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RI Induces the Tryptophan Degradation Pathway Involved in Regulating T Cell Responses1
Departments of
* Dermatology and
Psychiatry, University of Bonn, Bonn, Germany; and
Institut National de la Santé et de la Recherche Médicale Unité Etablissement Français du Sang-Alsace, Strasbourg, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI is suspected to play a pivotal role in the pathophysiology
of atopic disorders such as atopic dermatitis. In search for genes
differentially regulated by FcRI on APCs, a differential cDNA bank
of receptor-stimulated and unstimulated monocytes was established. By
means of suppression subtractive hybridization, we identified
kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase
(IDO) to be overexpressed in FcRI-activated monocytes. IDO is the
rate-limiting enzyme in the catabolism of the essential amino acid
tryptophan. We show that cross-linking of FcRI on monocytes results
in low tryptophan concentrations associated with impaired T cell
stimulatory capacity. Importantly, T cell suppression could be
prevented by the addition of tryptophan or inhibition of IDO. Moreover,
stimulation of T cells by FcRI-activated monocytes was increased
compared with T cell stimulation by nonactivated monocytes if exogenous
supply of tryptophan was available. We speculate that the expression of
IDO by FcRI+ APCs in vivo allows these cells to regulate
T cell responses in atopic disorders by inhibiting or stimulating T
cell proliferation, depending on the metabolic
environment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) is involved in IgE-mediated
allergic reactions (1). Besides its constitutive
expression on mast cells and basophils, FcRI can be detected on
certain APCs of atopic individuals (2, 3). On human
monocytes, FcRI has been identified both in donors with atopic
diseases such as atopic dermatitis
(AD)4 and in
clinically healthy individuals with an atopic family background
(4). There, FcRI has been shown to mediate efficient
IgE-dependent allergen uptake and presentation to T cells
(5).
The processes that activate and promote T cell-dependent inflammation
are better known than those that limit, turn off, or prevent T cell
proliferation. APCs are thought to mediate these profound effects on T
cells where kinetics of regulatory mechanisms seem to be highly
important (6, 7). The activation of FcRI almost
immediately results in the synthesis and secretion of proinflammatory
cytokines that contribute to the establishment of allergic inflammation
(8, 9). Recently, new immunomodulatory signals have been
described by the engagement of FcRI on human monocytes: the
activation of monocytes by FcRI mediates protection of these cells
against apoptosis, thereby prolonging the survival of monocytes
(10). In contrast, FcRI-mediated IL-10 production by
monocytes prevents their differentiation into myeloid dendritic cells
(DCs) (11), which are the most potent activators of T
cells (12). Apparently, there are differences in the
mobilization of defense mechanisms by the engagement of the receptor.
To clarify functional significances of FcRI, it is essential to
exactly define and investigate donors presenting similar clinical and
cellular appearance (atopic inflammatory disease or not; FcRI, CD23
cell surface expression; serum IgE levels).
To identify differentially regulated genes by the engagement of
FcRI, suppression subtractive hybridization was conducted of
FcRI-stimulated and unstimulated monocytes from a clinically healthy
person with an atopic family background. Interestingly, besides
sequences coding for proinflammatory cytokines or chemokines, we found
one clone whose sequence corresponded to kynurenine 3-monooxygenase, an
enzyme involved in the degradation of the amino acid tryptophan.
Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the
catabolism of tryptophan converting this amino acid into kynurenine.
Kynurenine is catabolized by kynurenine 3-monooxygenase into
3-hydroxy-kynurenine and quinolinic acid (13, 14). The
induction of cellular IDO is a common antimicrobial defense mechanism
for certain cells by depleting tryptophan from intracellular pools or
local microenvironments. The restriction of available tryptophan leads
to a condition in which cells become starved for tryptophan
(15). Recently, IDO production from macrophages and DCs
stimulated with IFN-
or IFN- and CD40 ligand (CD40L) has been
demonstrated to inhibit T cell proliferation in vitro (16, 17). The functional role of IDO has been demonstrated in vivo
where tryptophan depletion in the mammalian placenta is crucial for
establishing immune tolerance during pregnancy (18).
