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IMMUNOBIOLOGY
A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during
dendritic-cell maturation
Deborah Braun, Randy S. Longman, and Matthew L. Albert
Prostaglandins, a family of lipidic molecules released during inflammation, display immunomodulatory properties in
several models. One use includes exposure of monocyte-derived dendritic cells
(DCs) to a cocktail of cytokines that contains prostaglandin E2 (PGE2) for purposes
of maturation; such cells are currently being
used for cancer immunotherapy trials. Our
analysis of the transcription profile of DCs
matured in the presence of tumor necrosis
factor ␣ (TNF␣) and PGE2 revealed a strong
up-regulation of indoleamine 2-3 dioxygen-
ase (IDO), an enzyme involved in tryptophan
catabolism and implicated in both maternal
and T-cell tolerance. Using quantitative assays to monitor levels of IDO mRNA, protein
expression, and enzyme activity, we report
that PGE2 induces mRNA expression of IDO;
however, a second signal through TNF receptor (TNF-R) or a Toll-like receptor (TLR)
is necessary to activate the enzyme. Interestingly, use of TNF␣, lipopolysaccharide, or
Staphylococcus aureus Cowan I strain (SAC)
alone does not induce IDO. The effect of
PGE2 is mediated by activation of adenylate
cyclase via the Gs-protein–coupled receptor E prostanoid-2 (EP2). A better understanding of these regulatory mechanisms
and the crosstalk between TNF-R/TLR and
EP2 signaling pathways will provide insight
into the regulation of T-cell activation by
DCs and may help to improve existing immunotherapy protocols. (Blood. 2005;106:
2375-2381)
© 2005 by The American Society of Hematology
Introduction
Prostaglandin E2 (PGE2) is a catabolite of arachidonic acid and is
generated by the sequential activity of cyclo-oxygenase (COX) and
prostaglandin E synthase.1 Produced during inflammation, PGE2 is
believed to act as a counter-inflammatory agent, modulating
inflammatory responses and helping to restore tissue homeostasis.
For example, PGE2 has been reported to suppress T-cell proliferation,2,3 inhibit macrophage and dendritic-cell cytokine production
(eg, interleukin 12 [IL-12] and tumor necrosis factor ␣ [TNF␣]4,5),
and modulate antigen presentation by down-regulating expression
of major histocompatability complex (MHC) II.6 Additionally,
PGE2 may skew CD4⫹ T cells toward a T helper cell type 2 (TH2)
phenotype7,8 and B cells toward immunoglobulin E (IgE) production.5 In other studies, PGE2 has been shown to play a role in T-cell
development9 and may be an inhibitor of apoptosis in doublepositive thymocytes.10 Consistent with these reports, expression of
various prostaglandin biosynthetic enzymes and receptors has been
detected in the thymus.11
Dendritic cells (DCs) are considered to be the only antigenpresenting cell (APC) capable of priming naive T cells, and they
are also potent stimulators of recall responses.12 Briefly, DCs exist
in the periphery as immature cells where they serve as “sentinels,”
responsible for capturing antigen. Upon maturation, DCs migrate
to the draining lymphoid organs, where they may initiate immune
responses. This ability to traffic out of peripheral tissue with
captured antigen and enter the afferent lymph is unique to the DCs,
making them the appropriate carrier of tissue-restricted antigen to
lymphoid organs for the initiation of immunity.12 Their role in
priming T cells has also prompted much interest in discovering
strategies to efficiently use DCs carrying tumor antigen for
adoptive transfer and immunization of tumor-reactive T cells. Our
understanding of DC biology, however, has not resulted in overwhelming success; few DC-based studies have reported an effect
greater than the 10% rate achieved by Coley.13-15 Given that DCs
are capable of mediating both T-cell priming as well as T-cell
inactivation (tolerance), a deeper understanding of how to counter
DC tolerogenic programs may advance our efforts to achieve tumor
immunity. One area of active investigation has been the definition
of maturation programs for DCs, in an attempt to obtain large
numbers of stimulatory DCs for purposes of immunotherapy.
