at greater
risk of contracting the HIV and show increased incidence of HSV
reactivation and viral shedding (37, 38). A recent study has suggested that progesterone could be limiting the antiviral response
by inhibiting TLR7- and TLR9-induced IFN-a production by
plasmacytoid DCs (39). TLR3 is also known to be a vital component of antiviral responses by binding dsRNA and ssRNA molecules to induce not only IFNs but also proinflammatory cytokines, which have a key role to play in antiviral defense (32, 40,
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Poly I:C-induced BMDC expression of
CD40 (A), CD80 (B), and CD86 (C) preceded by
preincubation with the hormones progesterone, norgestrel, and dexamethasone. Cells were preincubated
in the presence or absence of progesterone, norgestrel
and dexamethasone for 24 h followed by a 16-h
stimulation with poly I:C. Unstimulated cells received
medium alone. Unstimulated cells treated with the
solvent vehicle of 0.04% chloroform (progesterone and
norgestrel) or 0.001% ethanol (dexamethasone) produced levels of CD40, CD80, or CD86 that were
comparable to medium controls (not shown). Stimulated control cells were treated with 10 mg/ml poly I:C.
Stimulated controls receiving 10 mg/ml poly I:C and
either 0.04% chloroform or 0.0001% ethanol produced
equivalent levels of CD40, CD80, and CD86 compared
with stimulated cells receiving poly I:C alone. pp ,
0.05 compared with stimulated controls using a Mann–
Whitney U test.
The Journal of Immunology
4531
41). We believe that our data demonstrate a further mechanism
through which progesterone could be functioning to limit the
antiviral response by reducing TLR3-induced inflammatory responses.
The release of proinflammatory cytokines, such as IL-6 and
IL-12, as induced by infectious disease is incompatible with
successful pregnancy, and, consequently, we examined the role of
progesterone on DC release of these cytokines (6). Progesterone
reduced the production of IL-6 and IL-12 by murine BMDCs
following stimulation with either LPS or poly I:C. Estrogen has
previously been shown to reduce cytokine and chemokine production following TLR3 stimulation in a human cell line (42).
However, to our knowledge, this is the first report to demonstrate
the role of progesterone in modulating TLR3-induced pathways
of IL-6 and IL-12 induction. Previous reports have described
a reduction in IL-12 levels following progesterone treatment of
lymphocytes from pregnant mice and murine splenic-derived
DCs (25, 43). However, the observed reduction in IL-6 production
is in contrast to studies carried out by Huck and colleagues (23),
who failed to show any significant effects of progesterone on production of this cytokine. Indeed, Yang et al. (25) reported increased
levels of intracellular IL-6 in murine DCs following progesterone
treatment, although this study differed from ours in that cells were
left unstimulated and were derived from spleen and not bone marrow. However, with regard to a successful pregnancy, it is the regulation of excessive inflammatory responses that is crucial.
Progesterone functions by binding to both the GR and PR
(44–46). Using the receptor-specific agonists dexamethasone and
norgestrel, we demonstrate in this study the ability of progesterone
to differentially use these receptors to cause downregulation of
IL-6 and IL-12 production. Whereas progesterone mediated a
reduction in TLR4- and TLR3-induced IL-6 production exclusively through the GR, inhibition of IL-12 production was mediated through the GR and PR. Although it has been shown that
glucocorticoids and progesterone can induce upregulation of the
anti-inflammatory cytokine IL-10 in human DCs (46), our studies
(results not shown) as well as one using rat-derived DCs treated
with progesterone did not show an upregulation of IL-10 (47). The
timing of LPS treatment relative to the seeding of DC cultures
in preparation of stimulation experiments is critical in defining
secretion patterns of IL-12 and IL-10 that likely explain the differences between such studies (48). Nonetheless, we show that
progesterone can reduce IL-12 levels in a manner independent
of IL-10.
Our studies provide further evidence of immunomodulatory activity through the PR by using the PR-specific agonist norgestrel.