Because AD is clinically and immunohistologically a cell-mediated
hypersensitivity reaction, positive and negative (tolerogenic)
FcRI-mediated events may induce immunomodulatory signals that
regulate lymphocyte proliferation (19).
In this study, we show that IDO and kynurenine 3-monooxygenase are
overexpressed in FcRI-activated monocytes. Functionally, this effect
seems to be time dependent starting after 
24 h of activation. Then,
these cells acquire the ability to suppress T cell proliferation in
vitro by degradation of tryptophan. T cell suppression can be prevented
by pharmacological inhibition of IDO or supplementation of tryptophan.
In comparison with nonactivated atopic monocytes, our studies imply an
increase of T cell proliferation with FcRI-activated monocytes in
vitro under nonlimiting conditions for tryptophan. This T cell
stimulatory capacity of atopic monocytes may mirror the
pathophysiological condition of atopic patients in vivo
(20).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All blood samples were obtained after informed consent from
volunteers in accordance with the local ethics committee. For the
generation of a FcRI-specific cDNA library, monocytes were isolated
by continuous flow centrifugation leukocytapheresis and counterflow
centrifugal elutriation as previously described (21).
Monocytes were determined to be >95% pure by cell surface markers.
For the following experiments, clinically healthy atopics were
determined by their positive atopic family background, i.e., family
members were known to have a history of atopic diseases such as
allergic rhinitis, allergic asthma, or AD (n = 4;
referred to as atopics). They disclosed IgE levels >100 kU/L in the
serum and were determined as positive for FcRI, when FcRI was
expressed on >15% of peripheral monocytes. Nonatopics were healthy
individuals without any atopic background (n = 4;
referred to as nonatopics); the serum IgE levels of nonatopics were
<100 kU/L and FcRI surface expression was <10%.
Flow cytometry
Surface labeling for FcRI, CD23, and CD40 was performed as
reported previously (22). Results are expressed as the
percentage of positive cells compared with the isotype control.
Stimulation of cells
Cells were incubated for 1 h with 4 µg/ml human (h)IgE or
10 µg/ml anti-FcRI mAb F(ab')2 22E7 (Dr.
J. Kochan, Department of Autoimmune Diseases, Hoffmann-LaRoche, Nutley,
NJ) or an isotype-matched control mouse (m)IgG1 at 37°C. After
washing with culture medium, 20 µg/ml rabbit anti-hIgE or 5
µg/ml goat anti-mouse IgG (GaMIgG) were added for the duration of
the culture. For RT-PCR, cells were stimulated in addition with IFN-
(1000 U/ml). Cells were incubated in culture flasks or six-well plates
(Nalge Nunc International, Roskilde, Denmark) at a density of
1 x 106 cells per milliliter unless
otherwise indicated.
Subtractive cDNA library
Monocytes were selectively stimulated by cross-linking FcRI
with hIgE and rabbit anti-human IgE as indicated above for 4
h. Polyadenylated mRNA was purified using the Oligotex mRNA kit from
Qiagen (Courtaboeuf, France) from 50 x 106
stimulated (tester population) and 150 x
106 unstimulated (driver population) monocytes.
One and 6.4 µg of poly(A)+ mRNA from stimulated
and unstimulated monocytes, respectively, were reverse transcribed and
processed by the use of PCR-Select cDNA Subtraction kit (Clontech
Laboratories, Burlingame, CA), which is based on the method of
suppression subtractive hybridization.
Cloning and analysis of the subtracted cDNA
Products from the subtracted cDNA were digested with the RsaI restriction enzyme and inserted into the EcoRV restriction site of pKS cloning vector (Stratagene, Amsterdam, The Netherlands). Plasmid DNAs were prepared (Flexiprep extraction kit; Amersham Pharmacia, Orsay, France). DNA sequencing reactions were performed using the BigDye terminator sequencing kit (PE Applied Biosystems, Fullerton, CA). Nucleic acid homology searches were performed using the basic local alignment search tool program at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD).
RT-PCR
RT-PCR was done as described previously (3).