On the basis of careful studies analyzing T-cell stimulatory
capacity, cell yield, DC migration studies, and safety concerns, the
majority of DC-based trials use Good Manufacturing Practice
(GMP)–grade cytokine cocktails (eg, TNF␣, IL-1␤, IL-6, and
PGE2) for the ex vivo maturation of monocyte-derived immature
DCs. The principal reason for the addition of PGE2 is based on the
observation that it regulates the migratory capacity of DCs by
sensitizing CC chemokine receptor 7 (CCR7) to its ligands, CC
chemokine ligand 19 (CCL19) and CCL21.16,17 Additionally, PGE2
enhances the effect of the maturation cocktail, making it possible to
use 20 to 100 times less TNF␣.18 In further support of its use, DCs
matured in the presence of PGE2 show no defect in the generation
of phenotypically mature DCs, the priming of naive allogeneic T
From the Institut Pasteur, Paris, France; and the Institut National de la Santé et
de la Recherche Médicale (INSERM) AV0201, Paris, France.
An Inside Blood analysis of this article appears at the front of this issue.
Submitted March 10, 2005; accepted May 12, 2005. Prepublished online as
Blood First Edition Paper, June 9, 2005; DOI 10.1182/blood-2005-03-0979.
Supported in part by grants from Fondation pour la Recherche Médicale (D.B.);
the National Institutes of Health (NIH) Medical Scientist Training Program
(MSTP) (grant GM07739) and The Albert Cass Fellowship (R.S.L.); Ligue
Nationale Contre le Cancer, Association pour la Recherche contre le Cancer,
and The Doris Duke Charitable Foundation (M.L.A.).
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
The online version of the article contains a data supplement.
Reprints: Matthew L. Albert, Institut Pasteur, 25, Rue du Dr ROUX, Paris,
France 75724; e-mail : [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
2375
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2376
BRAUN et al
cells, or the stimulation of antigen-specific CD4⫹ and CD8⫹ T-cell
responses.19,20 In prior studies, we have used TNF␣ and PGE2 to
mature DCs. Such cells possess the ability to both prime and
tolerize CD8⫹ T cells, depending on the immune context and the
route by which antigen is processed and presented.21
Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in
tryptophan catabolism. Its role in antimicrobial resistance is well
described; by actively depleting tryptophan, essential for the
growth of microorganisms, both within the infected cell and in the
surrounding milieu, IDO serves to suppress growth of invasive
bacteria. More recently, it has been established that IDO regulates
maternal tolerance and possibly more general aspects of T-cell
tolerance.22-26 IDO expression has been reported in placental
trophoblasts and interferon ␥ (IFN␥)–activated APCs (including
macrophages and DCs), reflecting its counter-inflammatory role.
An exciting advance for this field has been the discovery that IDO
is initially expressed as a proenzyme. Although little is known
regarding the biochemical signals responsible for transcriptional
activation of the IDO gene, it has been shown that reverse signaling
via B7 (CD80/CD86), as well as engagement of CD200R, serve to
regulate IDO enzyme activity.27-29
Herein, we characterize the mechanism by which IDO is
up-regulated in monocyte-derived DCs. We report that that PGE2
induces mRNA expression of IDO; however, the enzyme remains
inactive. Only in response to a second signal via TNF receptor
(TNF-R) or Toll-like receptor (TLR) do we observe bioactive IDO.
Surprisingly, use of TNF␣, lipopolysaccharide, or Staphylococcus
aureus Cowan I strain (SAC) alone does not induce IDO. This
study offers new insights into the immune-modulatory effects
of PGE2 as well as provides a better understanding as to how
IDO expression is achieved in inflammatory situations. Importantly, this work will impact the design of future DC-based
immunotherapy trials.
Materials and methods
Human subject materials
Human blood components were obtained from healthy donors (Etablissement Français du Sang [EFS], Rungis, France). Materials were stripped of
patient identifiers and shipped to Institut Pasteur in accordance with institutional
policy (no. HS2003-5720) and the tenets of the Helsinki protocol.
Reagents
TNF␣ (Endogen, Boston, MA) was used at a concentration of 100 ng/mL
and PGE2 (Sigma, St Louis, MO) at 5 ␮M, unless otherwise stated.