In addition, the action of hormones had no effect on cell metabolism in the current study as shown by a lack of any alteration to
alamarBlue reduction in treated cells. Therefore, progesterone is
directly modulating signaling pathways, leading to the production
of IL-6 and IL-12 by differential usage of the GR and PR. Immunomodulatory actions through the GR have been well documented. These include its ability to interact with the transcription
factor NF-kB (49). The PR has also been reported to physically
interact with NF-kB, linked to cytokine production (49, 50).
However, it would seem unlikely that the PR is inhibiting IL-12
production in this manner, as it would then be expected to induce
downregulation of IL-6. It has been suggested that steroid hor-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. The effect of progesterone, norgestrel, and dexamethasone on poly I:C-stimulated IRF-3 phosphorylation in BMDCs. BMDCs were treated
with vehicle, progesterone (62.5 mM), norgestrel (62.5 mM), or dexamethasone (62.5 nM) for 1 h pre-exposure to 25 mg/ml poly I:C for the indicated times.
Whole-cell extracts were prepared and IRF-3 phosphorylation and expression assessed by Western blotting using specific Abs as detailed in the Materials
and Methods section. Results in A are representative of three independent experiments, and associated data for experiments examining pretreatment with
progesterone, norgestrel, and dexamethasone were quantified by scanning densitometry and are depicted in B–D, respectively (n = 3).
4532
PROGESTERONE MODULATION OF DCs
mone receptors can differentially modulate genes at the promoter
level, which could explain our observations (50, 51). Alternatively,
divergences upstream of NF-kB in the individual signaling pathways leading to the synthesis of IL-6 and IL-12 may be targeted
by norgestrel to result in the specific inhibition of IL-12.
Related to these points, we sought to characterize further the
molecular mechanisms that underlie PR-mediated inhibition/suppression of LPS- and poly I:C-stimulated cytokine production.
In this study, as a first step toward pursuing the potential role of
signaling cascades in these processes, we examined the modulation
of key signaling molecules recognized to be sensitive to TLR3/4
activation (52). PG, norgestrel, and dexamethasone collectively
did not influence the phosphorylation status of the MAPKs and the
activation of the NF-kB cascade, commonly engaged by this receptor superfamily. This suggested these pathways were not influenced by steroid hormone pretreatment. It must be noted,
however, that in this study, experiments were limited to examination of the signaling cascades at the level of the MAPK/SAPKs
and the p65/IkBa complex directly. Further investigation is required to determine whether agonists at the GR and PR influence
events further downstream in the cascade—in particular, nuclear
functions.
It was noted, however, that progesterone and norgestrel pretreatment led to sustained IRF-3 phosphorylation that was specific
to the poly I:C–TLR3 response; this was not apparent for LPS–
TLR4 signaling. Numerous studies have described that elevated
IRF-3 activation leads to enhanced antiviral responses, including
gross expression of IFNs (53). Therefore, sustained phosphorylation may imply an expected elevation of cytokine production.
More recently, however, experiments in macrophages (54) have
detailed reduced expression of IL-12 p35 in response to viral infection due to constitutive activation of IRF-3 and its function as
a repressor of transcription via binding to bp 2172 to 2122 of the
p35 promoter (53). Whether this regulatory function is apparent in
BMDCs and can be correlated with IRF-3 protein phosphorylation
status remains to be determined.
The mechanism by which poly I:C-stimulated IRF-3 phosphorylation is sustained following progesterone and norgestrel
pretreatment also remains to be characterized thoroughly. This
may be a consequence of PR/GR-stimulated elevation of cellular
kinase activity; potentially via the inducible isoform of IkB kinase
and/or TRAF-associated NF-kB activator-binding kinase 1 as the
recognized upstream kinase regulators of this transcription factor (52). Alternatively, there may be a decrease in phosphatasecatalyzed dephosphorylation of IRF-3. Again, relevant candidate
phosphatases remain to be identified. GR-mediated immunosuppression has also been reported to be reliant on a glucocorticoid
receptor-interacting protein 1–IRF-3 interaction (54) in the formation of a protein complex capable of repressing, although the
influence of IRF-3 phosphorylation status upon this interaction has
not been detailed. Overall, from our studies to date, it appears that
these effects are specific to TLR3-mediated responses and as such
may represent a mechanism by which progesterone and norgestrel
mediate modulation of IRF-3 phosphorylation and therefore may
potentially influence transcription factor activation, downstream
cytokine production, and costimulatory molecule expression.