Specific primer sequences for each gene were as follows: human
-actin forward (5'-GAGCGGGAAATCGTGCGTGACATT-3') and reverse
(5'-GATGGAGTTGAAGGTAGTTTCGTG-3') (240 bp); human tryptase forward
(5'-CTCCCTCATCCACCCCCAGT-3') and reverse
(5'-GGATCCAGTCCAAGTAGTAG-3') (616 bp); human kynurenine
3-monooxygenase forward (5'-AGGCTGTGCAGCGTTGGCAT-3') and reverse
(5'-TGCACCCCCCAACCAGTAAT-3') (400 bp); human IDO forward
(5'-CTTCCTGGTCTCTCTATTGG-3') and reverse
(5'-GAAGTTCCTGTGAGCTGGTG-3') (430 bp). Amplification was performed on a
PerkinElmer GeneAmp PCR System 9600 thermocycler (PE Applied
Biosystems). The PCR cycle numbers were 30 for tryptase and 25 for
kynurenine 3-monooxygenase and IDO. -Actin was used to normalize
specific PCR amplifications. PCR fragments were separated on 1%
agarose gels and visualized using ethidium bromide staining. Identity
of RT-PCR products was confirmed by direct sequencing.
Kynurenine assay
The measurement of kynurenine levels in the media provides evidence of tryptophan degradation and functional IDO activity. Monocytes were cultured for 24 h at a density of 1 x 106cells/well in 96-well flat-bottom plates. The cells were washed and resuspended in HBSS containing 100 µM tryptophan (Sigma-Aldrich, St. Louis, MO) for an additional 4 h. Supernatants were harvested thereafter for quantification of tryptophan and kynurenine by HPLC. For measurements of tryptophan and kynurenine in monocyte and T cell cocultures, normal culture medium supernatants were collected 24 h after the addition of T cells to monocytes.
HPLC
Samples were deproteinized by the addition of 250 µl 5% sulfosalicylic acid to 250 µl cell culture supernatant (23). After incubation for 10 min at room temperature samples were centrifuged at 13,000 x g for 10 min at 8°C. L-Tryptophan and L-kynurenine were assayed by HPLC with UV detection (UV detector SPD-10A at 254 nm; Shimadzu, Duisburg, Germany).
Proliferation assays
T cell and monocyte isolations from peripheral blood of atopics
and nonatopics were done as described (13). Monocytes
were stimulated with hIgE/anti-IgE for 24 h or left untreated and
seeded in 96-well flat-bottom plates (1 x 106
cells/well, 200 µl). After 24 h, medium was replaced and
purified autologous T cells (2 x
105cells/well) were added along with anti-CD3
mAb (100 ng/ml; clone UCHT1; Beckman Coulter, Marseille, France) in the
presence or absence of different concentrations of the IDO inhibitor
1-methyl-DL-tryptophan (1-MT; 250, 500, and 1000 µM;
Aldrich Chemicals, Milwaukee, WI). To determine the role of
IFN- and IL-10, anti-IFN- mAb (500 ng/ml) and anti-IL-10
mAb (10 µg/ml) or an equivalent isotype control were added as
indicated at the same time with the T cells. L-Tryptophan
(1 µg/10 µl, equal to 1x normal tryptophan concentration of the
medium; Sigma-Aldrich) was added to cocultures after 24, 48, and
72 h as indicated. Seventy-two hours later, T cell proliferation
was measured using overnight incubation with
[3H]thymidine (0.5 µCi/well). Apoptotic and
nonviable monocytes were determined using FITC-labeled annexin V and
propidium iodide (Roche Molecular Biochemicals, Mannheim, Germany). In
all conditions (stimulated and unstimulated), monocyte viability was
>90% after 48 h.