Lipopolysaccharide (LPS), serotype 055:B5 (Sigma) was sonicated and
used at 50 ng/mL, SAC was used at 0.0025% wt/vol (Pansorbin, CalbiochemBehring, La Jolla, CA). L-tryptophan, L-kynurenine, and forskolin (Sigma)
were used as described in “Results”; prostaglandin E receptor EP1 subtype
(EP1), EP3, and EP4 agonists (L-335677, L-826266, and L-161982,
respectively) were provided by Merck Frost & Co (Québec, Canada) and
used at a concentration of 50 ␮M; butaprost and 19(R)-OH PGE2 (EP2
agonists), sulprostone (EP1⬎⬎3 agonist) were obtained from Cayman
Chemicals, Ann Arbor, MI, and used at 0.5 to 250 ␮M. The adenylate
cyclase inhibitor SQ22536 (Biomol International, LP, Plymouth Meeting,
PA) was titrated, and the optimal concentration to inhibit forskolin was
found to be 1 mM. H-89 (Sigma) was used at 10 to 50 ␮M for the inhibition
of protein kinase A (PKA).
Isolation and preparation of dendritic cells
Monocyte-derived DCs were prepared as described. Briefly, buffy coats
were obtained from healthy donors, and peripheral blood mononuclear cells
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
(PBMCs) were isolated by sedimentation over Ficoll-Hypaque (Pharmacia
Biotech, Piscataway, NJ). CD14-enriched and CD14-depleted fractions
were prepared using CD14 Miltenyi microbeads followed by magnetic cell
sorting according to the manufacturer’s instructions (Miltenyi Biotech,
Auburn, CA). Immature DCs were prepared from the CD14⫹ fraction by
culturing cells in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF; Berlix, Seattle, WA) and IL-4 (R&D Systems,
Minneapolis, MN) for 7 days. GM-CSF (1000 U/mL) and IL-4 (500 U/mL)
were added to the cultures on days 0, 2, and 4. To generate mature DCs, the
cultures were transferred to fresh wells on day 6 or 7, and the indicated
maturation stimulus was added for an additional 1 to 2 days. At days 6 to 7,
greater than 95% of the cells were CD14⫺CD83⫺HLA-DRlo DCs. After
maturation, on days 8 to 9, 70% to 95% of the cells were of the mature
CD14⫺CD83⫹HLA-DRhi phenotype (data not shown).
Quantitative analysis of IDO mRNA expression
RNA was extracted using Trizol (Invitrogen, Carlsbad, CA), and cDNA was
synthesized from 1 to 2 ␮g RNA using oligo-dT (Roche, Indianapolis, IN)
and Superscript reverse transcriptase (Invitrogen) according to manufacturers’ instructions. IDO-specific mRNA is quantified relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or TATA box binding protein
(TBP) using the following primers: IDO forward, 5⬘-AGAGTCAAATCCCTCAGTCC-3⬘; IDO reverse, 5⬘-AAATCAGTGCCTCCAGTTCC-3⬘;
GAPDH forward, 5⬘-ACTCCACGACGTACTCAGCG-3⬘; GAPDH reverse, 5⬘-GGTCGGAGTCAACGGATTTG-3⬘; TBP forward, 5⬘-GCACAGGAGCCAAGAGTGAA-3⬘; and TBP reverse, 5⬘-TCACAGCTCCCCACCATATT-3⬘. Primers for EP receptors are described in Kamphuis et al.30
Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR)
was performed using the SYBR Green JumpStart Taq ReadyMix (Sigma)
according to the manufacturer’s instructions. The reactions were run on a
PTC200 equipped with a Chromo4 detector (MJ Research, Boston, MA).
The analyses were performed with the Opticon Monitor software version
2.03 (MJ Research). All the measures were performed in duplicate and
validated when the difference in threshold cycle (Ct) between the 2
measures was less than 0.3. Amplification and dissociation curves of
increasing amounts of total PBMC cDNA allowed validation of our assay;
dissociation curves displayed a single peak, ruling out the presence of
primer dimers or parasitic products (Supplemental Figure S1A, available on
the Blood website; see the Supplemental Figures link at the top of the online
article). The ratio of gene of interest–housekeeping gene was calculated
according to the formula: ratio ⫽ 2⫺dCt (dCT ⫽ mean Ct gene ⫺ mean Ct
housekeeping). GAPDH and TBP were used to normalize for IDO and
EP-receptor mRNA expression, respectively.