In addition to the vital role that DCs play in initiating the innate
immune response by production of cytokines and chemokines,
these cells are critically important in triggering an effective adaptive immune response. During the maturation process, DCs up-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. The effect of progesterone, norgestrel, and dexamethasone on LPS-stimulated IRF-3 phosphorylation in BMDCs. BMDCs were treated with
vehicle, progesterone (62.5 mM), norgestrel (62.5 mM), or dexamethasone (62.5 nM) for 1 h pre-exposure to 1 mg/ml LPS for the indicated times. Wholecell extracts were prepared and IRF-3 phosphorylation and expression assessed by Western blotting using specific Abs as detailed in the Materials and
Methods section. Results in A are representative of three independent experiments, and associated data for experiments examining pretreatment with
progesterone, norgestrel, and dexamethasone were quantified by scanning densitometry and are depicted in B–D, respectively (n = 3). pp , 0.05, statistically significant versus agonist alone.
The Journal of Immunology
Disclosures
The authors have no financial conflicts of interest.
References
1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392: 245–252.
2. Belz, G., A. Mount, and F. Masson. 2009. Dendritic cells in viral infections.
Handb. Exp. Pharmacol. 188: 51–77.
3. Sher, A., E. Pearce, and P. Kaye. 2003. Shaping the immune response to parasites: role of dendritic cells. Curr. Opin. Immunol. 15: 421–429.
4. Ni, K., and H. C. O’Neill. 1997. The role of dendritic cells in T cell activation.
Immunol. Cell Biol. 75: 223–230.
5. Schuurhuis, D. H., N. Fu, F. Ossendorp, and C. J. Melief. 2006. Ins and outs of
dendritic cells. Int. Arch. Allergy Immunol. 140: 53–72.
6. Piccinni, M.-P., E. Maggi, and S. Romagnani. 2000. Role of hormone-controlled
T-cell cytokines in the maintenance of pregnancy. Biochem. Soc. Trans. 28: 212–
215.
7. Raghupathy, R., and J. Kalinka. 2008. Cytokine imbalance in pregnancy complications and its modulation. Front. Biosci. 13: 985–994.
8. Koch, C. A., and J. L. Platt. 2007. T cell recognition and immunity in the fetus
and mother. Cell. Immunol. 248: 12–17.
9. Wilczynski, J. R., J. Kalinka, and M. Radwan. 2008. The role of T-regulatory
cells in pregnancy and cancer. Front. Biosci. 13: 2275–2289.
10. Bachy, V., D. J. Williams, and M. A. Ibrahim. 2008. Altered dendritic cell
function in normal pregnancy. J. Reprod. Immunol. 78: 11–21.
11. Kammerer, U., A. Kruse, G. Barrientos, P. C. Arck, and S. M. Blois. 2008. Role
of dendritic cells in the regulation of maternal immune responses to the fetus
during mammalian gestation. Immunol. Invest. 37: 499–533.
12. Dörr, H. G., A. Heller, H. T. Versmold, W. G. Sippell, M. Herrmann,
F. Bidlingmaier, and D. Knorr. 1989. Longitudinal study of progestins, mineralocorticoids, and glucocorticoids throughout human pregnancy. J. Clin. Endocrinol. Metab. 68: 863–868.
13. Tai, P., J. Wang, H. Jin, X. Song, J. Yan, Y. Kang, L. Zhao, X. An, X. Du,
X. Chen, et al. 2008. Induction of regulatory T cells by physiological level estrogen. J. Cell. Physiol. 214: 456–464.
14. Beagley, K. W., and C. M. Gockel. 2003. Regulation of innate and adaptive
immunity by the female sex hormones oestradiol and progesterone. FEMS
Immunol. Med. Microbiol. 38: 13–22.