To study T cell proliferation without monocytes, anti-CD3 mAb was immobilized onto 96 flat-bottom tissue culture wells (10 µg/ml, 100 µl/well, according to the manufacturer’s protocol). Monocytes were activated or not for 24 h and the medium was replaced. After an additional 24 h of incubation, supernatants were collected. Sheep anti-mouse IgG magnetic beads (Dynabeads M-280; Dynal Biotech, Oslo, Norway) that were bound to mouse anti-hIL-10 mAb (3 µg/107 beads) were added to the supernatants (2 x 107 beads/ml) according to the manufacturer’s protocol. After 2 h of incubation at 4°C with gentle mixing, IL-10 was depleted by applying a magnet at least six times. Supernatants were controlled for IL-10 by ELISA and T cells were stimulated in IL-10-depleted monocytic supernatants.
Endotoxin levels in culture preparations were determined using a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) by an independent laboratory and were <0.025 IU/ml in all media.
Detection of cytokines
For ELISA, culture supernatants were centrifuged twice at 4°C
followed by immediate storage at -70°C. IL-10 production from
FcRI-activated (IgE/anti-IgE) and nonactivated monocytes was
assayed following culture of monocytes (1 x
106 cells per well; 200 µl) for 24 h,
replacement of medium, and incubation for another 24 h. For
quantification of IFN- in cocultures, supernatants were harvested
24 h after the addition of T cells. ELISA were conducted in
triplicate according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN).
IFN- bioassay
The human colonic adenocarcinoma cell line HT-29 (kindly
provided by Prof. N. Koch, Department of Zoophysiology, University of
Bonn, Bonn, Germany) was used to determine bioactive IFN-
24 h after neutralization of IFN- in cocultures. IFN-
induces the de novo synthesis of MHC class II molecules in a
concentration-dependent manner on the cell surface of HT-29 cells
(24). MHC class I molecules, which are constitutively
expressed on HT-29 cells, are sensitively up-regulated upon low doses
of IFN- (25). As a dose-response curve and positive
control, increasing amounts of human rIFN- was added in the
following concentrations: 10, 100, 250, 500, 1000, and 2000 pg/ml. As
negative control, the cells were incubated with normal culture medium.
All samples were in triplicate.
FACS analysis was performed after cultivation of these cells for 72 h. MHC class I-specific mAb W6/32 (mIgG2b, hybridoma culture supernatant), HLA-DR-specific mAb L243 (mIgG2b, hybridoma culture supernatant), and an isotype control Ab were used to analyze cellular fluorescence.
IL-10 bioassay
Ba/F3 cells, an IL-3-dependent pro-B cell line cotransfected with hIL-10R, were used to determine biologically active hIL-10 after supernatant depletion of IL-10 from activated monocytes. A standard curve with hIL-10, ranging from 10 to 100,000 pg/ml per well was prepared. Positive (10 ng/ml IL-3) and negative (culture medium) controls were included. Proliferation was done in 96-well plates in triplicate (final volume, 100 µl; 5 x 103 cells per well). After 72 h, [3H]thymidine (0.5 µCi/well) was added for another 24 h.
Data analysis
Data are mean ± SD. For T cell proliferation assays, different numbers of experiments were performed per experimental unit (donor). To account for the variable number of observations per experimental unit, the dependency of T cell proliferation on the experimental conditions was analyzed using general linear modeling techniques as implemented in the SAS procedure Genmod (SAS Institute, Cary, NC). Values of p < 0.05 are reported as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, FcRI expression on monocytes of the atopic donor of whom
the differential cDNA bank was established from has been determined by
flow cytometry. Monocytes revealed 72% FcRI expression compared
with the isotype control; the low-affinity receptor for IgE, CD23, was
found to be expressed on <10% of monocytes and CD40 expression was
>90%. The cells were stimulated for 4 h with hIgE/rabbit
anti-hIgE or left untreated. A differential cDNA bank was
constructed. About 100 clones were sequenced and analyzed for homology
in the GenBank and European Molecular Biology Laboratory
databases. One cDNA clone was found to correspond to human kynurenine
3-monooxygenase encoding mRNA, an enzyme involved in the degradation of
tryptophan along the kynurenine pathway. The overexpression of this
gene in FcRI-stimulated monocytes was verified by specific PCR run
in the tester (FcRI-stimulated) and driver (unstimulated) population
(data not shown).