Detection of IDO protein expression
Cell lysates were prepared from 106 DCs using RIPA buffer (20 mM Tris
[tris(hydroxymethyl)aminomethane] pH 7.5, 150 mM NaCl, 10% glycerol,
1% Nonidet P-40 [[Octylphenoxy]polyethoxyethanol], Complete [Roche;
protease inhibitor cocktail]). One third of the total protein lysate was
separated on 12% or 14% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE). After transfer to polyvinylidene difluoride
(PVDF) membranes, protein loading was monitored using Ponceau Red
staining. IDO protein was detected using a rabbit polyclonal antibody (Ab)
preparation (generous gift of David Munn, Medical College of Georgia,
Augusta, GA) and anti–rabbit IgG–horseradish peroxidase (HRP; Santa
Cruz Biotechnology, Santa Cruz, CA), and visualized by chemiluminescence (ECL; Amersham, Piscataway, NJ).
Determination of IDO enzymatic activity
To monitor enzyme activity, DCs were washed and resuspended in Hanks
Buffer (HBSS) containing 100 ␮M tryptophan (Life Technologies, Grand
Island, NY) and incubated for 4 hours. Supernatants were harvested and
assayed for the presence of kynurenine, the first stable catabolite downstream of IDO. Kynurenine was detected by either high-pressure liquid
chromatography (HPLC) or using a modified spectophotometric assay.
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BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
A TWO-STEP MECHANISM FOR THE ACTIVATION OF IDO
2377
HPLC was performed according to Yong and Lau31 with minor modifications. Briefly, 40 ␮L clarified sample was injected into an Amersham
reverse phase C2/C18 column and eluted with KH2PO4 buffer (0.01 M
KH2PO4 and 0.15 mM EDTA [ethylenediaminetetraacetic acid], pH 5.0)
containing 10% methanol at a flow rate of 1.0 mL/min. The spectrophotometer was set at 254 nm to detect both kynurenine and tryptophan. Retention
time was determined empirically using standard solutions of kynurenine
and tryptophan. IDO activity is reported as the concentration of kynurenine
produced. Alternatively, kynurenine were measured spectrophotometrically.32,33 The amount of 50 ␮L of 30% trichloroacetic acid was added to
100 ␮L culture supernatant, vortexed, and centrifuged at 8000g (10 000
rpm) for 5 minutes. Volume (75 ␮L) of the supernatant was then added to an
equal volume of Ehrlich reagent (100 mg P-dimethylbenzaldehyde, 5 mL
glacial acetic acid) in a microtiter plate well (96-well format). Optic density
was measured at 492 nm, using a Multiskan MS (Labsystems, Helsinki,
Finland) microplate reader. A standard curve of defined kynurenine
concentration (0-100 ␮M) permitted analysis of unknowns.
Results
IDO expression and activity are up-regulated
during DC maturation
The phenotypic and functional changes that occur during DC
maturation are critical for generating MHC/peptide complexes and
engaging T cells; however, the molecular definition of distinct
maturation programs has only recently been considered. As previously reported by others,34 we analyzed the transcriptional profile
of monocyte-derived DCs at the various stages of differentiation
using Affymetrix gene arrays (R.S.L. and M.L.A., unpublished data
collected 2003-2004). Strikingly, we observed a greater than
100-fold increase in expression of IDO as a result of DC
maturation. This has been reproduced in 4 of 4 donors, and the
relative signal intensity of IDO mRNA expression is shown, using
GAPDH expression as an internal reference (Figure 1).
On the basis of IDO’s proposed role in immune tolerance, we
evaluated the effect of different DC maturation stimuli on IDO
expression. Despite some published data in this area,35-37 a
thorough assessment of the expression and activity of IDO as
influenced by DC maturation had yet to be performed. We first
established assays to monitor IDO mRNA expression using realtime quantitative RT-PCR (q-PCR), and Western blot. RNA was
extracted from iDCs exposed to distinct maturation stimuli, and
IDO expression was quantified as described in “Materials and
methods.” Consistent with our gene array studies, iDCs matured
with TNF␣ and PGE2 up-regulated IDO mRNA (Figure 2A). In
contrast, iDCs exposed to LPS or SAC did not express measurable
levels of IDO mRNA. Evaluation of cell lysates using an IDO
rabbit pAb (generously provided by Dr David Munn, University of
Georgia) showed good correlation between protein expression and
the mRNA levels (Figure 2B).