15. Hughes, G. C., and E. A. Clark. 2007. Regulation of dendritic cells by female sex
steroids: relevance to immunity and autoimmunity. Autoimmunity 40: 470–481.
16. Amano, Y., S. W. Lee, and A. C. Allison. 1993. Inhibition by glucocorticoids of
the formation of interleukin-1 a, interleukin-1 b, and interleukin-6: mediation by
decreased mRNA stability. Mol. Pharmacol. 43: 176–182.
17. Elftman, M. D., C. C. Norbury, R. H. Bonneau, and M. E. Truckenmiller. 2007.
Corticosterone impairs dendritic cell maturation and function. Immunology 122:
279–290.
18. Mainali, E. S., and J. G. Tew. 2004. Dexamethasone selectively inhibits differentiation of cord blood stem cell derived-dendritic cell (DC) precursors into
immature DCs. Cell. Immunol. 232: 127–136.
19. Matyszak, M. K., S. Citterio, M. Rescigno, and P. Ricciardi-Castagnoli. 2000.
Differential effects of corticosteroids during different stages of dendritic cell
maturation. Eur. J. Immunol. 30: 1233–1242.
20. Pan, J., D. Ju, Q. Wang, M. Zhang, D. Xia, L. Zhang, H. Yu, and X. Cao. 2001.
Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class
II pathway. Immunol. Lett. 76: 153–161.
21. Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. E. Leone, C. Socci,
and V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentiation and maturation. J. Immunol. 162: 6473–6481.
22. Tuckermann, J. P., A. Kleiman, K. G. McPherson, and H. M. Reichardt. 2005.
Molecular mechanisms of glucocorticoids in the control of inflammation
and lymphocyte apoptosis. Crit. Rev. Clin. Lab. Sci. 42: 71–104.
23. Huck, B., T. Steck, M. Habersack, J. Dietl, and U. Kämmerer. 2005. Pregnancy
associated hormones modulate the cytokine production but not the phenotype of
PBMC-derived human dendritic cells. Eur. J. Obstet. Gynecol. Reprod. Biol.
122: 85–94.
24. Liang, J., L. Sun, Q. Wang, and Y. Hou. 2006. Progesterone regulates mouse
dendritic cells differentiation and maturation. Int. Immunopharmacol. 6: 830–838.
25. Yang, L., X. Li, J. Zhao, and Y. Hou. 2006. Progesterone is involved in the maturation of murine spleen CD11c-positive dendritic cells. Steroids 71: 922–929.
26. Jones, L. A., J. P. Anthony, F. L. Henriquez, R. E. Lyons, M. B. Nickdel,
K. C. Carter, J. Alexander, and C. W. Roberts. 2008. Toll-like receptor-4mediated macrophage activation is differentially regulated by progesterone via
the glucocorticoid and progesterone receptors. Immunology 125: 59–69.
27. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Rössner, F. Koch, N. Romani, and
G. Schuler. 1999. An advanced culture method for generating large quantities of
highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:
77–92.
28. McBride, J., P. R. Ingram, F. L. Henriquez, and C. W. Roberts. 2005. Development of colorimetric microtiter plate assay for assessment of antimicrobials
against Acanthamoeba. J. Clin. Microbiol. 43: 629–634.
29. MacKenzie, C. J., E. Ritchie, A. Paul, and R. Plevin. 2007. IKKalpha and
IKKbeta function in TNFalpha-stimulated adhesion molecule expression in
human aortic smooth muscle cells. Cell. Signal. 19: 75–80.
30. Thellin, O., B. Coumans, W. Zorzi, A. Igout, and E. Heinen. 2000. Tolerance to
the foeto-placental ‘graft’: ten ways to support a child for nine months. Curr.
Opin. Immunol. 12: 731–737.
31. Juretic, K., N. Strbo, T. B. Crncic, G. Laskarin, and D. Rukavina. 2004. An
insight into the dendritic cells at the maternal-fetal interface. Am. J. Reprod.