IDO and kynurenine 3-monooxygenase overexpression in
FcRI-stimulated monocytes
To confirm the above results, four additional clinically healthy
individuals with an atopic family background and four nonatopics were
investigated. Monocyte surface morphology, mRNA overexpression of IDO,
the rate-limiting enzyme in the tryptophan degrading pathway, and
kynurenine 3-monooxygenase transcript expression after FcRI
cross-linking were determined. Flow cytometric analysis for FcRI,
FcRII (CD23), and CD40 of freshly isolated atopic and nonatopic
monocytes (n = 4; >90% CD14 positive) was performed
(Fig. 1
). These two groups of donors were
chosen because they differed significantly only in their FcRI
surface expression. Basophil cell contamination was ruled out by
specific RT-PCR for tryptase (data not shown).
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RI
)/goat
anti-mouse IgG, mIgG1/GaMIgG, and IFN- was analyzed by RT-PCR
and compared with unstimulated cells. Total RNA was prepared after 4
and 24 h of incubation, converted into cDNA, and subjected to PCR.
In atopic monocytes after 24 h of induction, IDO and kynurenine
3-monooxygenase transcripts were significantly overexpressed in
FcRI-stimulated cells as compared with the isotype control and
unstimulated monocytes (Fig. 2). At
4 h of induction, 10 additional cycles each were necessary to
obtain the same levels of RT-PCR products corresponding to IDO and
kynurenine 3-monooxygenase (data not shown). In nonatopic
FcRI-activated and nonactivated monocytes, no specific transcripts
were seen applying the above PCR conditions for 4 h (data not
shown). At 24 h of induction, low levels of kynurenine
3-monooxygenase transcripts could be detected in nonatopic nonactivated
and activated monocytes, which was comparable to nonactivated atopic
monocytes. This baseline expression of kynurenine 3-monooxygenase
presumably results from the activation of cells by adherence onto
plastic and/or during isolation procedures. In contrast, IFN-
induced similar kynurenine 3-monooxygenase transcript band intensities
in atopic and nonatopic monocytes at 4 h (data not shown) and
24 h. At 24 h of IFN- stimulation, atopics showed an
enhanced IDO band intensity over nonatopics.
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RI ligation on
monocytes
To measure functionally active IDO, HPLC was done to determine the
breakdown product of tryptophan and kynurenine. Monocytes from atopics
and nonatopics were activated for 24 h with IgE/anti-IgE or
IFN- (1000 U/ml) or were left untreated. Cells were washed and
resuspended in HBSS containing 100 µM tryptophan and incubated at
37°C for an additional 4 h. Supernatants were harvested and
analyzed by HPLC for the presence of kynurenine (Table I). In nonatopic monocytes kynurenine was
detected significantly only upon stimulation with IFN-. In all but
one of the atopic donors, kynurenine was significantly detected after
FcRI stimulation and each donor responded with the induction of IDO
upon IFN- stimulation.
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Next we addressed the question of whether the degradation of
tryptophan by FcRI-activated atopic monocytes results in functional
abrogation of T cell proliferation. If so, the suppression should be
prevented by pharmacologic inhibition of IDO or supplementation of
tryptophan. 1-MT has been shown to be a competitive inhibitor for IDO
(26). Atopic monocytes were FcRI activated or left
untreated for 24 h and the medium was replaced. Autologous T cells
were added in the presence of different concentrations of 1-MT (Fig. 3, A and C).
Alternatively, cocultures were supplemented with 1 µg/10 µl of
tryptophan (which corresponds to 1x the tryptophan concentration found
in normal medium) 24, 48, and 72 h after T cell addition.
FcRI-activated atopic monocytes functionally suppressed T cell
proliferation, which could be prevented in a dose-dependent manner by
the addition of 1-MT. Atopic nonactivated monocytes did not show this T
cell-suppressive effect. The supplementation of
L-tryptophan to atopic activated monocytes and T
cells not only prevented T cell suppression but in addition increased T
cell proliferation compared with cultures with nonactivated monocytes.
In contrast, nonatopic monocytes did not show this suppressive T cell
effect (Fig. 3, B and D). To rule out an
immunostimulatory influence of 1-MT, T cell proliferation without
monocytes was done in different concentrations of 1-MT but no effect of
inhibitor was observed throughout the range of concentrations used
(data not shown).