In addition, it was important to monitor IDO enzymatic activity,
because there has been a reported discrepancy between IDO
expression and activity, suggesting possible posttranslational regulation of the enzyme.35,38 IDO activity may be assayed by
quantifying tryptophan catabolism as well as the generation of
kynurenine (the first catabolite in the metabolic pathway). After 36
to 48 hours of exposure to the distinct maturation stimuli, the DCs
were cultured in HBSS containing 100 ␮M tryptophan. After 4
hours, the concentration of tryptophan and kynurenine was determined by HPLC. As shown, the 2 products can be easily separated,
and the area under the curve correlates with the amount of the
respective analyte (Supplemental Figure S2A). Notably, we ob-
Figure 1. Dendritic-cell maturation induces IDO expression. Extracted data from
Affymetrix gene array studies (U133A chips) is shown for the 4 experiments.
Immature dendritic cells were differentiated from monocyte precursors using GMCSF and IL-4. These cells were then exposed to TNF␣ and PGE2 for 36 hours to
generate mature dendritic cells. The raw signal intensity reflects the relative
expression of IDO (solid line) and GAPDH (dotted line). Each line indicates an
independent donor.
served robust enzyme activity in DCs matured with TNF␣ and
PGE2, but no evidence of tryptophan catabolism was detected in
iDCs or in LPS- or SAC-matured DCs (Figure 2C). In our initial
studies, IFN␥-exposed DCs served as a positive control for IDO
expression and activity. DCs matured from multiple individuals
permitted evaluation of donor variability (Table 1).
Because of our interest in monitoring IDO functional activity in
several conditions for DC stimulation, we took advantage of a
medium-throughput (96-well assay) colorimetric assay for monitoring kynurenine.33 To validate this approach, we established a
standard curve for quantifying kynurenine and compared the
experimental values obtained using the colorimetric assay with
those from the HPLC analysis. A strong correlation was observed
(Supplemental Figure S2B-D), thus allowing us to use the colorimetric assay for monitoring IDO activity.
Prostaglandin E2 is responsible for the expression
of IDO mRNA
In an attempt to define the stimuli responsible for IDO expression,
we revealed a surprising result. Similar to treatment of iDCs with
LPS or SAC, when used alone TNF␣ did not up-regulate IDO
expression. Instead, it was exposure of PGE2 that accounted for
expression of IDO mRNA (Figure 3A-B). Although we observed a
50- to 200-fold increase in transcription of IDO, PGE2-treated DCs
lacked measurable IDO activity (Figure 3C).
This finding suggests that, although PGE2 induces transcription
of the IDO gene, a second signal, such as exposure to TNF␣, is
required to achieve active IDO enzyme. Similar to TNF-R engagement, TLR ligation also induced IDO activity when used in
combination with PGE2 (Figure 4A-B). Characterization of the
pAb used suggests that it may be selective for active enzyme (data
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2378
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
BRAUN et al
Figure 3. PGE2 triggers the transcription of IDO mRNA. (A-B) IDO mRNA
expression in DCs stimulated with TNF␣, PGE2, or a combination of both stimuli was
monitored by qRT-PCR. The PCR amplification curves from a representative
experiment, performed in duplicate, are displayed (A). The same data are reported as
a ratio of IDO/GAPDH, again normalized to expression in iDCs (set to a value of 1.0).
(B) The inset represents the level of IDO mRNA as a function of the dose of PGE2.
(C) DCs exposed to the conditions described above were monitored for their IDO
activity. Kynurenine production was measured using a colorimetric assay; known
concentrations were used for the establishment of a standard curve (Supplemental
Figure S2B). Values are the mean of triplicate wells, and error bars indicate standard
deviation. Data in Figure 3 are representative of 4 experiments.
Figure 2. The maturation stimulus used influences IDO expression and activity.
Human monocyte-derived iDCs were cultured in media alone or exposed to distinct
maturation stimuli as indicated. (A) The relative expression levels of IDO were measured
using quantitative RT-PCR. IDO expression is represented as a ratio of IDO/GAPDH as
compared with iDCs (set to a value of 1.0 to normalize the data). Each bar corresponds to
the mean of all donors assayed (n ⫽ 1-7). Error bars indicate standard error of the mean
(SEM). Non-normalized data are reported in Table 1. (B) IDO protein was detected by
Western blot in cell extracts using a polyclonal rabbit anti–human IDO Ab. The cell number
from which the protein was derived was normalized prior to loading the gel; and Ponceau
Red staining of the membrane confirmed that equivalent protein content was being
analyzed (data not shown). (C) Following the different culturing conditions, DCs were
washed well and incubated for 4 hours in HBSS containing 100 ␮M tryptophan.