Immunol. 52: 350–355.
32. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition
of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.
Nature 413: 732–738.
33. Doyle, S. E., R. O’Connell, S. A. Vaidya, E. K. Chow, K. Yee, and G. Cheng.
2003. Toll-like receptor 3 mediates a more potent antiviral response than Tolllike receptor 4. J. Immunol. 170: 3565–3571.
34. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon, M. Kelliher,
and J. Tschopp. 2004. RIP1 is an essential mediator of Toll-like receptor 3induced NF-kappa B activation. Nat. Immunol. 5: 503–507.
35. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz,
B. Monks, P. M. Pitha, and D. T. Golenbock. 2003. LPS-TLR4 signaling to IRF3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J. Exp. Med.
198: 1043–1055.
36. Le Goffic, R., V. Balloy, M. Lagranderie, L. Alexopoulou, N. Escriou, R. Flavell,
M. Chignard, and M. Si-Tahar. 2006. Detrimental contribution of the Toll-like
receptor (TLR)3 to influenza A virus-induced acute pneumonia. PLoS Pathog. 2:
e53.
37. Leclerc, P. M., N. Dubois-Colas, and M. Garenne. 2008. Hormonal contraception
and HIV prevalence in four African countries. Contraception 77: 371–376.
38. Cherpes, T. L., J. L. Busch, B. S. Sheridan, S. A. Harvey, and R. L. Hendricks.
2008. Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector
function and induces herpes simplex virus type 1 reactivation. J. Immunol. 181:
969–975.
39. Hughes, G. C., S. Thomas, C. Li, M. K. Kaja, and E. A. Clark. 2008. Cutting
edge: progesterone regulates IFN-alpha production by plasmacytoid dendritic
cells. J. Immunol. 180: 2029–2033.
40. Komastu, T., D. D. Ireland, and C. S. Reiss. 1998. IL-12 and viral infections.
Cytokine Growth Factor Rev. 9: 277–285.
41. Marshall-Clarke, S., J. E. Downes, I. R. Haga, A. G. Bowie, P. Borrow,
J. L. Pennock, R. K. Grencis, and P. Rothwell. 2007. Polyinosinic acid is a ligand
for toll-like receptor 3. J. Biol. Chem. 282: 24759–24766.
42. Lesmeister, M. J., R. L. Jorgenson, S. L. Young, and M. L. Misfeldt. 2005.
17Beta-estradiol suppresses TLR3-induced cytokine and chemokine production
in endometrial epithelial cells. Reprod. Biol. Endocrinol. 3: 74.
43. Par, G., J. Geli, N. Kozma, P. Varga, and J. Szekeres-Bartho. 2003. Progesterone
regulates IL12 expression in pregnancy lymphocytes by inhibiting phospholipase
A2. Am. J. Reprod. Immunol. 49: 1–5.
44. Kontula, K., T. Paavonen, T. Luukkainen, and L. C. Andersson. 1983. Binding of
progestins to the glucocorticoid receptor. Correlation to their glucocorticoid-like
effects on in vitro functions of human mononuclear leukocytes. Biochem.
Pharmacol. 32: 1511–1518.
45. Stimson, W. H. 1986. Sex steroids, steroid receptors and immunity. In Hormones
and Immunity. I. Berezi, and K. Kovacs, eds. MTP Press, Lancaster, United
Kingdom. p. 93–119.