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RI-activated cocultures
To confirm functional IDO activity in atopic activated
(IgE/anti-IgE and 22E7/GaMIgG) monocyte cocultures with T cells,
culture supernatants were harvested for tryptophan and kynurenine
24 h after the addition of T cells to stimulated or unstimulated
atopic monocytes (Fig. 4). Mean
tryptophan levels in the culture medium of stimulated (IgE/anti-IgE
and 22E7/GaMIgG) monocytes of three atopics decreased 37% of the
initial amount of tryptophan in the culture medium after 24 h,
whereas this amount was 7% for unstimulated monocytes with T cells.
IDO activity was detected in three of three cocultures with atopic
FcRI-activated monocytes (kynurenine with IgE/anti-IgE- and
22E7/GaMIgG-stimulated monocytes: mean, 11 ± 1.4 and 7 ± 4
µM, respectively), confirming the presence of functional IDO. In
unstimulated monocytes with T cells mean kynurenine levels were
0.7 ± 0.4 µM.
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and anti-IL-10 Ab
From previous studies it was known that IFN- is an inducer of
IDO in monocytes (27). In macrophages, even low amounts of
IFN- act in concert with CD40L from activated T cells to induce IDO,
whereas CD40L by itself does not seem to have an effect on the
degradation of tryptophan (18). Therefore, in our
cocultures, we had to rule out that IFN- was the main inducer of IDO
in monocytes. In addition, IL-10 was reported to be induced by FcRI
ligation on monocytes, a cytokine which is known to suppress T cell
proliferation (28). First, we determined the amount of
IFN- produced from cocultures of activated and nonactivated atopic
and nonatopic monocytes 24 h after the addition of T cells.
Moderate levels of IFN- were detected in atopic activated and
nonactivated cocultures (1133 ± 115 and 700 ± 400 pg/ml,
respectively). Lower levels were present in nonatopic activated and
nonactivated cocultures (85 ± 37 and 69 ± 28 pg/ml,
respectively). IL-10 production was determined from monocytes after
24 h of activation or nonactivation, medium replacement, and an
additional 24 h of incubation. Atopic activated monocytes produced
151 ± 46 pg/ml IL-10, whereas nonactivated atopics produced
57 ± 30 pg/ml. Nonatopic activated monocytes produced 102 ±
31 pg/ml and nonactivated nonatopics produced 30 ± 40 pg/ml. To
determine the role of IFN- and IL-10, neutralizing mAbs against
IFN- and IL-10 as well as a control mIgG1 Ab were added to atopic
activated cocultures (Fig. 5). Without
inhibitor of IDO (0 µM 1-MT), the suppression of T cell proliferation
by activated monocytes was not affected in the presence of either
anti-IFN- or IL-10 mAbs. IFN- activity was not detected
24 h after the addition of T cells, when neutralizing
anti-IFN- mAb was added to cocultures as determined by the
IFN- bioassay (data not shown). In the presence of 1000 µM 1-MT, T
cell proliferation was high in all conditions. These results imply that
in our cocultures the concentrations of IFN- and IL-10 do not seem
to influence IDO expression. The supplementation of these cultures with
tryptophan prevented T cell suppression in these conditions (data not
shown).
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RI-activated,
IL-10-depleted supernatants of atopic monocytes
It cannot be excluded that the amount of anti-IFN- Ab and
anti-IL-10 Ab in our coculture experiments could not have been
enough to neutralize these cytokines. Therefore, T cell proliferation
was studied without the influence of IFN- from T cells and IL-10
from activated monocytes (Table II).
Cultures of 24-h stimulated and nonstimulated atopic and nonatopic
monocytes were washed and incubated for an additional 24 h.