Supernatants were harvested, and the concentration of kynurenine was determined. The
mean concentration of kynurenine, as measured by HPLC, is represented (n ⫽ 1-6). Error
bars indicate SEM. Numeric values and the range observed in different donors are reported
in Table 1.
not shown); consequently, we are unable to determine whether
TNF-R/TLR regulates a transcriptional or posttranscriptional event.
What is clear from our data, based on Figures 2 to 4, is that there
exists a two-step regulation of IDO activity during DC maturation.
Signaling via EP2 triggers IDO expression
Eight human prostanoid receptors have been described, of which 4
bind PGE2 with high affinity (dissociation constant (Kd) in the
low-nM range). Of these 4 receptors, EP1 and EP3 are coupled to
inhibitory G proteins; whereas EP2 and EP4 signal via stimulatory
G proteins. On the basis of our microarray data and published
studies,39 only EP2 and EP4 are expressed by monocyte-derived
DCs (data not shown). We validated these findings in our culture
system using quantitative RT-PCR. As shown, iDCs express higher
levels of EP2 and low levels of EP4. After exposure to TNF␣ and
Table 1. IDO expression and activity
IDO mRNA (qPCR)*
Conditions
IDO activity (␮M kynurenine, HPLC)
n
Average
Range
SEM
n
Average
iDC
7
0.017
0.09-0.06
0.007
6
4.3
TNF␣ ⫹ PGE2
7
3.3
1.01-8.17
1.01
6
LPS
4
0.24
0.09-0.39
0.06
3
7.6
2.8-14
3.3
SAC
3
0.05
0-0.13
0.04
1
4.3
N/A
N/A
TNF␣ ⫹ PGE2 ⫹ IFN-␥
1
5.1
N/A
N/A
3
66.5
N/A indicates not applicable.
*Reported as a ratio of relative mRNA expression IDO/GAPDH. Non-normalized data are presented.
38
Range
SEM
2.8-7.7
0.8
19.3-68.9
7.6
60.4-74
4.0
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A TWO-STEP MECHANISM FOR THE ACTIVATION OF IDO
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Figure 4. TNF-R and TLR engagement triggers IDO enzymatic activity. DCs were
prepared as above, with maturation stimuli consisting of TNF␣, LPS, or SAC, either in
the presence or absence of PGE2. (A) IDO protein expression was detected by
Western blot, and (B) enzymatic activity was quantified by the colorimetric assay for
kynurenine, as described in the “Materials and methods.” Error bars indicate SEM.
PGE2, the pattern of expression is reversed (Figure 5A). Of note,
PGE2 seems to be responsible for the down-regulation of EP2,
whereas both TNF␣ and PGE2 are required to observe upregulation of EP4.
Next, we evaluated which EP receptor mediates IDO expression. This was done functionally, by exposing iDCs to agonists
specific for EP1 to EP4 in the presence of TNF␣, and IDO activity
was measured as described above. In 3 of 3 individuals, only the
EP2 agonist butaprost resulted in the induction of IDO activity
(Figure 5B). As EP2 is a G␣s-coupled receptor, we tested whether
ectopic activation of adenylate cyclase would mimic the effects of a
receptor agonist. When iDCs were treated with the adenylate
cyclase activator forskolin in combination with TNF␣, the IDO
activity was equivalent to that in TNF␣- and PGE2-matured DCs
(Figure 6A). We next directly tested the effect of adding either an
inhibitor of adenylate cyclase or the cAMP-triggered kinase PKA.
As shown, both inhibitors decreased the PGE2- and forskolininduced expression of IDO. Together, these results suggest that
during DC maturation, PGE2 acts through the EP2 receptor
expressed on iDC, which in turn activates adenylate cyclase
leading to PKA activation and an increase in the transcription of the
gene coding for IDO. The presence of a TNF-R or TLR agonist
enhances expression, and, importantly, this second signal facilitates
IDO enzymatic activity.