46. Sugino, N., C. M. Telleria, and G. Gibori. 1997. Progesterone inhibits 20alphahydroxysteroid dehydrogenase expression in the rat corpus luteum through the
glucocorticoid receptor. Endocrinology 138: 4497–4500.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
regulate the expression of cell surface molecules, such as CD40,
CD80, and CD86, that are important for interaction with B and
T cells and subsequent development of the adaptive immune response (1, 4). Expression of these maturation markers is significantly upregulated following treatment of BMDCs with the TLR3
ligand poly I:C and the TLR4 ligand LPS. Of particular note in
addition to the different sensitivities of these maturation factors to
progesterone depending on the particular TLR engaged was that
progesterone, but not dexamethasone or norgestrel, significantly
inhibited TLR4-induced CD80, whereas progesterone inhibited
TLR3-induced CD40 and CD80 to a significantly greater level
than ligation of either PR or GR alone. This would indicate that
progesterone can downregulate these molecules either by PR- or
GR-independent mechanisms. A non-PR and GR mechanism could
result from progesterone binding to membrane-associated receptors resulting in rapid, nongenomic effects on intracellular signaling that can subsequently affect nuclear translocation of transcription factors and protein synthesis (55, 56). This has not yet been
shown in DCs but has been demonstrated in T cells, where progesterone inhibits both Ca2+ and K+ channels and thus gene expression as induced by NFAT (57).
Therefore, overall, this study provides further verification that
progesterone has a role to play in fine-tuning the immune response.
However, more importantly, by targeting directly the inflammatory
armory of DCs, progesterone is likely vital in creating a favorable
immunological environment for fetal survival.
4533
4534
47. Butts, C. L., S. A. Shukair, K. M. Duncan, E. Bowers, C. Horn, E. Belyavskaya,
L. Tonelli, and E. M. Sternberg. 2007. Progesterone inhibits mature rat
dendritic cells in a receptor-mediated fashion. Int. Immunol. 19: 287–296.
48. Jiang, H. R., E. Muckersie, M. Robertson, H. Xu, J. Liversidge, and
J. V. Forrester. 2001. Secretion of interleukin-10 or interleukin-12 by LPSactivated dendritic cells is critically dependent on time of stimulus relative to
initiation of purified DC culture. J. Leukoc. Biol. 72: 978–985.
49. McKay, L. I., and J. A. Cidlowski. 1999. Molecular control of immune/
inflammatory responses: interactions between nuclear factor-k B and steroid
receptor-signaling pathways. Endocr. Rev. 20: 435–459.
50. Kalkhoven, E., S. Wissink, P. T. van der Saag, and B. van der Burg. 1996.
Negative interaction between the RelA(p65) subunit of NF-kappaB and the
progesterone receptor. J. Biol. Chem. 271: 6217–6224.
51. Ogawa, S., J. Lozach, C. Benner, G. Pascual, R. K. Tangirala, S. Westin,
A. Hoffmann, S. Subramaniam, M. David, M. G. Rosenfeld, and C. K. Glass.
2005. Molecular determinants of crosstalk between nuclear receptors and tolllike receptors. Cell 122: 707–721.
PROGESTERONE MODULATION OF DCs
52. Hiscott, J. 2007. Convergence of the NF-kappaB and IRF pathways in the regulation of the innate antiviral response. Cytokine Growth Factor Rev. 18: 483–490.
53. Dahlberg, A., M. R. Auble, and T. M. Petro. 2006. Reduced expression of IL-12
p35 by SJL/J macrophages responding to Theiler’s virus infection is associated
with constitutive activation of IRF-3. Virology 353: 422–432.
54. Reily, M. M., C. Pantoja, X. Hu, Y. Chinenov, and I. Rogatsky. 2006. The
GRIP1:IRF3 interaction as a target for glucocorticoid receptor-mediated immunosuppression. EMBO J. 25: 108–117.
55. Li, X., and B. W. O’Malley. 2003. Unfolding the action of progesterone
receptors. J. Biol. Chem. 278: 39261–39264.
56. Schmidt, B. M. W., D. Gerdes, M. Feuring, E. Falkenstein, M. Christ, and
M. Wehling. 2000. Rapid, nongenomic steroid actions: A new age? Front.
Neuroendocrinol. 21: 57–94.
57. Ehring, G. R., H. H. Kerschbaum, C. Eder, A. L. Neben, C. M. Fanger,
R. M. Khoury, P. A. Negulescu, and M. D. Cahalan. 1998. A nongenomic
mechanism for progesterone-mediated immunosuppression: inhibition of K+
channels, Ca2+ signaling, and gene expression in T lymphocytes. J. Exp. Med.
188: 1593–1602.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017