Supernatants were collected and depleted of IL-10 by magnetic
isolation (>90% of depletion in stimulated and nonstimulated atopics
as controlled by ELISA corresponding to 10 ± 4 and 7 ± 3
pg/ml, respectively; in nonatopics, IL-10 was below the limit of
detection). T cell proliferation induced by surface-bound anti-CD3
mAb was significantly inhibited in IL-10-depleted atopic
FcRI-activated supernatants, whereas the addition of tryptophan to
these supernatants prevented the T cell-suppressive effect. In
contrast, atopic nonactivated monocytic IL-10-depleted
supernatants supported T cell proliferation. In nonatopics, applying
these conditions, no influence of T cell proliferation on culture
conditions was seen. In theory, these results might indicate an
unanticipated immunostimulatory role for tryptophan itself. To exclude
this possibility, T cell proliferation was determined in normal medium
with and without the addition of tryptophan by surface-bound
anti-CD3 mAb. Applying these conditions, no additional
proliferative effect was observed by supplementing tryptophan (data not
shown). With a sensitive IL-10 bioassay, we could demonstrate that the
amounts of IL-10 after IL-10 supernatant depletion were below the
linear range of the dose-response curve and thus biologically inactive
(data not shown). In addition, exogenously added IL-10 in
concentrations of 10, 30, and 100 pg/ml did not suppress T cell
proliferation in control medium with surface-bound anti-CD3 (data
not shown). These results directly implicate the T cell suppressive
effect of 24-h FcRI-activated and 24-h cultivated monocytes by
degrading tryptophan from external supplies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-stimulated and unstimulated monocytes from a clinically healthy
person with an atopic family background and high FcRI expression on
monocytes. The results were extended to four additional atopic donors
of the same clinical criteria and serum parameters (high FcRI and
CD40; low CD23 monocyte surface expression; serum IgE levels >100
kU/L) and compared with donors without any atopic disease or atopic
family background (low FcRI and CD23; high CD40 monocyte surface
expression; serum IgE levels <100 kU/L). 1) We found IDO and
kynurenine 3-monooxygenase to be overexpressed in FcRI-activated
monocytes from atopic donors; 2) we demonstrated the functionality and
specificity of IDO expression in FcRI-stimulated atopic monocytes,
which results in the inhibition of T cell proliferation, whereas 3)
external supplies of tryptophan to atopic activated cocultures result
in an increase of T cell proliferation; and 4) we showed finally that
IL-10-depleted supernatants of 24-h FcRI-activated and 24-h
cultivated atopic monocytes do not support T cell proliferation as a
result of reduced tryptophan levels.
FcRI plays a central role in allergic diseases such as atopic
eczema/dermatitis syndrome and allergic asthma (29, 30).
In both conditions, immunohistochemical analysis is characterized by an
inflammatory T cell infiltrate. Kinetics of APC activation may exert
not only activatory functions on T cells but also negative or
tolerogenic signals that reduce lymphocyte proliferation. In line with
this complex interplay of cellular and metabolic factors, we found,
unexpectedly, the involvement of FcRI in the tryptophan degradation
pathway. IDO, the rate-limiting enzyme in the catabolism of tryptophan,
is a highly conserved enzyme which has been shown to be widely
distributed preferably in tissues that are involved in immune defense
or immunoprivilege (31). IDO production by
FcRI-activated monocytes could contribute to self-limitation of
immune responses. This is in line with our observation that only atopic
activated monocytes, which had been washed 24 h after stimulation
to eliminate proinflammatory cytokines (32, 33) or
supernatants thereof, mediate this T cell suppressive effect in vitro.
In addition, 4 h after FcRI cross-linking of atopic monocytes
no kynurenine was detected in culture supernatants by HPLC (data not
shown), whereas these levels increased significantly 24 h after
stimulation. The inhibition of IDO with increasing concentrations of
the IDO inhibitor 1-MT resulted in the proportional increase of T cell
proliferation. This confirms the central role of FcRI-induced IDO in
the regulation of T cells.
It has been reported that a metabolite of the tryptophan-degrading pathway, 3-hydroxyanthranilic acid, is able to induce apoptosis in monocytes/macrophages (34). However, in our studies, differences in the support for T cell proliferation between activated and nonactivated atopic monocytes was not due to differences in viability and apoptosis of monocytes as measured after 48 h of culture (data not shown).