Discussion
A novel mechanism by which PGE2 modulates
the immune system
Like many effectors of immune regulation, the actions of PGE2 are
complex and in some instances at apparent odds. With respect to its
Figure 5. PGE2 acts via EP2 to stimulate IDO expression. (A) We monitored EP2
and EP4 mRNA expression in DCs exposed to TNF␣, PGE2, or a combination of both
stimuli. TBP was used as a reference for our qPCR studies because its expression
levels matched that of the EP-Rs. Of note, no message could be detected for EP1
and EP3 by qPCR (data not shown). (B) PGE2 was replaced by different EP agonists
during DC maturation, and IDO activity was assessed after 48 hours. L-335677,
Butaprost, L-826266, and L-161982 were used to stimulate EP1 to EP4, respectively.
All were used at a concentration of 50 ␮M. In similar experiments, sulprostone was
used as an EP1⬎⬎ EP3 agonist and 19R-hydroxy PGE2 as an EP2 agonist, with
similar results (data not shown).
effect on T cells, the data indicate that PGE2 inhibits proliferation
and skews responses toward TH2 differentiation.2,7,40 And although
inhibition of TNF␣, IL-6, and IL-12p35 in antigen-presenting cells
would be consistent with a suppressive role for PGE2,4,41 recent
studies indicate a proinflammatory activity of PGE2, based on the
induction of bioactive IL-23.42,43 PGE2 has also been shown to act
as a survival signal for thymocytes and may be critical for T-cell
development.10 Furthermore, PGE2 is critical both for the generation of migratory DCs: monocyte-derived DCs demonstrate poor
migration potential to CCR7 ligands unless matured in the presence
of PGE216; in vivo, it has been demonstrated that mouse Langerhanscell migration to the draining lymph node is dependent on PGE2
signaling via EP4.44 The stimulation of DC migration has been
interpreted as proinflammatory, because migratory DCs are required for the trafficking of antigen to the T-cell area of lymph
nodes, thus permitting the initiation of an immune response.43
We report a novel mechanism by which PGE2 acts to counter
inflammatory responses. Our study indicates that PGE2 induces
mRNA expression of IDO by signaling via the Gs-protein–coupled
receptor EP2. This in turn activates adenylate cyclase, catalyzing
the formation of cAMP and activating PKA. Evidence to support
this mechanism is provided by the use of EP-receptor analogs and
the demonstration that only the EP2 agonist, butaprost, was able to
mimic the effects of PGE2 (Figure 5). Additionally, we demonstrate that
direct stimulation of adenylate cyclase triggered IDO expression;
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BRAUN et al
Figure 6. PGE2 signals via cyclic adenosine monophosphate (cAMP) and PKA
to trigger IDO. (A) The adenylate cyclase activator forskolin mimics PGE2 activation
of EP2/4 and induces IDO activity in the presence of TNF␣. Monocyte-derived DCs
(MoDCs) were matured during 48 hours in the presence of increasing doses of
forskolin with or without TNF␣. IDO activity was assessed spectrophotometrically
after 4 hours at the end of the culture. (B) The adenylate cyclase inhibitor SQ22536
and the PKA inhibitor H-89 block IDO expression. Monocyte-derived DCs were
cultured in the presence of PGE2 or forskolin, in addition to TNF␣ for 48 hours. The
effect of inhibiting adenylate cyclase during this culture using SQ22536, as well as the
effect of the PKA inhibitor H-89, were assessed on IDO mRNA levels.
whereas, treatment with an adenylate cyclase inhibitor or a PKA
inhibitor blocked EP2 signaling and inhibited PGE2-mediated induction
of IDO (Figure 6). Although this pathway accounted for a 50- to
200-fold increase in IDO expression, to our surprise, PGE2-treated DCs
displayed no active IDO enzyme as monitored by the catabolism of
tryptophan. Only when combined with a second signal via TNF-R or a
TLR did we observe the production of kynurenine (Figures 2-4).
Although use of TNF␣, lipopolysaccharide, or SAC alone does not
induce IDO, they are required to trigger enzymatic activity in PGE2treated DCs. These findings offer the first innate effector pathway for the
activation of IDO and suggest a link between DC migration and the
acquisition of mechanisms for immune tolerance. Indeed, such a
connection pushes one to consider the possibility that migratory DCs are
programmed so that its default pathway is tolerance.
Activation of indoleamine 2,3-dioxygenase
The actions of IDO offers an intriguing mechanism for achieving
T-cell tolerance (reviewed in Mellor and Munn23); it catabolizes the
essential amino acid tryptophan, resulting in the generation of a
putative proapoptotic agent, kynurenine, as well as other downstream immunomodulatory metabolites.45,46 IDO expression by
human monocyte-derived DCs and macrophages potentiates the
ability of these cells to inhibit T-cell proliferation. Furthermore, a
role for IDO has been shown critical in achieving maternal
tolerance, suppression of T cells reactive to haplotype-mismatched
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
allografts, and the control of autoreactive T cells.23 More recently,
it was demonstrated that the ectopic expression of IDO in tumor
cells confers immune evasion.47
Although there is strong support for IDO’s role in mediating T-cell
tolerance, less is known about the regulation of IDO expression and the
mechanism of enzyme activation. The only characterized trigger of IDO
expression is IFN␥. It has been demonstrated in some cell types that
IFN␥-induced gene expression can be enhanced or antagonized when
added in combination with other cytokines. For example in eosinophils,
it has been demonstrated that GM-CSF synergizes with IFN␥, whereas
IL-3 antagonizes IFN␥-mediated IDO expression.48 With respect to
stimulators of IDO activity, they include cytotoxic T-lymphocyte
antigen 4 (CTLA-4) and CD28, which have been shown to transmit
signals to the DCs by crosslinking the costimulatory molecules B7-1/
B7-2.27 Of note, IFN␥ also appears capable of achieving active IDO
enzyme; however, its contribution may need to be reevaluated in light of
data for B7-reverse signaling.49 If we are to interpret the findings that
IFN␥ and B7-reverse signaling are critical for IDO activation, it appears
that stimulation of the adaptive immune system is required to achieve
tolerance of the adaptive immune system. In identifying PGE2 as a
trigger for IDO expression, we offer a novel means by which the innate
immune system may regulate T-cell immunity.
Although previous studies have demonstrated the possibility of
posttranslational control for IDO,48,50 we are unable to conclude
whether our observation that TNF-R and TLR ligands are required
for achieving enzymatic activity is reflective of translational
control or a still undefined posttranslational event. Nonetheless, our
results help to clarify the signals responsible for IDO expression
and activity in monocyte-derived DCs. Given the interest in using
DCs for clinical immunotherapy and the sensitivity of IDO
expression to the culturing conditions,51,52 we hope that our
findings will help clarify some of the differences observed in the
various systems that are currently in use.
Implications for DC-based immunotherapy
With respect to DC-based immunotherapy protocols, it is important
to note that many use maturation cocktails containing PGE2. As
discussed above, this is in part due to its role in sensitizing DCs to
CCR7 ligands, thus enhancing DC migration to the draining lymph
nodes. Our finding that PGE2 is also sensitizing DCs to TNF␣-mediated
IDO activation predicates caution, as we may be inducing the opposite
of what is intended. One consideration would be to segregate the distinct
effects of PGE2. Migration appears to be mediated by EP4, whereas
IDO gene expression occurs in response to EP2 stimulation. Use of
EP4-specific agonists may allow such a separation to be achieved.
Alternatively, it may be possible to include an IDO inhibitor in the DC
maturation cocktail, thus generating a mature, CCR7-responsive DC
with an inactive IDO enzyme.
In sum, our findings illustrate a novel role for PGE2 and
important insight into the regulation of IDO. With a better
understanding of these regulatory mechanisms and the crosstalk
between TNF-R/TLR and EP2 signaling pathways, we hope to
uncover new insight into the regulation of T-cell activation by DCs
and the interplay between tumors and the immune system.
Acknowledgments
We thank Drs Olivier Lantz and Michael Lotze for their review of
the manuscript and helpful scientific discussions, and Dr Nathalie
Blachére and Elisabeth Fontan for their assistance with the HPLC.
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
A TWO-STEP MECHANISM FOR THE ACTIVATION OF IDO
2381
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From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
2005 106: 2375-2381
doi:10.1182/blood-2005-03-0979 originally published online
June 9, 2005
A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity
during dendritic-cell maturation
Deborah Braun, Randy S. Longman and Matthew L. Albert
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