M-CSF-derived macrophages respond to even low levels of IFN- in
combination with CD40L from activated T cells with the induction of
IDO, whereas monocytes were used as nonsuppressive accessory cells in
these experiments (18). In our experiments, CD40 surface
expression on monocytes was similar in the atopic and nonatopic group
and substantial IFN- production was observed in both cocultures.
Therefore, we blocked IFN- activity by adding anti-IFN- mAb
but still observed a T cell suppressive effect in atopic
FcRI-activated cocultures. Moreover, supernatants of 24-h
stimulated, washed, and 24-h incubated atopic activated monocytes
suppressed T cell activation which could be prevented by the addition
of tryptophan, further emphasizing that aggregation of FcRI alone is
sufficient to account for the T cell suppressive effect. In vivo, it is
conceivable that IFN- from atopic activated T cells plays a role in
the IDO induction of APCs other than monocytes. Macrophages and DCs are
the predominant APCs in microenvironments like the skin
(35) and are known to respond with the induction of IDO
upon IFN- exposure (16, 17).
We further ruled out the possibility that FcRI-induced IL-10 is the
suppressive agent for T cell proliferation in the cocultures. The
inhibitory function of IL-10 has been ascribed toward its
down-regulation of MHC class II and costimulatory molecules, which are
necessary in allogeneic or accessory function-dependent T cell
proliferation (36). We did not find a down-regulation of
costimulatory molecules on 48-h cultured atopic activated and
nonactivated monocytes such as CD80, CD83, CD86, CD40, and MHC class I
and II, which could account for the observed differences in T cell
proliferation (data not shown). In addition, by using an anti-IL-10
mAb in our FcRI-activated cocultures, we still observed inhibition
of T cell proliferation. The same was true when we neutralized both
IFN- and IL-10. Our data were confirmed by the fact that
IL-10-depleted, FcRI-activated monocyte supernatants suppressed T
cell proliferation, whereas this effect could be prevented by the
addition of tryptophan.
Interestingly, our experimental data further demonstrate an increase in
T cell proliferation with FcRI-activated monocytes if tryptophan is
in excess. However, this effect was pronounced only if FcRI surface
expression was high (>50%) and therefore not statistically
significant. An explanation could be that, in situations where
tryptophan concentrations are not limiting, proinflammatory cytokines
from activated monocytes meet optimal conditions to further accelerate
T cell proliferation. However, the supplementation of tryptophan does
not influence the expression of costimulatory molecules on monocytes
(data not shown).
In this study we provide the first evidence that FcRI induces the
degradation of tryptophan upon aggregation. The observation from our
proliferation studies suggests that FcRI+ APCs
communicate with T cells by regulating tryptophan concentrations in
defined metabolic compartments. T cells could respond with either
activation or arrest, depending on the level of tryptophan they
find.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dagmar von Bubnoff, Department of Dermatology, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. E-mail address: d.bubnoff{at}uni-bonn.de
3 H.d.l.S. and T.B. contributed equally to this work.
4 Abbreviations used in this paper: AD, atopic dermatitis; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-DL-tryptophan; h, human; m, mouse; GaMIgG, goat anti-mouse IgG; DC, dendritic cell; CD40L, CD40 ligand.
Received for publication February 19, 2002. Accepted for publication June 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and nonactivated (
) atopic monocytes
(1 x 106) along with or without anti-CD3 mAb
(
) after replacement of the medium. T cells were added in the
presence or absence of various concentrations of the IDO inhibitor
1-MT. Alternatively, cocultures were supplemented with tryptophan (1
µg/10 µl) 24 and 48 h after T cell addition. Seventy-two hours
later, T cell proliferation was measured. Data are expressed as the
mean ± SD of data from three experiments per donor. Each
experiment was done in triplicate (* p < 0.001).
Data are representative of four atopic donors. B and
D, T cell stimulation in vitro with anti-CD3 mAb and
autologous nonatopic monocytes. Data in both B and
D represent one donor. The experimental design was the
same as in A and C. By general linear
modeling analysis, no effects of nonatopic monocytes on T cell
proliferation were found applying these experimental conditions.
